InteractiveFly: GeneBrief

twins : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Before any discussion of Twins function, it will prove useful to have a look at its somewhat complicated enzymatic structure. Twins is the B-subunit (PR55) of a heterotrimeric protein phosphatase known as 2A (PP2A). The trimer is composed of three subunits -- the so-called A and B subunits, plus a catalytic subunit: mammalian PP2A then, is composed of a catalytic 36-kD subunit, a 65kD regulatory subunit (the A subunit or PR65) and the B-subunit. The dimeric core complex (catalytic and A-subunit) may be combined with one of a number of regulatory subunits (the B-subunits) ranging in size from 54 to 74kD. The activity and substrate specificity of the core complex is modulated by association with a B-subunit. In Drosophila, genes for the catalytic and PR65 (A) subunits of PP2A were cloned by sequence similarity to their mammalian counterparts (Mayer-Jaekel, 1992). Cloning of the B subunit PR55, coding for Twins protein, was accomplished through homology to the human homolog (Mayer-Jaekel, 1993 and Uemura, 1993).

Twins protein and the catalytic subunit of PP2A, the two proteins for which mutations have been isolated, have a multitude of functions, suggesting involvement in a number of pathways. Initially implicated as a regulator of mitotic progression (see Effects of mutation and Evolutionary homologs sections) , Twins has subsequently been implicated in three additional roles.

In the first of these, twins mutation causes a peripheral nervous system defect similar to that observed in numb and musashi mutations. How does this defect manifest itself? At this point, a quick look at the non-mutated phenotype is in order. Adult mechanosensory bristles normally consist of four cells: two cells of neural derivation (neuron and glial or thecogen cells), and two support cells ( a socket cell [tormogen] and a shaft cell [trichogen]). These four cells are derived from a single precursor called the sensory organ precursor (SOP). Mutations in twins (Shiomi, 1994), musashi (Nakamura, 1994) and numb result in an increase in the ratio of support cells, at the expense of neural cells.

One additional digression will clarify Twins's involvement in such defects, with respect to numb, taking us by way of tramtrack: mutation of tramtrack results in the the transformation of socket and shaft cells to neuron and glial cells. Ectopic expression of ttk has just the opposite effect: the transformation of neuron and glial cells into socket and shaft cells. Numb protein is asymmetrically distributed to neural precursor cells in the first division of the SOP, and Numb targets tramtrack, which then acts as a repressor of support cell fate in neural and glial progeny (Rhyu, 1994 and Guo, 1995). Interestingly, an N-terminal domain of Numb protein consists of residues predictive of a phosphotyrosine binding domain (PTB domain) (Uemura, 1989 and Zhong, 1996). It is possible that Twins protein regulates a Numb phosphoprotein target, and in this manner is involved in the peripheral nervous system defect.

A second function for Twins is found in wing morphogenesis. Mutation of twins causes a pattern duplication in Drosophila imaginal discs. Inactivation of twins induces the formation of extra wing blade anlagen in the posterior compartment. The duplication is mirror symmetrical, and the line of symmetry does not correspond to any of the known compartment borders (Uemura, 1993).

A third function for Twins has to do with eye morphogenesis. Mutations in the catalytic subunit of PP2P interfere with Ras pathway function in eye morphogenesis. PP2P mutation exacerbates defects caused by unregulated Ras function and diminishes defects cause by unregulated Raf function. Ras and Raf are both components of the EGF-receptor signaling pathway required for determination of photoreceptor fate in the compound eye (Wassarman, 1996).

These four defects, associated with mutant PP2A (defective mitosis, defective cell fate determination in the PNS, alteration of patterning in the wing, and interferance in Ras pathway function in eye morphogenesis) do not seem to share any common denominator, other than the involvement of PP2A. It is likely that PP2A substrates are different for each of these defects.

One of the most exciting aspects of PP2A research is PP2A involvement in differentiation. In terms of understanding developmental roles, Drosophila is the organism of choice, but in terms of an understanding of biochemical pathways, a multitude of other organisms are proving useful. Organisms as diverse as yeast, Acetabularia, Xenopus and mammals reveal an intimate interaction between PP2A in the cytoskeleton and cyclinB/cdc2. Different PP2A subunits in mammals react differently with respect to subcellular location, suggesting cell type specific functions (McCright, 1996). Other work shows that PP2A is involved in positively and negatively regulating response to neuropeptides with different responses depending on receptor subtype (Huang, 1996). Finally, PP2A plays a key role in the differentiation-dependent expression of the neurofilament gene, suggesting an involvement in neural cell fate (Sasahara, 1996). It is clear that PP2A takes on many developmental roles, and only the combination of investigative genetics and the resources of developmental biology coupled with biochemical techniques will reveal the complexities of PP2A's functions.

PP2A and GSK-3β act antagonistically to regulate active zone development

The synapse is composed of an active zone apposed to a postsynaptic cluster of neurotransmitter receptors. Each Drosophila neuromuscular junction comprises hundreds of such individual release sites apposed to clusters of glutamate receptors. This study shows that protein phosphatase 2A (PP2A) is required for the development of structurally normal active zones opposite glutamate receptors. When PP2A is inhibited presynaptically, many glutamate receptor clusters are unapposed to Bruchpilot (Brp), an active zone protein required for normal transmitter release. These unapposed receptors are not due to presynaptic retraction of synaptic boutons, since other presynaptic components are still apposed to the entire postsynaptic specialization. Instead, these data suggest that Brp localization is regulated at the level of individual release sites. Live imaging of glutamate receptors demonstrates that this disruption to active zone development is accompanied by abnormal postsynaptic development, with decreased formation of glutamate receptor clusters. Remarkably, inhibition of the serine-threonine kinase GSK-3beta completely suppresses the active zone defect, as well as other synaptic morphology phenotypes associated with inhibition of PP2A. These data suggest that PP2A and GSK-3beta function antagonistically to control active zone development, providing a potential mechanism for regulating synaptic efficacy at a single release site (Viquez, 2009).

This study demonstrates that the serine-threonine phosphatase PP2A is required in the presynaptic neuron for normal development and maturation of presynaptic release sites. This action of PP2A is opposed by the serine-threonine kinase GSK-3β, suggesting that this phosphatase/kinase pair co-regulate the phosphorylation state and activity of proteins that are required for proper synaptic development (Viquez, 2009).

At the Drosophila NMJ, the synaptic terminal of a motoneuron is a branched chain of synaptic boutons whose gross structure is strongly influenced by the cytoskeleton. Within each synaptic terminal, there are hundreds of individual synapses, neurotransmitter release sites with an active zone directly apposed to a cluster of postsynaptic glutamate receptors. Most studies in Drosophila have focused on genes controlling synaptic terminal development. However with the recent development of antibodies to the active zone component Bruchpilot and the essential glutamate receptor DGluRIII, a genetic analysis of active zone and postsynaptic density development is now feasible. Previous studies have demonstrated that PP2A acts in the motoneuron to control synaptic terminal morphology likely via regulation of microtubules. This study demonstrate that PP2A is also essential for the proper development of the individual synaptic unit, the active zone and glutamate receptor dyad (Viquez, 2009).

Presynaptic inhibition of PP2A impairs synaptic transmission, leading to a large decrease in quantal content. While investigating potential morphological explanations for defective transmitter release, it was observed that many glutamate receptor clusters are unapposed to the active zone protein Bruchpilot. This is not due to retraction of the presynaptic terminal, since apposed and unapposed GluR clusters are intermingled throughout the terminal in a salt and pepper pattern, and presynaptic structures such as synaptic vesicles are still apposed to the entire extent of the postsynaptic specialization. Instead, there is a defect at the level of the individual synapse. These GluR clusters may be unapposed to active zones, or may be apposed to abnormal active zones lacking Bruchpilot. Two lines of evidence suggest that these GluR clusters may be unapposed to active zones. First, Bruchpilot is required for the localization of T-bars to the active zone, so if many active zones are missing Bruchpilot, then there should be a decrease in the proportion of active zones with T-bars. However when PP2A is inhibited no change was seen in the proportion of active zones with T-bars. Second, with PP2A inhibition the number of Brp puncta is down, as is the density of active zones as defined by ultrastructural analysis. This suggests that there is not a large pool of active zones without Brp. Both of these findings suggest that there are fewer active zones, and that those active zones that form do contain Brp. If this is so, then why are GluR clusters present that are unapposed to active zones? This could be due either to a problem with synapse formation/maturation or maintenance. While it is not known which is the case, the model that there is a defect in the formation or maturation is preferred for the following reasons. First, unapposed receptors are more prevalent in the distal regions of the NMJ where new synapses tend to be added. Second, the unapposed receptors form quite small clusters, while newly forming GluR clusters in wild type are also quite small. Finally, live imaging reveals that fewer GluR clusters form late in larval development, demonstrating a defect in synapse formation (Viquez, 2009).

A model is proposed in which PP2A activity is required for the maturation phase of synapse development. In this view, at a wild type synapse a signal would initiate synapse formation, leading to postsynaptic clustering of glutamate receptors as well as transsynaptic interactions that form the tightly apposed pre- and postsynaptic membranes as seen in electron micrographs. Later, additional active zone components such as Brp would be recruited to the active zone, a process known to occur after GluR clustering. With PP2A inhibition, this unknown signal would still initiate synapse formation and induce GluR clusters. However, at some fraction of nascent synapses the maturation process would fail. The GluR clusters could be trapped in their small, immature state or lost, while the transsynaptic process leading to the tight apposition of pre- and postsynaptic membranes would also fail and Brp would not be recruited. The alternate model that synaptic maintenance is disrupted, and that unapposed GluR clusters are the remains of synapses at which the presynaptic terminal has been lost, cannot, however, be ruled out. Regardless of the precise mechanism, these data demonstrate that PP2A is required to ensure the correct apposition of structurally normal active zones and glutamate receptors at the synapse (Viquez, 2009).

PP2A is one of the major serine/threonine phosphatases in the cell, so inhibiting its function likely leads to hyperphosphorylation of many proteins. Hence, phenotypes could be due to the pleiotropic effects of misregulating numerous pathways. The data, however, argue for a good deal of specificity in the function of PP2A for the synaptic morphology phenotypes assayed. Inhibiting PP2A in the neuron leads to misapposed GluR clusters, a disrupted synaptic cytoskeleton, and an altered bouton morphology. Each of these phenotypes is suppressed when GSK-3β is inhibited. This suggests that these synaptic phenotypes are due to the misregulation of a pathway that is antagonistically regulated by PP2A and GSK-3β. Opposite phenotypes are not seen, however, when PP2A is overexpressed, suggesting that hyperphosphorylation affects this pathway more than hypophosphorylation. While genetic studies cannot prove that this phosphatase/kinase pair act directly on the same substrate, the simplest interpretation of the data is that PP2A and GSK-3β co-regulate the phosphorylation state and activity of a protein or proteins that are required for the proper development of active zones and the synaptic cytoskeleton. While these PP2A phenotypes are all suppressed by inhibition of GSK-3β, there is no suppression of the accumulation of synaptic material in the axon, a phenotype consistent with defects in axonal transport. Decreased transport of active zone material such as Brp is a plausible mechanism for the active zone defects in this mutant. However, the failure of GSK-3β inhibition to suppress the axonal transport phenotype demonstrates that the active zone maturation and axon transport phenotypes are genetically separable. Hence, the accumulation of Brp in the axon cannot be responsible for the defects in synaptic maturation (Viquez, 2009).

The identity of the pathway regulated by PP2A and GSK-3β is not known. One candidate substrate is APC2, which binds to and stabilizes the plus end of microtubules and which is a characterized substrate of both PP2A and GSK-3β. In hippocampal cells phosphorylation of APC by GSK-3β inhibits APC function and so disrupts microtubule stability and axon outgrowth. It was shown that loss of APC2 dominantly enhances the PP2A phenotype, which is consistent with the model from hippocampal cells that phosphorylating APC decreases its function. However, if APC2 were the key substrate, then it would be predicted that homozygous APC2 mutants, where all APC2 function is lost, should replicate the PP2A phenotype. However a synaptic apposition phenotype is not seen in recessive mutants for APC2 or in APC1/APC2 double mutants. Instead, the enhancement of the PP2A phenotype by the loss of APC2 suggests that APC2 promotes PP2A function, possibly in its role as a scaffolding molecule. Wnt signaling is candidate pathway for mediating these synaptic phenotypes because wnt signaling is required for normal Drosophila NMJ development and because GSK-3β and PP2A regulate the phosphorylation state of β-catenin in canonical wnt signaling. Inhibition of PP2A would be predicted to lead to hyperphosphorylation and destruction of β-catenin, thereby blocking wnt signaling. However it is unlikely that the PP2A synaptic phenotype is due to loss of canonical wnt signaling. First, this study found that expression of a constitutively active β-catenin does not suppress the PP2A synaptic phenotype but instead has a slight tendency to enhance the cytoskeletal defect. Second, APC functions as part of the destruction complex that leads to degradation of β-Catenin and block of wnt signaling, however APC mutants enhance rather than suppress the PP2A phenotype. These results are inconsistent with the model that the phenotype is due to decreased canonical wnt signaling through β-Catenin. However, the data are consistent with a role for β-catenin-independent wnt signaling. A third candidate substrate is Futsch, since it can be phosphorylated by GSK-3β and the effect of reduction of PP2A activity on Futsch structure is suppressed by reduction in GSK-3β levels. Continued genetic analysis may lead to the identification of the relevant substrate(s) that are antagonistically regulated by PP2A and GSK-3β to control synaptic development (Viquez, 2009).

There are interesting parallels between the function of PP2A and GSK-3β in the developing Drosophila neuromuscular system and in the pathogenesis of neurodegenerative diseases such as Alzheimer's. In Drosophila, PP2A antagonizes GSK-3β function to stabilize the synaptic cytoskeleton and promote synapse formation. In models of Alzheimer's Disease, PP2A and GSK-3β also act antagonistically, for example in regulating the phosphorylation state of tau. In addition, disruptions to the axonal cytoskeleton and synapse loss are early events in Alzheimer's pathogenesis. Characterizing the function of PP2A/GSK-3β in regulating cytoskeletal and synaptic integrity during development may provide insights into their role in regulating cytoskeletal and synaptic integrity during disease (Viquez, 2009).

Greatwall-phosphorylated Endosulfine is both an inhibitor and a substrate of PP2A-B55 heterotrimers

During M phase, Endosulfine (Endos) family proteins, that serve as protein phosphatase inhibitors, are phosphorylated by Greatwall kinase (Gwl), and the resultant pEndos inhibits the phosphatase PP2A-B55 (Twins), which would otherwise prematurely reverse many CDK-driven phosphorylations. This study shows that PP2A-B55 is the enzyme responsible for dephosphorylating pEndos during M phase exit. The kinetic parameters for PP2A-B55's action on pEndos are orders of magnitude lower than those for CDK-phosphorylated substrates, suggesting a simple model for PP2A-B55 regulation that is called inhibition by unfair competition. As the name suggests, during M phase PP2A-B55's attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. When Gwl is inactivated during the M phase-to-interphase transition, the dynamic balance changes: pEndos dephosphorylated by PP2A-B55 cannot be replaced, so the phosphatase can refocus its attention on CDK-phosphorylated substrates. This mechanism explains simultaneously how PP2A-B55 and Gwl together regulate pEndos, and how pEndos controls PP2A-B55 (Williams, 2014).

A model is presented for the function of the Gwl --> pEndos ---| PP2A-B55 module in cell cycle transitions. The major driver for transitions between interphase and M phase is the cyclic activation and degradation of M phase-promoting factor (MPF: Cdk1-Cyclin B). When activated (in part through a feed-forward autoregulatory loop involving the kinases Myt1 and Wee1 and the phosphatase Cdc25), MPF phosphorylates many substrates (CDKSs) that play key roles in M phase events. One such MPF substrate is the kinase Greatwall (Gwl), which in its active form phosphorylates Endosulfine (Endos). Phosphorylated Endos binds to and inhibits the phosphatase PP2A-B55. This inhibition protects MPF substrates, including components of the autoregulatory loop, from premature dephosphorylation during M phase entry. During M phase exit, MPF is inactivated by degradation of its Cyclin B component, and PP2A-B55 becomes reactivated to dephosphorylate many MPF substrates. M phase exit also requires the dephosphorylation and inactivation of both Gwl and Endos. This study show that PP2A-B55 catalyzes Endos dephosphorylation. The activities responsible for the inactivation of Gwl currently remain unknown (Williams, 2014).

Three independent lines of evidence indicate that the major activity contributing to the dephosphorylation of Gwl-phosphorylated Endos is a phosphatase that includes the three subunits of the PP2A-B55 heterotrimer. (1) Inhibitor specificities: all of the anti-Endos activity (whether in M phase or interphase) is sensitive to okadaic acid and calyculin A, and the majority is highly sensitive to fostriecin, suggesting that the catalytic subunit of the anti-Endos phosphatase is PP2A or its less abundant relatives PP4 or PP6. Furthermore, the facts that tautomycetin and the S68D Drosophila phosphomimetic Endos protein are such weak inhibitors of anti-Endos can be most easily explained if the anti-Endos phosphatase has a very low K_m for pEndos-below that for either inhibitor-as is the case for PP2A-B55. (2) Ablation of phosphatase activities: depletion of PP2A-A or PP2A-C from S2 tissue culture cells removes most anti-Endos from extracts, while depletion of PP4 from HeLa cells does not affect this activity. Drosophila larvae mutant for twins, which encodes the sole B55-type regulatory subunit of PP2A in flies, exhibit very little anti-Endos, while mutations in genes for other PPP family catalytic and regulatory subunits do not impede pEndos dephosphorylation in larval extracts. (3) Biochemical purification of the activity: anti-Endos copurifies with the anti-CDKS activity previously ascribed to PP2A-B55 heterotrimer, while on the same column it resolves away from other PPP phosphatase subunits. Furthermore, highly purified PP2A-B55 heterotrimer displays robust anti-Endos activity whose properties match that of the predominant anti-Endos activity in extracts (Williams, 2014).

Because most of the experiments were performed with extracts prepared from unsynchronous mammalian or Drosophila cultured cells (the large majority of which are in interphase), it is conceivable that a phosphatase other than PP2A-B55, that is activated only for a very short period following anaphase onset, is the enzyme responsible for dephosphorylating pEndos during M phase exit. This possibility is thought to be extremely remote for several reasons. (1) the inhibition by unfair competition mechanism that is proposed is sufficiently fast to account for the observed rapidity of M phase exit. (2) The characteristics of the M phase and interphase anti-Endos activities measured in Xenopus egg extracts are very similar; PP2A-B55's ability to dephosphorylate pEndos is therefore constitutive with respect to the cell cycle. (3) Because of its extremely low K_m, pEndos is so tightly bound to PP2A-B55 during M phase that no other hypothetical phosphatase would be able to inactivate it in a reasonable time frame; a theoretical simulation illustrates this point. There is no reason to postulate the existence of such a transiently activated phosphatase, and this possibility is incompatible with the observed relationship between PP2A-B55 and pEndos (Williams, 2014).

Some extracts contain a secondary activity (~10 to 30% of the total) that also targets the Gwl site in Endos. This minor activity was not characterized in detail, but some evidence suggests that it may be a form of PP1: it is relatively resistant to fostriecin, and RNAi depletion of PP1-87B, the most abundant form of the PP1 catalytic subunit, removes ~20 to 25% of anti-Endos activity from Drosophila S2 cell extracts (Williams, 2014).

A straightforward model is proposed for the relationship between pEndos and PP2A-B55. In essence, pEndos competes with a large class of CDK-catalyzed mitotic phosphosites (of which pCDKS and pSer50 Fizzy are examples) for access to the phosphatase active site. pEndos can successfully compete for the site because its affinity for PP2A-B55 is much higher than those of the competing substrates; that is, the K_m for pEndos is extremely low. However, PP2A-B55 dephosphorylates pEndos at a much slower rate (low k_cat) than the CDK phosphosites, so the tight interaction of PP2A-B55 and pEndos would be prolonged. Thus, pEndos would soon sequester the phosphatase from competing substrates, protecting such substrates against premature dephosphorylation. pEndos in this way permits M phase to begin and to be maintained as long as Gwl kinase is active. This mechanism is called 'inhibition by unfair competition'. In its essence, the model clarifies how PP2A-B55 can be 'off' during M phase for CDK-phosphorylated substrates, but 'on' at the same time for the Gwl-phosphorylated pEndos substrate (Williams, 2014).

In the context of the cell, the inhibition by unfair competition mechanism requires that pEndos be present in molar excess over PP2A-B55 in mitosis to account for the near-total absence of anti-CDKS activity observed during M phase. Considerable evidence exists that this requirement is met in many cell types. From quantitation on Western blots, it is estimated that the intracellular concentration of the PP2A B55 subunit to be between 100 and 250 nM and that of Endos to be between 500 nM and 1 μμ, depending on the cell type. The 5:1 ratio of Endos to PP2A-B55 observed is roughly consistent with estimates from other laboratories and with estimates from mass spectrometry studies of whole cell extracts, after accounting for all Endos and PP2A-B55 family members. For example, the Pax-DB database integrating the results of many mass spectrometry experiments on a variety of tissue types estimates that the abundance of Endos in Drosophila cells is 295 ppm while that of Twins (B55) is 123 ppm; for human cells, the integrated datasets yield values of 83.2 ppm for Endos-family proteins (ENSA and ARPP-19) and 23.1 ppm for B55-family proteins. Although this study did not determined the fraction of the total Endos protein that is phosphorylated during M phase, other investigators have found that this proportion is roughly 50% in extracts of synchronized mammalian tissue culture cells (perhaps some of which may not have been in M phase), and approaches 100% in frog egg M phase extracts. Sufficient 'headroom' thus exists to conclude that the molar concentration of pEndos during M phase in fact exceeds that of the PP2A-B55 phosphatase (Williams, 2014).

A major virtue of the inhibition by unfair competition model is that it explains not only how pEndos inhibits PP2A-B55 dephosphorylation of pCDKS-class substrates, but also how pEndos can itself become inactivated: the system has an automatic reset that is intrinsic to the mechanism that inactivates PP2A-B55 phosphatase in the first place. When Gwl is inactivated at anaphase onset, the pEndos dephosphorylated by PP2A-B55 can no longer be replaced. PP2A-B55 is now free to work on CDK-phosphorylated substrates and thus to promote the M phase-to-interphase transition. Experiments in which non-radioactive pEndos and radioactive pCDKS were added to the same tube of PP2A-B55, provide a direct in vitro test of the proposed mechanism by mimicking the events that occur during M phase exit. The results show that PP2A-B55 targets pCDKS only after the enzyme has dephosphorylated almost all of the pEndos (Williams, 2014).

M phase exit is surprisingly rapid given the large number of phosphorylations which must be reversed; in Xenopus cycling extracts, for example, the M phase-to-interphase transition is completed within less than 5 min. Even though the turnover rate of pEndos dephosphorylation by PP2A-B55 is quite slow (∼0.05 s⁻¹), the inhibition by unfair competition mechanism is nevertheless compatible with the rapidity of M phase exit. The reason is that Endos is present in cells only in at most a fivefold stoichiometric excess with respect to the phosphatase, as was just discussed. Each molecule of PP2A-B55 thus needs to dephosphorylate only a few molecules of pEndos to effect the M phase-to-interphase transition (Williams, 2014).

To explore this idea quantitatively, the dynamics of a simplified system was modeled, consisting solely of Endos and PP2A-B55 at estimated physiological conditions. Because the K_m of PP2A-B55 for pEndos is four-to-five orders of magnitude smaller than the K_m for other substrates, typified by pCDKS, and the pEndos concentration during M phase is in excess of the phosphatase concentration, it is suggested that the approximation of ignoring the binding of PP2A-B55 to other substrates is appropriate, and that this simplified model should capture the essence of the regulatory process. The calculation begins at the end of M-phase (t = 0) with the inactivation of Gwl. Almost all the PP2A-B55 is sequestered by pEndos until PP2A-B55-catalyzed dephosphorylation causes the pEndos concentration to decrease from 1 μM to ∼250 nM, the intracellular PP2A-B55 concentration. Beyond this point, PP2A-B55 is rapidly released from sequestration; half is available to act on other substrates within 74 s (Williams, 2014).

Cells likely harbor a pEndos-targeting phosphatase other than PP2A-B55; because this secondary activity is fostriecin-resistant, it is speculated that it may be a form of PP1. The time courses of pEndos dephosphorylation and PP2A-B55 desequestration are modified if this second phosphatase (again called PPX) is present at the same concentration as PP2A-B55 and acts on pEndos with kinetic parameters matching the activity of PP2A-B55 on pCDKS. In this case, the lag in PP2A-B55 desequestration is shortened, because PPX can dephosphorylate the pEndos that is not bound to PP2A/B55. However, the presence of PPX makes very little difference once the pEndos concentration decreases to the PP2A-B55 concentration of 250 nM, since almost all the remaining pEndos is bound to PP2A-B55. In this scenario, half of the PPA-B55 is released from pEndos by 38 s, producing a modest acceleration in M phase exit (Williams, 2014).

Our identification of PP2A-B55 as the anti-Endos phosphatase poses a dilemma in terms of M phase exit: the inhibition by unfair competition model describes events that must occur downstream of the inactivation of Gwl kinase, but what then can inactivate Gwl upon anaphase onset? According to the model, the rate-limiting Gwl-inactivating phosphatase cannot be PP2A-B55, because the system would be futile. Gwl inactivation could not proceed until pEndos was depleted, but pEndos cannot be inactivated as long as Gwl is active. An argument can be made that PP2A-B55 should be the Gwl-inactivating phosphatase because Gwl is activated by CDKs. Indeed, one group has recently reported some evidence in favor of this idea. However, it should be emphasized that PP2A-B55 does not act on all, or even perhaps the majority, of CDK phosphosites. The obvious resolution of this dilemma is therefore that a phosphatase other than PP2A-B55 inactivates Gwl during M phase exit. Preliminary evidence in support of this idea in two ways. First, purified PP2A-B55 in vitro is very inefficient in inactivating Gwl. Second, when active Gwl is added to interphase extracts from a variety of cell types, it becomes rapidly inactivated and dephosphorylated in a process that is insensitive to okadaic acid (and cannot therefore be controlled by PP2A-B55). Some evidence in favor of the existence of an okadaic acid-resistant anti-Gwl phosphatase has also recently been obtained by another group; the identity of this Gwl-inactivating enzyme is currently unknown (Williams, 2014).

PP2A-B55 promotes nuclear envelope reformation after mitosis in Drosophila

As a dividing cell exits mitosis and daughter cells enter interphase, many proteins must be dephosphorylated. The protein phosphatase 2A (PP2A) with its B55 regulatory subunit plays a crucial role in this transition, but the identity of its substrates and how their dephosphorylation promotes mitotic exit are largely unknown. This study conducted a maternal-effect screen in Drosophila melanogaster to identify genes that function with PP2A-B55/Tws in the cell cycle. Eggs that receive reduced levels of Tws and of components of the nuclear envelope (NE) often fail development, concomitant with NE defects following meiosis and in syncytial mitoses. Mechanistic studies using Drosophila cells indicate that PP2A-Tws promotes nuclear envelope reformation (NER) during mitotic exit by dephosphorylating BAF and suggests that PP2A-Tws targets additional NE components, including Lamin and Nup107. This work establishes Drosophila as a powerful model to further dissect the molecular mechanisms of NER and suggests additional roles of PP2A-Tws in the completion of meiosis and mitosis (Mehsen, 2018).

The molecular mechanisms mediating an orderly mitotic exit and return into interphase are much less understood than the mechanisms of mitotic entry. Moreover, while phosphatases are known to play crucial roles in promoting the mitosis to interphase transition, their specific contributions to the various events of this process remain largely unknown. This study has used the Drosophila system to search for and dissect the molecular events controlled by the PP2A-B55/Tws phosphatase in the cell cycle. Second-site noncomplementation screens have been used in various model organisms to identify functionally linked genes. This work builds on the power of second-site noncomplementation maternal-effect screens in Drosophila to identify close collaboration between genes in cell cycle regulation (Mehsen, 2018).

The genetic screen uncovered a strong link between PP2A-Tws and NER at the end of M phase. Simultaneously reducing the levels of Tws and Lamin in eggs using heterozygous mutations in mothers causes major defects in NER after meiosis II or after mitosis for embryos that initiated syncytial nuclear divisions. This result is striking considering that Lamin is not an essential protein in several cell types. Hypomorphic lamin mutants develop to adulthood, despite showing nuclear migration defects in photoreceptors and being female sterile. In lamin null mutants, neuroblasts continue to proliferate in the absence of detectable Lamin. In mice, the orthologous B-type lamins are dispensable for cell viability and proliferation, at least in keratinocytes; however, B-type lamins are essential in neurons. In general, B-type lamins may play a crucial role in structuring nuclei and withstanding force in cells where nuclear migration/positioning is essential. Such cell types include Drosophila eggs, where pronuclei must converge before fusing, and syncytial embryos, where nuclei migrate toward the cortex (Mehsen, 2018).

Using cells in culture, this study found that PP2A-Tws promotes the recruitment of several NE components after mitosis, namely BAF, Lamin, and Nup107. In Drosophila oogenesis, BAF phosphorylation by NHK-1 promotes the detachment of chromatin from the germinal vesicle during karyosome formation. The current work found that BAF requires NHK-1 phosphorylation sites to dissociate from chromatin during NEB, as in C. elegans. The current genetic, biochemical and imaging results suggest that phosphorylation of BAF by NHK-1 is reversed by PP2A-Tws to promote its recruitment on chromatin at the onset of NER. This is consistent with results in C. elegans that showed a role for PP2A in this process, although the relevant phosphorylation sites in BAF were not investigated and the PP2A adaptor subunit involved was unclear. Recent work shows that BAF plays a crucial role in holding chromosomes together just after anaphase to promote the assembly of the NE around a single nucleus (Samwer, 2017). The current findings suggest that PP2A-Tws dephosphorylates BAF to promote this function. The results also suggest that regulation of BAF phosphorylation by NHK-1 and PP2A-Tws regulates its ability to form complexes with Lamin. In vertebrates, BAF is known to interact with lamins via LEM domain proteins at the NE. Human BAF phosphorylation by VRK1/NHK-1 decreases its ability to interact with a LEM domain. LEM-domain proteins have also been shown to be phosphorylated to negatively regulate their ability to interact with BAF in X. laevis extracts. Thus, PP2A-B55 could dephosphorylate LEM proteins to further promote their association with BAF during NER, and this possibility should be investigated (Mehsen, 2018).

By inducing the recruitment of BAF on reassembling nuclei, PP2A-Tws likely promotes the recruitment of multiple downstream NE components. Nevertheless, PP2A-Tws likely has other targets in NER, possibly including Lamin and Nup107. Both proteins contain multiple CDK phosphorylation motifs, and PP2A-B55 enzymes have been shown to dephosphorylate many such sites efficiently. Moreover, it was observed that Lamin and Nup107 both associate with Tws. This study found that mutation of all CDK consensus sites in Lamin prevents lamina disassembly in mitosis and, although the attempted phospho-mimetic mutation of all sites did not disrupt lamina assembly in live cells, it increases Lamin solubility in cell lysates. The CDK sites on Lamin are grouped in two clusters flanking the coiled region, and some of these sites have already been shown to negatively regulate homotypic interactions of Lamin. The effect of mutating CDK sites in Nup107 was not examined. However, Nup107 is rapidly dephosphorylated at multiple sites during mitotic exit in human cells and dephosphorylation of at least one CDK site in Nup107 was shown to depend on PP2A-B55. However, although PP2A-B55 is capable of dephosphorylating several CDK sites, many of these sites are probably regulated mainly by another phosphatase in vivo. Moreover, numerous examples of PP2A-B55-dependent, non-CDK sites were recently identified. This is further exemplified in this study by the dephosphorylation of BAF by PP2A-Tws at a NHK-1 site, which cannot be a CDK site, as it lacks the proline residue in position +1. Nevertheless, with its positively charged amino acid residues in positions +2 to +4, this site resembles the recently defined PP2A-B55 consensus motif and a consensus motif for sites lacking a Pro residue at position +1 but that are rapidly dephosphorylated during mitotic exit (Mehsen, 2018).

Overall, PP2A-B55 appears to target multiple proteins, dephosphorylating them at various sites that depend on multiple kinases, to promote NER cooperatively. A recent phosphoproteomic study found that several proteins of the NE are particularly prone to rapid dephosphorylation during mitotic exit, in a process that likely involves other phosphatases. Much work remains to be done to fully dissect the mechanisms at play. The fact that NER is only delayed and not completely prevented when PP2A-Tws is silenced in cell culture could be due to an incomplete inactivation of PP2A-Tws inherent to the RNAi approach. Alternatively, other phosphatases may partially compensate for the loss of PP2A-Tws activity. Protein phosphatase 4 (PP4) may function in this way as it has been shown to dephosphorylate BAF in human cells. In addition, protein phosphatase 1 enzymes likely contribute to NER in Drosophila, as they promote this process through multiple mechanisms in vertebrates, including the dephosphorylation of Lamin B (Mehsen, 2018).

The screen results point at other functions of PP2A-Tws in the completion of M phase that remain to be explored, although some of the genetic interactions identified could reflect roles of PP2A-Tws unrelated to mitotic regulation. Preliminary, unpublished results suggest that the genetic interaction between tws and CycB3 reflects their collaboration in the completion of meiosis. Interestingly, this study uncovered genetic interactions between tws and genes that encode nucleocytoplasmic transport factors. Mutations in the gene for Cse1/CAS, which transports importin α back to the cytoplasm to promote its function in nuclear import, enhances tws-dependent embryonic lethality. Conversely, mutations in embargoed (emb), which encodes the nuclear export factor Crm1, rescues tws-dependent embryonic lethality. These results suggest that active nuclear import plays an important role in NER or other aspects of the establishment of interphase nuclei after mitosis and/or meiosis, presumably by promoting the nuclear localization of crucial enzymes or structural factors. Defining these factors and the regulation of their nucleocytoplasmic transport during mitotic exit should be the topic of future investigations (Mehsen, 2018).

This work has used a genetic strategy to search for the roles of PP2A-Tws in the cell cycle in vivo. PP2A-Tws was found to promote NER, and studies have begun to dissect the mechanisms at play. This study opens the door to the use of Drosophila to gain a better mechanistic understanding of NER at the molecular level. Moreover, it will be a powerful system to further dissect the functions of PP2A-Tws and other phosphatases in the coordination of mitotic exit (Mehsen, 2018).

Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis

In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase in flies, worms and vertebrates. It has been hypothesized that CycB3-Cdk1 may be responsible for APC/C activation in meiosis but this remains to be determined. Using Drosophila, this study found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, CycB3 was found to promote APC/C activity and anaphase in cells in culture. CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. These results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis (Garrido, 2020).

Mitosis and meiosis (collectively referred to as M-phase) are distinct modes of nuclear division resulting in diploid or haploid products, respectively. In animals, both require the breakdown of the nuclear envelope, the condensation of chromosomes and their correct attachment on a microtubule-based spindle, where chromosomes are under tension and chromatids are held together by cohesins. Progression through these initial phases requires multiple phosphorylation events of various protein substrates by mitotic kinases including Cyclin-Dependent Kinases (CDKs) activated by their mitotic cyclin partners. M-phase completion from this point (mitotic exit) requires the degradation of mitotic cyclins, and the dephosphorylation of several mitotic phosphoproteins by phosphatases including Protein Phosphatase 2A (PP2A). Mitotic exit begins with the segregation of chromosomes in anaphase. In mitosis, sister chromatids segregate. In meiosis I, replicated homologous chromosomes segregate, and in the subsequent meiosis II, sister chromatids segregate. Nuclear divisions are completed with the reassembly of a nuclear envelope concomitant with the decondensation of chromosomes. How mitosis and meiosis are alike and differ in the molecular mechanisms of their exit programs is not completely understood (Garrido, 2020).

Chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase. By catalysing the addition of ubiquitin chains on the separase inhibitor securin, the APC/C targets it for degradation by the proteasome. As a result, separase cleaves cohesins, allowing separated chromosomes to migrate towards opposing poles of the spindle. Activation of the APC/C in mitosis requires its recruitment of its co-factor Cdc20. This recruitment can be prevented by the Spindle-Assembly Checkpoint (SAC), a complex mechanism that allows the sequestration of Cdc20 until all chromosomes are correctly attached on the spindle. Cdc20 binding to the APC/C is also inhibited by its phosphorylation at CDK sites. Phosphatase activity is then required to dephosphorylate Cdc20 and allow its binding of the APC/C for its activation of anaphase. In addition, phosphorylation of the APC/C itself is required to allow Cdc20 binding. Phosphorylation of APC3/Cdc27 and APC1 is key to this process. Phosphorylation of APC3 at CDK sites promotes the subsequent phosphorylation of APC1, inducing a conformational change in APC1 that opens the Cdc20 binding site. However, the precise identity of the kinase(s) involved in this process in vivo is unknown (Garrido, 2020).

At least 3 types of cyclins contribute to M-phase in animals: Cyclins A, B and B3. The Cyclin A type (A1 and A2 in mammals) can activate Cdk1 or Cdk2 and is required for mitotic entry, at least in part by allowing the phosphorylation of Cdc20 to prevent its binding and activation of the APC/C. This allows mitotic cyclins to accumulate without being ubiquitinated prematurely by the APC/C and degraded. The Cyclin B type (B1 and B2 in mammals) also promotes mitotic entry and is required for mitotic progression by allowing the phosphorylation of several substrates by Cdk1. Mammalian Cyclin B3, which can associate with both Cdk1 and Cdk2, is required for meiosis but its contribution to mitosis is less clear in view of its low expression in somatic cells. Drosophila possesses a single gene for each M-phase cyclin: CycA (Cyclin A), CycB (Cyclin B) and CycB3 (Cyclin B3) that collaborate to ensure mitotic progression by activating Cdk1. Genetic and RNAi results suggest that they act sequentially, CycA being required before prometaphase, CycB before metaphase and CycB3 at the metaphase-anaphase transition. CycA is the only essential cyclin, as it is required for mitotic entry. CycB and CycB3 mutants are viable, but mutations of CycB and CycB3 are synthetic-lethal, suggesting redundant roles in mitosis. However, mutation of CycB renders females sterile due to defects in ovary development, and mutant males are also sterile (Garrido, 2020).

Drosophila CycB3 associates with Cdk1 and is required for female meiosis (Jacobs, 1998). In Drosophila, eggs normally stay arrested in metaphase I of meiosis until egg laying triggers entry into anaphase I and the subsequent meiosis II. However, CycB3 mutant eggs predominantly stay arrested in meiosis I (Bourouh, 2016). In addition, silencing CycB3 expression in early embryos delays anaphase onset during the syncytial mitotic divisions (Yuan, 2015). Cyclin B3 is also required for anaphase in female meiosis of vertebrates and worms. In mice, RNAi Knock-down of Cyclin B3 in oocytes inhibits the metaphase-anaphase transition in meiosis I. Recently, two groups independently knocked out the Cyclin B3-coding Ccnb3 gene in mice and found that they were viable but female-sterile due to a highly penetrant arrest in meiotic metaphase I. In C. elegans, the closest Cyclin B3 homolog, CYB-3 is required for anaphase in meiosis and mitosis (Garrido, 2020).

How Cyclin B3 promotes anaphase in any system is unknown. One possibility is that it is required for Cdk1 to phosphorylate the APC/C on at least one of its activating subunits, APC3 or APC1. This has not been investigated. Another possibility is that inactivation of Cyclin B3 leads to an early mitotic defect that activates the SAC. This appears to be the case in C. elegans, because inactivation of the SAC rescues normal anaphase onset in the absence of CYB-3. However, in Drosophila, inactivation of the SAC by the mutation of mad2 did not eliminate the delay in anaphase onset observed when CycB3 is silenced in syncytial embryos. Similarly, in mouse oocytes, silencing Mad2 does not rescue the meiotic metaphase arrest upon Cyclin B3 depletion. In other studies, SAC markers on kinetochores did not persist in metaphase-arrested Ccnb3 KO oocytes, and SAC inactivation by chemical inhibition of Mps1 did not restore anaphase. Finally, it is also possible that Cyclin B3 is required upstream of another event required for APC/C activation, for example the activation of a phosphatase required for Cdc20 dephosphorylation and subsequent recruitment to the APC/C (Garrido, 2020).

This study has investigated how CycB3 promotes anaphase in Drosophila. Several lines of evidence are reported indicating that CycB3 directly activates the APC/C in both meiosis and mitosis (Garrido, 2020).

Altogether, the results strongly suggest that CycB3-Cdk1 directly activates the APC/C by phosphorylation, promoting its function at the metaphase-anaphase transition in meiosis and in both maternally driven early embryonic mitoses and somatic cell divisions. This regulation is likely mediated by the phosphorylation in the activation loop of APC3 by CycB3-Cdk1 that ultimately promotes the recruitment of the Cdc20-type co-activators Fizzy and Cortex. Previous work has shown that APC3 phosphorylation and APC/C activation by cyclin-CDK complexes require their CKS subunit (see Cks30A). CKS subunits can act as processivity factors that bind phosphorylated sites to promote additional phosphorylation by the CDK. Thus, phosphorylation of APC3 would prime the binding of a cyclin-CDK-CKS complex to promote the additional phosphorylation of APC1, allowing for Cdc20 binding. It has been shown that mutation of phosphorylation sites into Asp or Glu residues cannot substitute for the presence of phosphate in the CKS binding site. Therefore, it was not possible to generate a mutation in APC3 that would have mimicked phosphorylation at S316 to enhance cyclin-CDK-CKS binding. Such a mutation in APC3, if it were possible, would have potentially rescued APC/C activity in the absence of CycB3 according to this model. However, it is likely that this analysis did not detect all phosphorylation sites in the APC/C. Thus, the possibility cannot be exclustion that other phosphorylation events, mediated by CycB3-Cdk1 or another kinase, may be required for complete APC/C activation. For example, other phosphorylation events have been proposed to regulate APC/C localization. It is even formally possible that CycB3-Cdk1 is required to activate another proline-directed kinase that phosphorylates APC3 at S316. The interdependence between CycB3 and Tws that this study uncovered may reflect a role of PP2A-Tws in the recruitment of Cdc20 co-activators to the APC/C. Cdc20 must be dephosphorylated at CDK sites before binding the APC/C, and in human cells both PP2A-B55 and PP2A-B56 promote this event (Garrido, 2020).

CycB3 is strongly required for APC/C activation in meiosis and in the early syncytial mitoses, and to a lesser extent in other mitotic divisions, despite the presence of two additional mitotic cyclins, CycA and CycB, capable of activating Cdk1. There are many possible reasons for this requirement. Overexpression of stabilized forms of CycA or CycB can block or slow down anaphase, suggesting that they may interfere with APC/C function in this transition. However, under normal expression levels, CycA or CycB or both may contribute to activate the APC/C like CycB3. CycB3 mutant flies develop until adulthood, which implies that the APC/C can be activated to induce anaphase in at least a vast proportion of mitotic cells, and this activation could be mediated by CycA and/or CycB. CycA is essential for viability and CycB mutants show strong female germline development defects, complicating the examination of potential roles for these cyclins at the metaphase-anaphase transition. Thus, in principle, the requirements for CycB3 in female meiosis, in embryos and in mitotic cells in culture could merely reflect the need for a minimal threshold of total mitotic cyclins. This possibility is considered unlikely because CycB3 is expressed at much lower levels than CycB in early embryos. Moreover, while maternal heterozygosity for mutations in CycB3 and tws causes a metaphase arrest in embryos, heterozygosity for mutations in CycB and tws does not cause embryonic defects. In fact, genetic results suggest that the function of CycB is antagonized by PP2A-Tws in embryos, while CycB3 and PP2A-Tws collaborate for APC/C activation in embryos. Thus, although it is possible that CycA and CycB can participate in APC/C activation, CycB3 probably has some unique feature that makes it particularly capable of promoting APC/C activation (Garrido, 2020).

By what mechanism could CycB3 be particularly suited for APC/C activation? Cyclins can play specific roles by contributing to CDK substrate recognition or by directing CDK activity in space and time. This study did not investigate the precise nature of the molecular recognition of the APC/C by CycB3. It may be that CycB3 possesses a specific binding site for the APC/C that is lacking in CycA and CycB. Another possibility is that differences in localization between cyclins dictate their requirements. In particular, while CycA and CycB are cytoplasmic in interphase, CycB3 is nuclear. It is surmised that the nuclear localization of CycB3 may help concentrate CycB3 in the spindle area upon germinal vesicle breakdown, when the very large oocyte enters meiosis. In future studies, it will be interesting to compare the ability of different mitotic cyclins to activate the APC/C and to determine the molecular basis of potential differences (Garrido, 2020).

In any case, the results show that CycB3 activates the APC/C and that this regulation is essential in Drosophila. Cyclin B3 has been shown to be required for anaphase in female meiosis of insects (Drosophila), worms (C. elegans) and vertebrates (mice). It is tempting to conclude that the activation of the APC/C is a function of Cyclin B3 conserved in all these species. However, in C. elegans embryos, the metaphase arrest upon CYB-3 (Cyclin B3) inactivation requires SAC activity. The underlying mechanism and whether it also occurs in other systems remain to be determined. However, CYB-3 plays roles in C. elegans that have not been detected for Cyclin B3 in flies or vertebrates, including a major role in mitotic entry, where CYB-3 mediates the inhibitory phosphorylation of Cdc20. In this regard, C. elegans CYB-3 may be more orthologous to Cyclin A. Yet, given that Cyclin B3 is required for anaphase in a SAC-independent manner in flies and mice, it seems reasonable to suggest that the direct activation of the APC/C by Cyclin B3 is conserved in vertebrates (Garrido, 2020).

The AMPK-PP2A axis in insect fat body is activated by 20-hydroxyecdysone to antagonize insulin/IGF signaling and restrict growth rate

In insects, 20-hydroxyecdysone (20E) limits the growth period by triggering developmental transitions; 20E also modulates the growth rate by antagonizing insulin/insulin-like growth factor signaling (IIS). Previous work has shown that 20E cross-talks with IIS, but the underlying molecular mechanisms are not fully understood. This study found that, in both the silkworm Bombyx mori and the fruit fly Drosophila melanogaster, 20E antagonized IIS through the AMP-activated protein kinase (AMPK)-protein phosphatase 2A (PP2A) axis in the fat body and suppressed the growth rate. During Bombyx larval molt or Drosophila pupariation, high levels were found of 20E activate AMPK, a molecular sensor that maintains energy homeostasis in the insect fat body. In turn, AMPK activates PP2A, which further dephosphorylates insulin receptor and protein kinase B (AKT), thus inhibiting IIS. Activation of the AMPK-PP2A axis and inhibition of IIS in the Drosophila fat body reduced food consumption, resulting in the restriction of growth rate and body weight. Overall, this study revealed an important mechanism by which 20E antagonizes IIS in the insect fat body to restrict the larval growth rate, thereby expanding understanding of the comprehensive regulatory mechanisms of final body size in animals (Yuan, 2020).

This study has discovered that in the insect fat body, 20E activates AMPK in two ways: By up-regulating the mRNA levels of all three AMPK subunits and by inducing energy stress to activate AMPK (Yuan, 2020).

The transcription levels of all three AMPK subunits, the protein level of AMPKα, and the phosphorylation level of AMPKα were all elevated in the Bombyx fat body at ∼4M and in the Drosophila fat body during pupariation, showing developmental profiles that were consistent with those of 20E signaling. Both the gain-of-function and loss-of-function experiments further demonstrate that AMPK is transcriptionally activated by 20E signaling. According to preliminary data, 20E-EcR-USP does not directly induce the expression of the AMPK-PP2A subunit genes, and further studies should be performed to investigate the detailed mechanisms whereby the 20E-triggered transcriptional cascade is involved in this transcriptional activation (Yuan, 2020).

20E is well known to act through the insect larval central nervous system (CNS) to induce wandering behavior and escape from food. Moreover, 20E slowly reduces insect feeding behavior and, thus, food consumption. Nevertheless, both the induction of wandering behavior and the reduction of feeding behavior can cause energy stress, such as sugar starvation, which ultimately increases the cellular AMP/ATP ratio, leading to the activation of AMPK. According to the Bombyx fat body results at ~4M, such a poor nutrition status promoted AMPK activity and inhibited IIS. Altogether, 20E slowly induces a sugar starvation-like condition to activate AMPK in the fat body by modulating CNS-controlled feeding behavior and wandering behavior in insects (Yuan, 2020).

IIS is an anabolic pathway, while AMPK accounts for catabolism, thus it naturally exists a mutual inhibition between IIS and AMPK. AMPK and PP2A might affect each other, and the AMPK-PP2A axis has been documented in mammalian cells. This study confirmed that the AMPK-PP2A axis exists in the Drosophila fat body, linking the antagonism of IIS by 20E (Yuan, 2020).

In addition to the dephosphorylation of AKT by PP2A, PP2A also dephosphorylates S6K, playing a key role in the attenuation of IIS and its downstream TORC1 activity. These studies determined that PP2A not only dephosphorylates AKT and inhibits TORC1 activity but also dephosphorylates InR and inactivates PI3K, showing that PP2A inhibits IIS starting from the dephosphorylation of InR. It is hypothesized that PP2A might dephosphorylate InR, PI3K, and AKT and, thus, inhibit IIS in an integrative manner (Yuan, 2020).

Finally, this study demonstrated that 20E activates the AMPK-PP2A axis to antagonize IIS in the insect fat body. After blocking either AMPK or PP2A, 20E no longer antagonizes IIS in the fat body. In summary, the AMPK-PP2A axis in the insect fat body is activated by 20E to antagonize IIS (Yuan, 2020).

Considering the similar regulatory functions in the antagonism of IIS by 20E, the possible relationship between miR-8/Ush and AMPK-PP2A was examined. Via bioinformatics prediction, it was found that miR-8 does not target AMPK or PP2A transcripts. Meanwhile, preliminary data showed that overexpression of AMPKCA or PP2ACA did not affect Ush expression in the fat body. Thus, it is supposed that AMPK-PP2A should function in parallel with miR-8 in the antagonism of IIS by 20E. It is concluded that the AMPK-PP2A axis is a crucial, but not a unique, pathway linking 20E to IIS (Yuan, 2020).

Previous studies and the current results together indicate that, similar to the inhibition of IIS in the larval fat body, activation of the fat body AMPK-PP2A axis reduces food consumption and thus restricts growth rate and body size in Drosophila. In other words, the AMPK-PP2A axis and IIS in the fat body play opposite developmental roles in regulating the larval growth rate and body size, and one crucial reason should be the modulation of feeding behavior and thus food consumption (Yuan, 2020).

The insect fat body, which is analogous to the mammalian liver, functions as an energy reservoir and nutrient sensor to regulate developmental timing. Fat body-derived amino acid signals, which involve Slimfast (the amino acid transporter) and TORC1 signaling, reactivate quiescent neuroblasts and finally control larval growth by regulating the synthesis and release of insulin/IGF. In addition to amino acid-dependent signals, certain other growth-promoting factors, such as CCHamide-2 and Unpaired 2, secreted from the fat body also affect the brain to remotely control insulin/IGF secretion in Drosophila. Moreover, IIS acts as the center of energy and nutrition response and positively regulates the larval growth rate partially by inhibiting autophagy in the Drosophila fat body. In contrast, 20E negatively regulates the larval growth rate by impeding IIS in the Drosophila fat body. Interestingly, preliminary results suggest that the AMPK-PP2A axis had little effect on fat body autophagy during normal feeding conditions and that TORC1 in the fat body plays little role in regulating the larval growth rate (Yuan, 2020).

It is likely that activation of the AMPK-PP2A axis and the inhibition of IIS in the fat body might affect the nutritional and endocrinal functions of this tissue. These changes in the fat body should cause the reduction of food consumption, resulting in the restriction of growth rate and body size. Investigating the detailed molecular mechanisms of how food consumption and its related feeding behavior and wandering behavior are regulated by hormonal and nutritional signals in the fat body might open a new window for understanding the regulatory mechanisms of final body size in insects. In future, it is worthwhile to examine whether the CNS. as well as neuropeptides and neurotransmitters, are involved in this regulation. Taking these data together, a model is proposed in which 20E antagonizes IIS by activating the AMPK-PP2A axis in the fat body to restrict the larval growth rate in insects. This study expands understanding of the comprehensive regulatory mechanisms underlying final body size determination in animals (Yuan, 2020).

REGULATION

Twins/PP2A regulates aPKC to control neuroblast cell polarity and self-renewal

Asymmetric cell division is a mechanism for generating cell diversity as well as maintaining stem cell homeostasis in both Drosophila and mammals. In Drosophila, larval neuroblasts are stem cell-like progenitors that divide asymmetrically to generate neurons of the adult brain. Mitotic neuroblasts localize atypical protein kinase C (aPKC) to their apical cortex. Cortical aPKC excludes cortical localization of Miranda and its cargo proteins Prospero and Brain tumor, resulting in their partitioning into the differentiating, smaller ganglion mother cell (GMC) where they are required for neuronal differentiation. In addition to aPKC, the kinases Aurora-A and Polo also regulate neuroblast self-renewal, but the phosphatases involved in neuroblast self-renewal have not been identified. Thus study reports that aPKC is in a protein complex in vivo with Twins, a Drosophila B-type protein phosphatase 2A (PP2A) subunit, and that Twins and the catalytic subunit of PP2A, called Microtubule star (Mts), are detected in larval neuroblasts. Both Twins and Mts are required to exclude aPKC from the basal neuroblast cortex: twins mutant brains, twins mutant single neuroblast mutant clones, or mts dominant negative single neuroblast clones all show ectopic basal cortical localization of aPKC. Consistent with ectopic basal aPKC is the appearance of supernumerary neuroblasts in twins mutant brains or twins mutant clones. It is concluded that Twins/PP2A is required to maintain aPKC at the apical cortex of mitotic neuroblasts, keeping it out of the differentiating GMC, and thereby maintaining neuroblast homeostasis (Chabu, 2009).

Drosophila aPKC regulates neuroblast cell polarity and neuroblast self-renewal, however understanding of how aPKC is regulated is far from complete. Several kinases regulate neuroblast cell polarity and cell fate, but the identity of opposing phosphatases have remained elusive. This study identified Twins as part of a protein complex containing aPKC. Twins is a regulatory subunit of PP2A, and this study also shows that the catalytic subunit of PP2A, Mts, is immunoprecipitated by aPKC. Furthermore, mts and twins mutants have similar defects in neuroblast cell polarity and expansion in neuroblast numbers. This strongly suggests that the Twins/PP2A complex regulates neuroblast polarity and self-renewal (Chabu, 2009).

The primary defect in twins mutant neuroblasts is an expansion of aPKC from the apical cortex to the basal cortex, and this ectopic aPKC is active based on its ability to exclude Miranda from the basal cortex. Twins/PP2A may promote apical Baz localization, similar to the role of PP2A in promoting Baz/Par-3 apical localization in epithelia; a reduced level of apical Baz in neuroblasts may lead to failure to localize all cortical aPKC at the apical cortex and hence ectopic basal aPKC. Alternatively, PP2A may keep active aPKC from the basal cortex by directly dephosphorylating aPKC at its N-terminus, consistent with the role of mammalian PP2A in dephosphorylating aPKCλ/ζ (Nunbhakdi-Craig, 2002). In support of this model, overexpression of aPKC lacking its N-terminus (aPKCΔN) displaces Miranda from the basal cortex into the cytoplasm, similar to twins mutant neuroblasts (Chabu, 2009).

How does Twins regulate neuroblast self-renewal? Ectopic active aPKC causes formation of supernumerary neuroblasts, as does reduced levels of the basal cortical protein Miranda. twins mutant neuroblasts have both ectopic basal cortical aPKC and a loss of basal cortical Miranda. It is likely that the primary defect causing supernumerary neuroblasts is ectopic aPKC, because reducing aPKC levels in twins mutants can rescue both basal Miranda targeting and the formation of supernumerary neuroblasts. This is in contrast to the role of another phosphatase, PP4, in regulating Miranda localization independent of aPKC (Sousa-Nunes, 2009; Chabu, 2009 and references therein).

It has been shown that Dap160, a protein related to mammalian Intersectin, is apically localized and required to anchor aPKC at the apical cortex (Chabu, 2008). This study has shown that Twins is also required for tight apical localization of aPKC. A major difference, however, is that Dap160 directly stimulates the activity of aPKC, so that in dap160 mutant neuroblasts the ectopic basal aPKC is inactive and unable to exclude Miranda from the cortex. In contrast, twins mutants have ectopic basal aPKC that remains active and thus can drive Miranda off the cortex. This supports the conclusion, from biochemical experiments, that Twins does not stimulate aPKC activity. However, the possibility that another regulatory subunit can target PP2A to aPKC in the absence of Twins cannot be excluded (Chabu, 2009).

Neuroectoderm cells of the optic lobe undergo a progressive differentiation to adopt a neuroblast fate. twins mutant optic lobes show a dramatic increase in optic lobe neuroblast numbers, suggesting that Twins normally functions to inhibit precocious neuroblast fate in the optic lobe neuroectoderm cells. How does Twins normally suppress precocious neuroectodermal-to-neuroblast differentiation? This study has show that at least one pathway utilizes aPKC to regulate neuroectoderm differentiation; twins mutant optic lobe with reduced active aPKC has a less severe phenotype compared to their twins mutant counter parts. Another pathway that has been implicated in the differentiation of neuroectoderm cells to neuroblast is the Janus Kinase/Signal transducer and activation of transcription (JAK/STAT) pathway. JAK/STAT signaling functions in neuroectoderm cells inhibits expression of proneural genes, thereby blocking precocious neuroblast differentiation. Twins/PP2A could act positively at any point in the JAK/STAT–proneural pathway, or in an independent pathway in promoting the neuroectodermal-to-neuroblast transition in the optic lobe (Chabu, 2009).

Protein Interactions

The 55 kDa regulatory subunit of Drosophila protein phosphatase 2A is located in the cytoplasm at all cell cycle stages, by the criterion of immunofluorescence. No significant changes were detected in protein phosphatase activity during the nuclear division cycle of syncytial embryos. However, cell cycle function of the enzyme is suggested by the mitotic defects exhibited by two Drosophila mutants, aar¹ and twins^P, defective in the gene encoding the 55 kDa subunit. The reduced levels of the 55 kDa subunit correlate with the loss of protein phosphatase 2A-like, okadaic acid-sensitive phosphatase activity of brain extracts against caldesmon and histone H1 phosphorylated by p34cdc2/cyclin B kinase, but not against phosphorylase a. Thus the mitotic defects of aar¹ and twins^P are likely to result from the lack of dephosphorylation of specific substrates by protein phosphatase 2A (Mayer-Jaekel, 1994).

Sex combs reduced (SCR) is a Drosophila Hox protein that determines the identity of the labial and prothoracic segments. In search of factors that might associate with SCR to control its activity and/or specificity, a yeast two-hybrid screen was performed. A Drosophila homolog of the regulatory subunit (B'/PR61) of serine-threonine protein phosphatase 2A (dPP2A,B') specifically interacts with the SCR homeodomain. The N-terminal arm within the SCR homeodomain has been shown to be a target of phosphorylation/dephosphorylation by cAMP-dependent protein kinase A and protein phosphatase 2A, respectively. In vivo analyses reveal that mutant forms of SCR mimicking constitutively dephosphorylated or phosphorylated states of the homeodomain are active or inactive, respectively. Inactivity of the phosphorylated mimic form is attributable to impaired DNA binding. Specific ablation of dPP2A,B' gene activity by double-stranded RNA-mediated genetic interference results in embryos without salivary glands, an SCR null phenotype. These data demonstrate an essential role for Drosophila PP2A,B' in positively modulating SCR function (Berry, 2000).

PP2A exists as a multisubunit enzyme complex in a variety of organisms and cell types. The enzyme complex is composed of a catalytic and a scaffold subunit, which together form a core dimer that then associates with one of a number of regulatory subunits to constitute a trimeric enzyme complex. Regulatory subunits of PP2A are encoded by at least three unrelated gene families: B (PR55), B' (PR61) and B" (PR72). Each family consists of several members, which in addition can give rise to a number of splice variants, thereby greatly increasing the variety of distinct trimeric enzyme complexes. Several lines of evidence suggest that the regulatory subunits of PP2A may serve as specific adaptors that confer substrate specificity to the core domain of PP2A. Specific interaction of dPP2A,B' with the SCR homeodomain, as documented here, therefore reflects its potential of reversibly recruiting SCR into the PP2A complex (Berry, 2000).

The two phosphorylatable residues (T and S) within the N-terminal arm of the SCR homeodomain appear to be conserved, since at least one such site has been found in all SCR homologs from other species, except for PS12-B of Atlantic salmon. The homeodomain of PS12-B in fact seems more closely related to ANTP than to SCR. In vivo results suggest that in developing embryos, SCR is functionally inactive when the N-terminal arm of its homeodomain is phosphorylated and is active upon dephosphorylation. These results may have important implications for the functional specificity of homeotic proteins in general: since ANTP has a glutamine instead of threonine at position 6 (which is well conserved in all the SCR homologs), it is proposed that the differential modification of this residue plays an important role in determining the functional specificity of these two homeotic proteins (Berry, 2000).

The data from the functional knockout of dPP2A,B' by dsRNA interference prove unequivocally that expression of dPP2A,B' is essential for the functional activity of SCR. Genetic studies in Drosophila have shown that Ras-1 activity positively modulates the function of Hox proteins such as proboscipedia (PB) and SCR -- a finding that suggests that covalent modifications triggered by Ras-1-mediated signals might influence the activity of PB and SCR. The catalytic subunit of dPP2A has been identified as a component operating downstream of Ras-1. The observation that the functional activity of SCR is dependent upon the presence of dPP2A,B' seems to provide a missing link, suggesting that Ras-1 might influence the activity of SCR via dPP2A (Berry, 2000).

A model is proposed to describe the regulation of SCR activity: in a cell, where SCR function is not required continuously, the protein is locked in an inactive state by phosphorylation of residues 6 and/or 7 within the N-terminal arm of the homeodomain. The fact that, in older embryos, SCR is present but is no longer able to induce the expression of its target gene forkhead, may be a case in point. In response to positive signals, SCR-specific protein phosphatase (dPP2A) becomes activated, possibly through a signaling cascade involving Ras-1. In the absence of positive signals, or when negative signals, e.g. DPP and SP1 prevail, specific dPP2A activity is inhibited and, as a result, SCR can no longer be maintained in its dephosphorylated state. PKA or PKA-like enzymes will phosphorylate residues 6/7 of the SCR homeodomain, thus abrogating the ability of SCR to bind to its target genes. A delicate balance between the activities of SCR-specific PP2A and specific protein kinases would thus allow a cell to fine-tune SCR activity (Berry, 2000).

Planar cell polarization requires Widerborst, a B' regulatory subunit of protein phosphatase 2A

widerborst (wdb), a B' regulatory subunit of PP2A, located at 98A6-8 and distinct from Protein phosphatase 2A at 85F (the B subunit of PP2A), has been identified as a conserved component of planar cell polarization mechanisms in both Drosophila and in zebrafish. The German name Widerborst means something stubborn or recalcitrant (derived from wider, meaning against, and borst, meaning bristle). PP2A is a holoenzyme that consists of a catalytic (C) subunit, an A regulatory subunit and one of a large family of B, B' or B'' subunits. The latter subunits are thought to regulate the activity of the C subunit and provide substrate specificity. In metazoans, the B' subunits have diverged into two related subclasses. The central regions of these proteins are strongly conserved, but they differ at their N and C termini. The protein encoded by widerborst is more closely related to the human alpha, ß and epsilon subunits (62%-66% identity) than to the ß or gamma subunits (52%-59% identity). Its sequence suggests that wdb might influence tissue polarization by regulating PP2A activity with respect to specific targets (Hannus, 2002).

In Drosophila, wdb acts at two steps during planar polarization of wing epithelial cells. It is required to organize tissue polarity proteins into proximal and distal cortical domains, thus determining wing hair orientation. It is also needed to generate the polarized membrane outgrowth that becomes the wing hair. Widerborst activates the catalytic subunit of PP2A and localizes to the distal side of a planar microtubule web that lies at the level of apical cell junctions. This suggests that polarized PP2A activation along the planar microtubule web is important for planar polarization. In zebrafish, two wdb homologs are required for convergent extension during gastrulation, supporting the conjecture that Drosophila planar cell polarization and vertebrate gastrulation movements are regulated by similar mechanisms (Hannus, 2002).

Widerborst is unique in that it does not colocalize with other tissue polarity proteins at the cell cortex. Instead, as cortical polarization is beginning (18-24 hours apf), it is found on microtubules on the distal side of each wing epithelial cell. Furthermore, it localizes there before obvious organization of proximodistal cortical domains, and its polarization is independent of them. Strikingly, at earlier developmental stages (7-9 hours apf), Wdb polarity is not distal but proximal. These dynamic shifts in Wdb polarity and their independence from previously described tissue polarity genes suggest the existence of a novel polarization mechanism (Hannus, 2002).

How might Wdb operate to specify cortical polarity? When Wdb activity is reduced, components of the cortical domains like Dsh and Fmi accumulate uniformly around the cell cortex at high levels. By contrast, disruption of Frizzled signaling interferes with the accumulation of Dsh and Fmi at the cell cortex. This suggests that Wdb is not required to activate Frizzled signaling, but rather is important for making it asymmetric (Hannus, 2002).

The genetic data indicate that Wdb exerts its activity by activating the catalytic subunit of PP2A with respect to specific substrates, and the localization of Wdb suggests that it does so on the distal side of the planar microtubule web. Which proteins might be targeted for dephosphorylation by Widerborst? One possibility is Dishevelled. Heterozygosity for wdb strongly suppresses the mwh phenotype of dsh¹ suggesting that, during tissue polarization, these two proteins act antagonistically. Dishevelled cortical localization correlates with hyperphosphorylation, and the cortical localization of Dsh is certainly expanded in Wdb dominant-negative expressing cells. Supporting this possibility, two-hybrid experiments have indicated that Dishevelled can physically interact with a Xenopus B' regulatory subunit. If Wdb normally acted by antagonizing Dsh, then the dominant-negative might overactivate Frizzled signaling and cause defects in tissue polarity. This model is not easily reconcilable with a role for the distal localization of Wdb; one might naïvely expect an antagonist of Frizzled signaling to accumulate proximally instead of distally. Nevertheless, although the early distal localization of Wdb is suggestive, it has not been proven that distal localization is relevant to cortical polarization; for example, Wdb might have a role in transducing the Frizzled signal, for which distal localization is not required (Hannus, 2002).

What might be the importance of Wdb binding to the distal microtubule web? Binding to the cytoskeleton might simply allow stable distal localization of an otherwise diffusible cytosolic molecule. More interesting, this association raises the possibility that Widerborst directs the dephosphorylation of a microtubule-associated protein. Consistent with this idea, the structure of the planar microtubule web is disrupted by dnWdb expression. PP2A activity is important for the accumulation of stable microtubules, presumably through the effects of PP2A on the phosphorylation state of MAPs. Microtubule stability can affect the binding of microtubule motor proteins and can contribute to polarized protein delivery. In the wing, microtubules have been suggested to play important roles in hair polarity; depending on the time at which vinblastine is added, vinblastine treatment of pupal wings causes either failure of hair outgrowth or the formation of multiple wing hairs. Polarized dephosphorylation of MAPs within the planar microtubule web might bias the transport of vesicles containing components of the proximodistal cortical domains. At later stages, it might also help direct transport of components of the hair formation machinery to the distal side of the cell, or promote the stability of microtubules in the outgrowing hair. This model for Widerborst action could provide a single explanation for its effects on hair outgrowth and on cortical polarity. Identification of the relevant Widerborst substrate(s) should greatly advance understanding of the cell biology of tissue polarization (Hannus, 2002 and references therein).

The data also support other studies indicating that B' alpha/epsilon regulatory subunits antagonize the classical Wnt signaling pathway. Experiments in Xenopus embryos and tissue culture cells have shown that increasing the level of a B' alpha subunit inhibits Wnt signaling and causes ventralization. Consistent with this, experiments in zebrafish show that reducing Wdb levels causes dorsalization of embryos. Although Wdb, like Frizzled and Dishevelled, is a shared component of both planar polarization and classical Wnt signaling pathways, it probably has different functions in each; during classical Wnt signaling, the B' alpha is thought to act downstream of Dishevelled, forming part of a ß-catenin degradation complex that plays no role in planar polarity signaling (Hannus, 2002).

The observation that widerborst is needed both for distal polarization of Drosophila wing hairs and for convergent extension movements during zebrafish gastrulation points to a conserved role for Wdb in regulating tissue polarity in development. Furthermore, it provides additional evidence supporting the conjecture that components of the planar polarization pathway in Drosophila are also used to control cell polarity and movement during vertebrate gastrulation. To date, the evidence for this is based on analysis of various dsh constructs and, more recently, on the analysis of vang/stbm and rhoA during vertebrate gastrulation. The identification of Wdb as another shared component provides further evidence that this signaling cascade is indeed conserved between Drosophila and vertebrates. Additional experiments will have to address the precise function(s) of vertebrate wdb homologs and where wdb acts in the genetic pathway regulating vertebrate gastrulation movements (Hannus, 2002).

A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway

Members of the Hedgehog (Hh) family of signaling proteins are powerful regulators of developmental processes in many organisms and have been implicated in many human disease states. This study reports the results of a genome-wide RNA interference screen in Drosophila cells for new components of the Hh signaling pathway. The screen identified hundreds of potential new regulators of Hh signaling, including many large protein complexes with pleiotropic effects, such as the coat protein complex I (COPI), the ribosome and the proteasome. The multimeric protein phosphatase 2A (PP2A) and two new kinases, the D. melanogaster orthologs of the vertebrate PITSLRE and cyclin-dependent kinase-9 (CDK9) kinases, were identified as Hh regulators. A large group of constitutive and alternative splicing factors, two nucleoporins involved in mRNA export and several RNA-regulatory proteins were identified as potent regulators of Hh signal transduction, indicating that splicing regulation and mRNA transport have a previously unrecognized role in Hh signaling. Finally, it was shown that several of these genes have conserved roles in mammalian Hh signaling (Nybakken, 2005).

Phosphorylation is associated with the activities of at least five components of the Hh pathway: Fu, Cos, Smo, Su(fu) and Ci. Little is known about the kinases that phosphorylate Su(fu) and Fu, but at least two sites in Cos are phosphorylated by Fu, and several kinases are involved in phosphorylating Ci and Smo, including PKA-C1, CkIalpha and Sgg. But no phosphatase has been implicated in Hh signaling, and a previous RNAi screen did not identify any phosphatases involved in Hh signaling. The screen identified microtubule star (mts), which encodes the D. melanogaster PP2A catalytic subunit, as a gene that substantially reduced Hh signaling when targeted by RNAi. PP2A is a multimeric enzyme that consists at minimum of the catalytic subunit, a regulatory A subunit (encoded by CG33297 in D. melanogaster) and a B subunit principally involved in substrate selection. The B-subunit family in D. melanogaster is represented by the gene twins (tws), the B' family by the genes widerborst (wdb) and PP2A-B', and the B" family by CG4733. All the PP2A component dsRNAs were obtained and tested from a dsRNA library and additional, distinct dsRNAs to these components were generated and tested. In addition to confirming the mts result, it was found that both the original-library dsRNA and three new, unique dsRNAs targeting wdb all reduced Hh signaling. This indicates that Wdb is likely to be the B subunit that targets Mts to its substrate in the Hh signaling pathway. This hypothesis is in agreement with recent findings from Xenopus laevis, where the wdb ortholog encoding B56e has been found to regulate Hh signaling. In addition, some PP2A-B' amplicons cause a reduction in reporter activity averaging ~30%, indicating that they may have a partially redundant role in targeting PP2A to its Hh pathway substrate (Nybakken, 2005).

To determine whether PP2A acts on Cos, whether overexpression of cos and mts results in similar phenotypes was examined. When overexpressed in Hh-stimulated clone 8 cells, cos completely abrogates Hh signaling, reducing it to near uninduced levels, whereas overexpression of mts reduces Hh signaling by 40%. Thus, Mts and Cos have different overexpression profiles and do not seem to regulate Hh signaling in the same way. The overexpression phenotype of mts was compared with those of cos and 14 other hits from the screen, including the fu, Cdk9 and Pka-C1 kinases. Overexpressing cos in uninduced cells further reduces background signaling, whereas mts overexpression doubled reporter activity, although these levels are still very low compared with the Hh-activated state. Of the 18 other genes tested, only Pka-C1 overexpression had an effect on Hh reporter activity similar to that of mts: doubling of reporter activity in the Hh-uninduced state and a 50% reduction of activity in the Hh-stimulated state. It is therefore possible that PKA-C1 and Mts act on similar substrates. Because several studies have identified Ci as a substrate of PKA-C1, Mts could also be acting on Ci, perhaps removing inhibitory phosphates in response to Hh stimulation (Nybakken, 2005).

This screen allowed the grouping of the ribosome, proteasome, COPI complex and PP2A phosphatase as important regulators of Hh signaling, none of which had been identified as Hh regulators in vivo. Notably, some of the components identified in the screen had already been implicated in aspects of Hh signaling. For instance, the gene encoding eRF1, a translational regulator, was identified in a screen for modifiers of a gain-of-function smo allele, and polyhomeotic and additional sex combs have both been shown to modify ectopic hh expression phenotypes. These results open many new avenues for investigation of Hh signaling. In particular, elucidation of the Hh pathway substrates affected by PP2A will be important in defining the role of dephosphorylation in Hh signaling. Finally, the paradigm of Hh signaling would change substantially if further investigation determines that alternative splicing and mRNA regulation do have vital roles in Hh signaling (Nybakken, 2005).

The protein phosphatase PP2A-B' subunit Widerborst is a negative regulator of cytoplasmic activated Akt and lipid metabolism in Drosophila

Inappropriate regulation of the PI3-kinase/PTEN/Akt kinase-signalling cassette, a key downstream target of insulin/insulin-like growth factor signalling (IIS), is associated with several major human diseases such as diabetes, obesity and cancer. In Drosophila, studies have recently revealed that different subcellular pools of activated, phosphorylated Akt can modulate different IIS-dependent processes. For example, a specific pool of activated Akt within the cytoplasm alters aspects of lipid metabolism, a process that is misregulated in both obesity and diabetes. However, it remains unclear how this pool is regulated. The protein phosphatase PP2A-B' regulatory subunit Widerborst (Wdb), which coimmunoprecipitates with Akt in vivo, selectively modulates levels of activated Akt in the cytoplasm. It alters lipid droplet size and expression of the lipid storage perilipin-like protein LSD2 in the Drosophila ovary, but not in epithelial cells of the eye imaginal discs. It is concluded that isoforms of PP2A-B' can act as subcellular-compartment-specific regulators of PI3-kinase/PTEN/Akt kinase signalling and IIS, potentially providing new targets for modulating individual subcellular pools of activated Akt in insulin-linked disease (Vereshchagina, 2008).

The signalling cassette involving Class I phosphatidylinositol 3-kinase (PI3K), phosphatase and tensin homologue on chromosome 10 (PTEN) and Akt (also known as protein kinase B or PKB) is part of a major intracellular kinase cascade that regulates multiple cellular functions including metabolism, growth, proliferation and survival. It responds to a variety of stimuli, such as insulin, other growth factors including PDGF and FGF, and attachment to the extracellular matrix. Upon activation, PI3K catalyses the formation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃] from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂]. PtdIns(3,4,5)P₃ is a lipid second messenger, which in turn recruits the PH-domain-containing Akt protein kinase from the cytosol to the plasma membrane. Here it is activated through phosphorylation at Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and at Ser473 (or Ser505 in the unique Drosophila Akt kinase, Akt1) by PDK2, which is thought to be the rictor-mTOR complex. Once activated, Akt subsequently phosphorylates multiple targets, leading to its numerous downstream effects (Vereshchagina, 2008).

Misregulation of Akt and its cellular targets is linked to several major human diseases. For example, cellular insulin resistance is associated with reduced signalling by the PI3K/PTEN/Akt cassette and is an important defect in individuals suffering from Type 2 diabetes. By contrast, hyperactivation of this cassette, most notably through loss-of-function mutations in the tumour suppressor PTEN, which converts PtdIns(3,4,5)P₃ back to PtdIns(4,5)P₂, is strongly associated with many forms of human cancer (Vereshchagina, 2008 and references therein).

Molecular genetic studies in Drosophila have resulted in several fundamental insights into the regulation and functions of the PI3K/PTEN/Akt-signalling cassette. Not only has this work highlighted the central importance of nutrient-regulated insulin/insulin-like growth factor signalling (IIS) in controlling the activity of this cassette and cell growth, but it has also revealed a critical downstream link with the nutrient-sensitive mTOR-signalling cascade, which regulates several cellular processes including protein translation and autophagy. Furthermore, studies in invertebrates have indicated roles for PI3K/PTEN/Akt and mTOR in ageing, cell polarity and neurodegeneration, functions that all appear to be conserved in mammals and which might involve a combination of cellular and metabolic defects (Vereshchagina, 2008).

If the role of PI3K/PTEN/Akt in insulin-linked diseases is to be fully understood, it is essential to determine how this single signalling cassette regulates so many different cellular functions. One important part of the explanation is presumably the existence of cell-type-specific downstream-signalling targets that perform different roles. However, recent work, much of it again initiated in flies, has indicated that Akt activity can also be differentially regulated in specific subcellular domains and that these subcellular pools of activated Akt can control different processes. For example, precise regulation of Akt activity at the apical membrane of epithelial cells by localised PTEN is required for normal apical morphology in higher eukaryotes. By contrast, cytoplasmic activated Akt appears to be required for transcription of specific IIS target genes and regulation of lipid metabolism and droplet size in nurse cells of the Drosophila female germ line (Vereshchagina, 2006). These observations have highlighted the importance of finding the molecules that regulate different pools of activated Akt in vivo, because their modulation might alter specific functions of IIS in health and disease more selectively (Vereshchagina, 2008).

In a screen for novel phosphatase regulators of IIS, Widerborst (Wdb), one of the B' regulatory subunits of the protein phosphatase PP2A, was identified as a negative regulator of the PI3K/PTEN/Akt-signalling cassette. Although wdb is essential for cell viability in some tissues, wdb mutant cells in the germ line and follicular epithelium of the ovary are viable and display phenotypes that are similar to those seen in PTEN mutant ovaries. This study shows that Wdb and Drosophila Akt1 physically interact in the ovary, and that within this tissue, Wdb regulates the subcellular pool of activated Akt1 in the cytoplasm. This study therefore highlights an important new function for PP2A-B' subunits in selectively modulating certain IIS-dependent processes by controlling signalling in a specific subcompartment of the cell (Vereshchagina, 2008).

Several lines of evidence confirm that Wdb controls IIS activity and Akt1 phosphorylation state. First, when overexpressed, wdb genetically modifies phenotypes produced by altered IIS signalling, rescuing a lethal PTEN mutant combination and modifying the effects of FOXO in the eye. Second, loss-of-function wdb mutations produce very similar phenotypes to PTEN mutations in nurse cells, elevating levels of cytoplasmic pAkt1 and LSD2 [a Perilipin/ADRP homologue that regulates lipid metabolism, and inducing an abnormal accumulation of lipid droplets. Third, although wdb mutations do not independently appear to have strong effects on growth, they do suppress growth phenotypes produced by reduced Akt1 signalling both in mutant follicle cells homozygous for the Akt1¹ allele and in animals carrying a hypomorphic viable combination of Akt1 alleles. Genetic interactions with the PP2A catalytic subunit Mts in the eye indicate that these effects are dependent on the PP2A regulatory activity of Wdb (Vereshchagina, 2008).

Coimmunoprecipitation experiments revealed that Akt1 and Wdb form a complex in ovaries, the tissue in which the most obvious effects of wdb on pAkt1 levels are seen. The data suggest that one isoform of Wdb affects IIS within a complex containing Akt1, presumably by directly modulating the phosphorylation state of this molecule. This regulatory interaction appears to be evolutionarily conserved, because several studies in mammalian cell culture have shown that a PP2A-type activity controls Akt phosphorylation at Ser473, the equivalent position to Ser505 in Drosophila Akt1. PP2A-B' activity has been implicated in this process. Furthermore, mammalian PP2A can dephosphorylate Akt in vitro. The phosphorylation state of Thr308 might also be affected by PP2A. However, current tools do not allow determination of the phosphorylation state of Thr342 (the equivalent position to Thr308 in mammalian Akt) in wdb mutant cells in ovaries. Nevertheless, this study adds to the current understanding of the effects of PP2A on Akt by showing for the first time that at least one PP2A-B' isoform can act as a pool-specific suppressor of activated Akt. It is thought that that this property is likely to be shared by some mammalian PP2A-B' isoforms (Vereshchagina, 2008).

Unlike several other previously characterised components of the IIS cascade, the effects of wdb mutations on IIS appear to be tissue specific. Although pAkt1 levels are strongly upregulated in wdb mutant nurse cells and follicle cells, they appear unaffected in clones within the eye. PP2A is a broad-specificity protein phosphatase, which is selectively targeted to specific signalling molecules by regulatory subunits such as Wdb. Wdb has already been shown to be involved in several signalling events, including those regulating apoptosis and the Hedgehog (Hh) pathway, pathways that might be implicated in the wdb mutant phenotype observed in the eye imaginal disc (Vereshchagina, 2008).

How can Wdb have such a central IIS-regulatory role in the ovary, but show no detectable effect on this pathway in the developing eye? It seems unlikely that wdb mutant cells in the eye die too rapidly to observe changes in Akt1 phosphorylation, because wdb clones are seen in posterior positions within eye imaginal discs, which must have formed many hours previously. The IIS cascade is active in this tissue, because mutations altering IIS produce significant effects on growth in the eye disc. However, unlike in nurse cells, activation of IIS in the developing eye primarily leads to cell surface accumulation of pAkt1, at least in pupae. Surface-localised activated Akt1 may normally be sufficient to promote eye growth, since a myristoylated membrane-anchored form of Akt1 dominantly induces overgrowth in this and other tissues. One possible explanation for these data is therefore that cytoplasmic pAkt1 levels in the eye are restricted by other unknown molecules in addition to Wdb in this tissue, so loss of wdb here has little effect, whereas increased expression can still modify the FOXO phenotype (Vereshchagina, 2008).

In this context, at least two other phosphatases might be involved in Akt1 regulation. First, there is a second isoform of PP2A-B' in flies [called PP2A-B', CG7913 or Well-rounded (Wrd); that is most closely related to mammalian PP2A-B'γ isoforms. Simian virus 40 small t antigen acts as a specific inhibitor of mammalian PP2A-B'γ, stimulating phosphorylation of Akt and other targets, and thereby promoting growth. Reduced PP2A-B'γ activity has also been linked to the establishment and progression of melanomas (Vereshchagina, 2008).

Surprisingly, a recent report suggests Wrd is nonessential. Unless it acts redundantly with Wdb, it cannot therefore play a significant role in growth regulation). Analysis of the PP2A catalytic subunit Mts, using a dominant-negative construct, indicates that this enzyme enhances the effects of FOXO and is important in normal growth regulation in the eye, perhaps consistent with the idea that the two PP2A-B' isoforms do act redundantly. Alternatively, Mts may perform some of its growth regulatory functions independently of PP2A-B' (Vereshchagina, 2008 and references therein).

A second candidate negative regulator of Akt is the novel phosphatase PHLPP, which directly dephosphorylates human Akt at Ser473 and Drosophila Akt1 at Ser505 in cell culture, a function that may be disrupted in some tumours. Drosophila PHLPP could therefore control pAkt1 accumulation at the cell surface and perhaps reduce the amount of pAkt1 that can diffuse into the cytoplasm in tissues such as the eye. Since loss of wdb in either follicle cells or nurse cells is sufficient to elevate levels of cytoplasmic pAkt1, PHLPP presumably does not play such an important role in these cell types (microarray data suggest that PHLLP is not expressed at detectable levels in the adult ovary) (Vereshchagina, 2008).

Interestingly, the data in the ovary suggest further variable tiers of pAkt1 control. In nurse cells, loss of PTEN leads to accumulation of pAkt1 and LSD2 in the cytoplasm, but most PTEN mutant follicle cell clones do not show these phenotypes, presumably because other pAkt1 regulators such as Wdb play a more dominant role in these cells. No good explanation is available for how genetically identical clones can show such phenotypic variability. There is no obvious correlation with clone size or position in the small minority of PTEN-mutant follicular clones where pAkt1 and LSD2 upregulation is observed (Vereshchagina, 2008).

Because perilipin, the mammalian LSD2 orthologue, is thought to be regulated via insulin-dependent transcriptional and post-translational mechanisms, it is proposed that the increased LSD2 expression seen in PTEN mutant nurse cell clones results from similar effects of IIS on this molecule in flies. An alternative explanation is that increased IIS promotes excess triacylglyceride (TAG) synthesis and that LSD2 is only indirectly upregulated to permit proper packaging of these triacylglycerides into lipid droplets. Analysis of wdb mutant follicle cell clones does not support this latter model, since these clones strongly upregulate LSD2 expression, but do not show obvious changes in lipid droplet accumulation (Vereshchagina, 2008).

When wdb is overexpressed in the differentiating eye, the external structure of the eye becomes more disorganised and there is a slight reduction in overall eye size. Since this effect is not noticeably suppressed by co-overexpressing Akt1, it seems unlikely to be caused by reduced IIS. Unlike PTEN mutant follicle cells, wdb mutant follicle cells are not noticeably larger than their wild-type neighbours. Furthermore, although low level constitutive expression of Wdb in a pupal-lethal PTEN mutant background can rescue these flies to viability, the rescue may be explained by altered metabolism, because the rescued flies are still larger than normal. All these observations are consistent with the model that Wdb modulates cytoplasmic pAkt1 and has less of an effect on cell surface pAkt1, which is thought to be the primary regulator of normal growth. Wdb shows a relatively strong genetic interaction with the IIS-regulated transcription factor FOXO and this is completely suppressed by Akt1, raising the possibility that low levels of pAkt1 in the cytoplasm may play an important part in controlling FOXO activity (Vereshchagina, 2008).

Although wdb does not appear to modulate growth significantly under normal IIS-signalling conditions, mutations in wdb do enhance growth when Akt1 activity is reduced. Viable Akt1 mutant animals are larger in the presence of a heterozygous wdb mutation, while the Akt1¹ recessive growth phenotype in follicle cells is strongly suppressed by wdb. Interestingly, it has been reported that mutations in foxo have no effect on growth in otherwise normal animals, but that when IIS is reduced in chico mutants, which produce small adults, this phenotype is partially suppressed by loss of foxo function. The current data are consistent with this result, and may indicate that growth regulation in chico flies relies more on cytoplasmic pAkt1 and its effects on downstream targets like FOXO than it does in normal flies (Vereshchagina, 2008).

In conclusion, the identification of a PP2A-B' subunit as a novel cell-type-specific regulator of IIS within a specific subcellular compartment highlights the importance of studying the subcellular control of this signalling pathway in multiple cell types in vivo. Akt activation also promotes lipid synthesis and droplet formation in many mammalian cell types. This is likely to involve similar regulatory control mechanisms for cytoplasmic pAkt to those uncovered in flies. This work therefore raises new issues concerning the underlying causes of IIS-associated disease. For example, excess accumulation of lipid and obesity could be linked to selective changes in cytoplasmic pAkt control and might therefore be modulated by specific PP2A-B' subunits. Developing a better understanding of this form of regulation could therefore suggest new strategies for disease-specific treatments of IIS-linked disorders in the future (Vereshchagina, 2008).

A screen for modifiers of Hedgehog signaling in Drosophila melanogaster identifies swm and mts

Signaling by Hedgehog (Hh) proteins shapes most tissues and organs in both vertebrates and invertebrates, and its misregulation has been implicated in many human diseases. Although components of the signaling pathway have been identified, key aspects of the signaling mechanism and downstream targets remain to be elucidated. An enhancer/suppressor screen was performed in Drosophila to identify novel components of the pathway and 26 autosomal regions were identified that modify a phenotypic readout of Hh signaling. Three of the regions include genes that contribute constituents to the pathway: patched, engrailed, and hh. One of the other regions includes the gene microtubule star (mts) that encodes a subunit of protein phosphatase 2A. mts is necessary for full activation of Hh signaling. A second region includes the gene second mitotic wave missing (swm). swm is recessive lethal and is predicted to encode an evolutionarily conserved protein with RNA binding and Zn⁺ finger domains. Characterization of newly isolated alleles indicates that swm is a negative regulator of Hh signaling and is essential for cell polarity (Casso, 2008).

This screen identified twenty-six autosomal regions that modified a smo hypomorphic phenotype in a dosage-sensitive manner. Two aspects of its design were key to its success. First, its two-generation crossing scheme eliminated background effects by homogenizing the genetic backgrounds of both experimental and control flies. It also generated reasonably large numbers of both classes of progeny so that a good estimate of an average phenotype could be obtained. These features allowed monitoring of subtle variations in wing vein morphology, despite the significant strain differences among the many lines tested. Second, its high scoring threshold rendered it relatively insensitive to changes in Hh signaling strength, thereby helping to submerge weak influences. Key to this property was the ptcGAL4 driver that was used to express smo RNAi; it functioned in part as a 'genetic buffer.' Since ptcGAL4 is itself responsive to Hh, a modifier that increased Hh signaling would also be predicted to increase the expression of ptcGAL4 and smo RNAi, while a modifier that decreased Hh signaling might be expected to decrease the expression of the ptcGAL4 and smo RNAi. ptcGAL4 therefore buffered against changes in signaling strength and decreased the effects of genetic factors that enhance or suppress signaling; as a consequence, only highly penetrant and consistent phenotypes were scored (Casso, 2008).

The screen netted many of the known core components of the Hh signaling pathway, including smo, ptc, hh, and en. mts and swm were two genes whose haplo-insufficiency phenotypes were sufficiently strong to score above the threshold set by the genetic tests. Many other known regulators of Hh signaling were not identified in this screen. There are perhaps multiple reasons, including the high scoring threshold of the smo RNAi screen, or the possibility that not all pathway regulators have haplo-insufficiency phenotypes. skinny hedgehog or suppressor of fused were not included among those identified in the screen, despite the fact that deficiencies that removed them interacted with smo RNAi. The reason is that mutant alleles of these genes that were tested did not yield similar interaction phenotypes. Since many examples were observed of interaction between null alleles of Hh pathway regulators and smo RNAi but consistent failure of hypomorphic alleles to interact, lack of interaction is not viewed as evidence against a gene being a smo RNAi enhancer/suppressor. The possibility that stronger alleles might interact cannot be discounted. It was surprising that hemizygosity of cos2 did not show an interaction with smo RNAi. This could be because it is not haplo-insufficient in the particular assay or because of the complex positive and negative roles cos2 plays in Hh signaling. Finally, there was no apparent overlap between the regions identified and the mutant lines that were identified in previous screens for modifiers of Hh phenotypes; the smo RNAi assay may be less sensitive but more specific (Casso, 2008).

mts lies within 1 of 16 regions that enhanced the smo RNAi phenotype, suggesting that its wild-type function augments the Hh response. mts encodes the catalytic (C) subunit of PP2A, a heterotrimeric phosphatase that has two regulatory subunits, B and B'. It was previously identified as a Hh pathway regulator in CL8 cells (Nybakken, 2005); the current study provides in vivo evidence for a role in Hh signaling during development. Three proteins in Hh signal transduction have been shown to be functionally phosphorylated. Phosphorylation of the Smo C terminus is induced by Hh and is required for surface accumulation of Smo and normal activation of the pathway. Thus, reduction of PP2A activity and increased phosphorylation of Smo would not be expected to decrease Hh signaling and enhance the smo RNAi phenotype. Other possible targets of Mts are Ci and Cos2. Phosphorylation of Ci by PKA, casein kinase 1α, and GSK3β is required to convert Ci from its full-length form to its transcriptional repressor form, Ci-75. Hh signaling blocks this proteolytic transformation and also promotes conversion of Ci to an activator form. A decrease in phosphatase activity might increase levels of phosphorylated Ci to effect enhanced conversion to Ci-75 and reduced levels of Ci activator. Levels of Hh signaling would be predicted to decrease. Alternatively, Mts might control phosphorylation of Cos2 by Fu. Phosphorylation of Cos2 prevents its binding to Smo and release of Smo from Cos2 increases the cell surface accumulation of Smo that is necessary for pathway activation. Therefore, a reduction of Smo on the plasma membrane due to loss of PP2A activity might attenuate Hh pathway activation (Casso, 2008).

While the catalytic subunit of PP2A carries enzymatic phosphatase activity, the substrate specificity of PP2A is directed by its regulatory subunits. The phenotypes of mutants in genes that encode the B and B' regulatory subunits of PP2A, twins and widerborst (wdb), respectively, are interesting to consider in the context of Hh signaling. Wing discs in the twins^P mutant have mirror symmetrical posterior compartment duplications that are associated with ectopic compartment borders. Symmetric wing duplications have also been observed after ectopic expression of Hh or Dpp, or after loss of en/inv induces an ectopic compartment border. Since loss of PP2A function should reduce Hh signaling, it is not obvious how loss of the B twins regulatory subunit leads to an ectopic signaling center. Understanding this interesting aspect of the twins phenotype warrants further investigation (Casso, 2008).

Misexpression of PP2A can cause cell planar polarity defects in the wing. Misexpression of mts, wdb, or mutant alleles of these genes disrupted wing hair polarity. Like mts, reducing wdb expression with RNAi reduced Hh signaling in CL8 cells (Nybakken, 2005). This evidence, as well as the wing hair polarity phenotype of swm mutants, raises the possibility that PP2A links Hh signaling with cell polarity. The PCP and Hh pathways may be parallel and independent if PP2A activity is simply common to both, but evidence that Hh is required to establish PCP in the Drosophila embryonic and adult epidermis has recently been described. The phenotype of swm mutants provides additional evidence for an association of Hh signaling with cell polarity (Casso, 2008).

swm was first identified as l(2)37Dh in a screen for recessive lethal alleles within Df(2L)E55 (37D2-38A1). It was shown to exhibit synthetic lethality as an enhancer of Minutes. Among the mutant chromosomes from the current screen that failed to complement swm, one had a Minute-like phenotype. No changes in the swm coding sequence were found in this mutant; rare escapers that eclosed as heterozygotes with the verified swm alleles had a variety of phenotypes including loss of ocelli, thin macrochetae, and deformed legs. In contrast to swm mutant escapers, however, both their eyes and wings were phenotypically normal (Casso, 2008).

swm was identified as a suppressor of the roughex eye phenotype. Alleles of ptc were also isolated in this screen. These interactions between rux, ptc, and swm were confirmed. Since Ptc is a negative regulator of the Hh pathway and ptc mutations are therefore likely to elevate Hh signaling, and since Hh plays a key role in eye morphogenesis, the rux phenotype is apparently sensitive to Hh levels. Therefore the identification of both ptc and swm mutants as rux suppressors is interpreted as a consequence of the same mechanism -- an increase in Hh signaling caused by a decrease in the level of a negative regulator (Casso, 2008).

The results provide several additional lines of evidence that swm negatively regulates Hh signaling. swm mutants dominantly suppress smo hypomorphic phenotypes (smo RNAi and smo^5A, enhance a Hh gain-of-function phenotype (hh^Mrt), and increase targets of Hh signaling such as Ptc and Ci. These effects on Hh signaling seem to occur through swm activity in the anterior compartment since swm RNAi expressed in these cells is sufficient to suppress smo RNAi. Although these interactions implicate Swm, it has not been determined how and where Swm impacts signal transduction or what its molecular function might be. Swm protein has features suggestive of a function in nucleic acid metabolism -- it has a putative RRM RNA binding domain and a CCCH Zn⁺ finger, and a GFP-Swm fusion that was examined localized to nuclei in cultured cells. Presumably, Swm affects expression, production, or presentation of proteins involved in Hh signaling or signal transduction. However, swm function is not specific to Hh signaling, since many aspects of the phenotype (e.g., ectopic venation, wing hair polarity, cell size, and interaction with Minutes) are not attributable to defects in Hh signaling (Casso, 2008).

swm is expressed broadly in both embryos and larvae, and in wing discs, it appears to be required in all cells. Null alleles, which are cell lethal in a swm/+ background, share some, but not all characteristics of Minute ribosomal protein mutants. Although swm mutants do not have thin bristles as is characteristic of Minutes, they are recessive lethal and developmentally delayed, and they interact genetically with Minutes and Minute-like loci. The wings of the Minute locus RpL38 have defects which are similar to swm wings -- extra venation, expanded distance between veins 3 and 4, wing hair polarity abnormalities, and increased cell size. Although RpL38^45-72, Df(2R)M41A10, and M41^A4 suppressed hh^Mrt, they did not interact with smo RNAi or smo5A (Casso, 2008).

While the interactions between swm and Minutes, as well as the similar phenotypes of swm and the RpL38 genes, might indicate a direct role in ribosome function, both Drosophila Swm and one of its two vertebrate homologs (RBM-27) are nuclear. The presence of RRM sequences in Swm and its homologs might suggest a role in RNA binding or metabolism, and the RRM of RBM-27 binds RNA. However, RRMs can have a structural role in protein-protein interactions independent of RNA binding, so the molecular function of Swm and its homologs cannot be determined by genetic methods alone. The fact is intriguing that the other vertebrate homolog, RBM-26, was identified as se70-2, an autoantigen that is recognized by sera of cutaneous T-cell lymphoma patients and has been used as a diagnostic marker for this tumor. In addition, the mouse RBM-26/se70-2 locus was identified as one of four genes deleted in a region required for normal murine skeletal, cartilage, and craniofacial development. Perhaps the roles of Hh that extend beyond pattern formation to cell cycle regulation, growth control, and cell polarity signify that Hh signal transduction integrates inputs from all three pathways. The pleiotropy of swm and mts may reflect these multiple inputs (Casso, 2008).

PR130 is a modulator of the Wnt-signaling cascade that counters repression of the antagonist Naked cuticle

The Wnt-signaling cascade is required for several crucial steps during early embryogenesis, and its activity is modulated by various agonists and antagonists to provide spatiotemporal-specific signaling. Naked cuticle is a Wnt antagonist that itself is induced by Wnt signaling to keep Wnt signaling in check. Little is known about the regulation of this antagonist. It has been shown that the protein phosphatase 2A regulatory subunit PR72 is required for the inhibitory effect of Naked cuticle on Wnt signaling. The present study shows that PR130, which has an N terminus that differs from that of PR72 but shares the same C terminus, also interacts with Naked cuticle but instead functions as an activator of the Wnt-signaling pathway, both in cell culture and during development. PR130 modulates Wnt signal transduction by restricting the ability of Naked cuticle to function as a Wnt inhibitor. These data establish PR130 as a modulator of the Wnt-signaling pathway and suggest a mechanism of Wnt signal regulation in which the inhibitory activity of Naked cuticle is determined by the relative level of expression of two transcripts of the same protein phosphatase 2A regulatory subunit (Creyghton, 2006).

Non-requirement of a regulatory subunit of Protein Phosphatase 2A, PP2A-B', for activation of Sex comb reduced activity in Drosophila melanogaster

The Drosophila HOX transcription factor, Sex combs reduced (SCR), is required for determining labial and the first thoracic segmental identity. A Protein Phosphatase 2A holoenzyme assembled with the PP2A-B′ regulatory subunit is proposed to specifically interact with, and dephosphorylate, the SCR homeodomain activating SCR protein activity. To test this hypothesis further, a null mutation was created in the PP2A-B′ gene, PP2A-B′^Δ, using Flip-mediated, site-specific recombination. The number of sex comb bristles, salivary gland nuclei and pseudotracheal rows are SCR-dependent and were counted as a measure of SCR activity in vivo. Adults and larvae homozygous for PP2A-B′^Δ showed no decrease in SCR activity. In addition, no evidence of functional redundancy of PP2A-B′ with other regulatory subunits, Twins (TWS) and Widerborst (WDB), for dephosphorylation and activation of SCR activity was observed. In conclusion, a PP2A holoenzyme containing the PP2A-B′ regulatory subunit has no role in the dephosphorylation and activation of SCR, and analysis of functional redundancy of PP2A regulatory subunits uncovered no evidence supporting a role of PP2A activity in dephosphorylation and activation of SCR (Moazzen, 2009).

Although the gene that encodes PP2A-B' is dispensable for viability, PP2A-B' is functional. The analysis of functional redundancy between PP2A-B' and TWS/WDB showed that removal of PP2A-B' in a genetic background deficient for one or both of the tws and wdb loci significantly increased the number of sex comb bristles. This suggests that the PP2A holoenzyme containing either TWS, the B regulatory subunit, or WDB, a B' regulatory subunit, may functionally substitute for the loss of PP2A-B'. Although PP2A-B' is dispensable for development, it may have an essential and specific role in biological processes not assayed in this study like the immune response, mating behaviour or circadian rhythm (Moazzen, 2009).

Sequential phosphorylation of Smoothened transduces graded Hedgehog signaling

The correct interpretation of a gradient of the morphogen Hedgehog (Hh) during development requires phosphorylation of the Hh signaling activator Smoothened (Smo); however, the molecular mechanism by which Smo transduces graded Hh signaling is not well understood. This study shows that regulation of the phosphorylation status of Smo by distinct phosphatases at specific phosphorylated residues creates differential thresholds of Hh signaling. Phosphorylation of Smo was initiated by PKA and further enhanced by casein kinase I (CKI). Protein phosphatase 1 (PP1) directly dephosphorylates PKA-phosphorylated Smo to reduce signaling mediated by intermediate concentrations of Hh, whereas PP2A specifically dephosphorylates PKA-primed, CKI-phosphorylated Smo to restrict signaling by high concentrations of Hh. A functional link was established between sequentially phosphorylated Smo species and graded Hh activity. Thus, a sequential phosphorylation model is proposed in which precise interpretation of morphogen concentration can be achieved upon versatile phosphatase-mediated regulation of the phosphorylation status of an essential activator in developmental signaling (Su, 2011).

The conversion of a gradient of the morphogen Hh into distinct transcriptional responses is essential for cell-fate decisions and tissue patterning during development. This study has provided genetic and biochemical evidence to support a model in which sequential phosphorylation of Smo, which is established by distinct kinases and phosphatases on specific serines, transduces graded Hh signaling. A basal extent of Smo activity, regulated by as yet unknown kinases and phosphatases, was sufficient to transduce low-threshold Hh signaling. PKA and PP1 collaborate to sustain PKA-phosphorylated Smo to transduce intermediate-threshold Hh signaling, whereas CKI and PP2A facilitate high-threshold Hh signaling by maintaining PKA-primed, CKI-phosphorylated Smo (Su, 2011).

Wdb-PP2A directly and specifically acts on CKI-pSmo to restrict high-threshold Hh signaling. Apart from PP2A, another phosphatase, PP1, specifically dephosphorylated PKA-phosphorylated Smo. This collaborative regulation between different phosphatases on the same substrate also functions in other cellular processes. For example, PP1 and PP2A dephosphorylate Par-3 to regulate cell polarity in the specification of neuroblast cell fate. Similarly, PP2A and PP4 respond to different DNA damage signals to dephosphorylate γ-H2AX to facilitate the repair of DNA double-strand breaks (Su, 2011 and references therein).

The activity of several Hh signaling components, including Smo, Ci, and Cos2, is regulated by phosphorylation. For example, PKA- and CKI-mediated phosphorylation of Ci leads to its destabilization. PP2A is implicated in regulating Ci activity in flies. This study confirmed that the catalytic PP2A subunit Mts associates with both Smo and Ci in cl-8 cells. Consistent with the substrate specificity of PP2A being conferred by its obligate regulatory subunits, this study found that Wdb specifically regulates the signaling potential of Smo. Another regulatory subunit, Twins , may direct PP2A activity toward Ci, which may potentially promote the translocation of CiFL to the nucleus, thereby activating Hh signaling. The use of distinct PP2A regulatory subunits in the same developmental process was also observed in transforming growth factor-β (TGF-β) signaling. The effect of the regulatory Bα subunit on PP2A activity activates Smad2 signaling, whereas the Bδ subunit inhibits Smad2 activity. The elaborate regulation of these two signaling systems by PP2A highlights a potential paradigm in which differential PP2A activity plays an essential role in developmental signaling. PP2A is a strong tumor suppressor; thus, modulation of PP2A activity provides an additional route by which development and tumorigenesis might be controlled (Su, 2011).

Another phosphatase, PP4, may play a role in inhibiting Smo; however, inhibiting PP4 alone is not sufficient to promote constitutive cell surface localization of wild-type Smo, unless Hh protein is provided. The surface localization of Smo is tightly linked to PKA- and CKI-dependent enhanced phosphorylation of Smo. PP4-specific RNAi further increases the extent of constitutive surface localization of Smo mutants that mimic PKA- and CKI-mediated phosphorylation, which suggests that PP4 may act on sites other than those in the PKA-CKI clusters (Su, 2011).

To delineate mechanisms whereby PP2A and PP4 might act on Smo, the expression of Hh signaling components as well as of Hh targets were systematically examined in wing discs expressing pp4 RNAi. In addition to the Smo stabilization, it was found that pp4 RNAi reduced the abundance of Cos2 protein. This might be as a consequence of the increased Smo abundance, because smo RNAi stabilized Cos2. Alternatively, PP4 might regulate Cos2 directly, because phosphorylated Cos2 is not stable. To distinguish between these two possibilities, the genetic relationship between smo and pp4 was examined by monitoring the stabilization of Cos2. Cos2 abundance was still reduced in wing discs containing both pp4 and smo RNAi. This effect is similar to the effect of pp4 RNAi alone, thus placing pp4 downstream of smo in regulating Cos2. Consistent with this, reduced expression of pp4 compromised Ptc and Collier/Knot (Col) expression at the AP boundary. The expanded area containing Ptc, albeit at a reduced abundance, away from the AP boundary has been observed previously. These experiments are consistent with a positive role of Cos2 in mediating maximal activation of Hh signaling in cl-8 cells as well as in wing discs; Ptc and Col expression are reduced in cos2 clones abutting the AP boundary. These data, together with the observation of a direct interaction between Cos2 and PP4 (Jia, 2009), argue that PP4 might also directly affect the extent of Cos2 phosphorylation (Su, 2011).

Smo contains three PKA-CKI phosphorylation clusters, with one PKA and two CKI consensus serines in each cluster. A previous study compared the signaling potential of phosphorylation-defective Smo by mutating PKA consensus serines to alanines in one, two, or three of the PKA-CKI clusters and concluded that at least six serines in Smo are required to fully induce the expression of ptc-lacZ, whereas only three serines are needed for the expression of dpp-lacZ. Another study further demonstrated that PKA- and CKI-mediated phosphorylation, which results in the generation of negatively charged residues, counteracts the positive charges conferred by nearby arginine clusters, thus enabling Smo to adopt a conformational change required to activate Hh signaling. These two studies support a model of collective Smo phosphorylation such that the identity of the phosphorylated serines in the PKA-CKI consensus clusters is probably not important; rather, the resulting negative charges collectively carried by these residues after phosphorylation are critical to determine the signaling strength of Smo (Su, 2011).

On the basis of this model, a variant Smo (Smo^-CKI) in which the CKI, but not the PKA, consensus serines are mutated to alanines, thus rendering Smo^-CKI resistant to CKI-mediated phosphorylation, would be anticipated to have the same signaling potential as Smo^-PKA23, a variant containing a single intact PKA-CKI cluster, because both mutants contain three serines that can be phosphorylated. In smo loss-of-function clones, Smo^-PKA23 is sufficient to drive expression of dpp-lacZ; however, Smo^-CKI fails to rescue dpp-lacZ expression. The discrepancy between the effects of Smo^-PKA23 and Smo^-CKI on dpp-lacZ expression cannot be simply explained by the collective phosphorylation model. Moreover, these experiments reveal a functional distinction between different phosphorylated residues: Three PKA consensus serines in Smo^-CKI may have less signaling activity than the single PKA and two CKI consensus serines in Smo^-PKA23. The distinct signaling potentials of the two phosphorylation variants of Smo may be caused by different negative charge densities being carried by individual PKA-CKI clusters, or they may reflect intrinsic properties of sequential phosphorylation within each cluster (Su, 2011).

Indeed, the hierarchy of importance among individual PKA-CKI clusters in Smo has been revealed. Cluster 2 (also known as region V) is more prevalent than the other two clusters in activating ptc-luc reporter in smo-depleted cl-8 cells. Whether this functional distinction among PKA-CKI clusters also holds true in wing discs is unclear, because neither the hierarchical importance of individual clusters nor the relative importance of specific PKA and CKI phosphorylation events in neutralizing nearby arginine clusters has been directly studied. Nevertheless, when the relative importance of the three serines in cluster 2 was examined, the PKA-primed, CKI consensus sites (that is, sequential phosphorylation) were essential for Hh activation in cl-8 cells. The differential ability of Smo^CKI-SA and Smo^PKA-SA variants to activate dpp transcription in wing discs uncovered in this study is consistent with results obtained in cl-8 cells. Both observations challenge the model of collective Smo phosphorylation by arguing that the signaling potential of individual serines between each PKA-CKI cluster, as well as within a cluster, is most probably not equal. It is believed that regulated phosphorylation at specific serines may therefore contribute to graded Smo signaling (Su, 2011).

The collective phosphorylation model does not distinguish between the contributions of individual phosphorylated residues in PKA-CKI clusters. This study of phosphorylation-defective Smo variants revealed a Smo activity gradient in which phosphorylation at the PKA consensus sites and phosphorylation at the PKA-primed, CKI consensus sites were required for intermediate- and high-threshold Hh signaling, respectively. This activity gradient of Smo was directly visualized with the α-Smo-pS⁶⁶⁷ antibody. The abundance of PKA-phosphorylated Smo species, which is uncovered in Hh-stimulated fly cells by mass spectrometric analysis, increased initially but then declined sharply in response to Hh. As predicted from the model, phosphorylated Smo in response to intermediate-threshold Hh signaling was sensitive to dephosphorylation by PP1 but much less so to PP2A. Together, these data highlight the importance of sequential Smo phosphorylation to the transduction of graded Hh signaling. Sequential phosphorylation may be required to initialize graded Smo signaling activity. In addition, collective phosphorylation between different clusters may reinforce and maximize the Smo signaling potential to ensure the appropriate Hh signaling outcome (Su, 2011).

The presence of up to 26 serine or threonine residues in Smo that can be phosphorylated in response to Hh resembles the composition of residues found in the Kv2.1 potassium channel. Variable calcineurin-dependent dephosphorylation of Kv2.1 at 16 phosphorylated residues generates an activity gradient for channel gating and neuronal firing. The opposing actions of kinases and phosphatases on a multisite substrate are known through mathematical modeling to efficiently generate a range of stable phosphorylated forms. The spectrum of such distributions can be further increased with the number of phosphorylated sites. Two additional kinases, CK2 and G protein (heterotrimeric guanosine 5'-triphosphate-binding protein)-coupled receptor kinase 2 (GRK2), phosphorylate sites in Smo other than those targeted by PKA and CKI. Thus, the complex composition of phosphorylated residues in the cytoplasmic tail of Smo, coupled with versatile dephosphorylation by distinct phosphatases, provides an efficient and reliable mechanism to precisely convert the concentration thresholds of Hh into a graded signaling activity (Su, 2011).

The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification

Centriole duplication is a tightly regulated process that must occur only once per cell cycle; otherwise, supernumerary centrioles can induce aneuploidy and tumorigenesis. Plk4 (Polo-like kinase 4) activity initiates centriole duplication and is regulated by ubiquitin-mediated proteolysis. Throughout interphase, Plk4 autophosphorylation triggers its degradation, thus preventing centriole amplification. However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown. This paper shows that PP2A [Protein Phosphatase 2A(Twins)] counteracts Plk4 autophosphorylation, thus stabilizing Plk4 and promoting centriole duplication. Like Plk4, the protein level of PP2A's regulatory subunit, Twins (Tws), peaks during mitosis and is required for centriole duplication. However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification. Paradoxically, expression of tumor-promoting simian virus 40 small tumor antigen (ST), a reported PP2A inhibitor, promotes centrosome amplification by an unknown mechanism. ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification (Brownlee, 2011).

Plk4 protein is maintained at near-undetectable levels for the majority of the cell cycle by ubiquitin-mediated proteolysis. The ubiquitin ligase SCF^Slimb is responsible for Plk4 degradation and recognizes an extensively phosphorylated degron situated immediately downstream of the kinase domain (KD; ~50 amino acids containing the Slimb-binding domain)KD. Slimb is appropriately positioned on centrioles throughout the cell cycle to promote rapid Plk4 destruction, but centrioles are not required for its activity. In any case, Plk4 degradation is critical in blocking all pathways of centriole amplification. Unlike other Polo kinase members, Plk4 is a homodimer capable of autophosphorylating its downstream regulatory element (DRE), a serine-rich region surrounding its SBD, in trans to promote Slimb binding. Autoregulation is a conserved feature of Plk4. Moreover, a RNAi screen of the fly kinome suggests that no other kinase is required for Plk4 degradation. The continuous and efficient degradation of Plk4 indicates that Plk4 is immediately active when expressed and that control of Plk4’s protein level is key to regulating its activity (Brownlee, 2011).

However, surprisingly little is known about the converse event: how Plk4 is activated. The results reveal the existence of a previously unknown facet of the regulation of centriole duplication, a process which transiently stabilizes and activates Plk4 specifically during mitosis. Serine/threonine phosphatases were investigated as possible effectors to counteract Plk4 autophosphorylation. PP2A is an excellent candidate to fulfill this role as it has important functions in mitosis and localizes to mitotic centrioles in cultured fly cells and centrosomes in dividing Caenorhabditis elegans embryos. A previous study found that the number of γ-tubulin foci in mitotic S2 cells was diminished after PP2A RNAi, but whether this resulted from a bona fide loss of centrioles or instead reflects a requirement for PP2A for centrosome maturation was not determined. Subsequently, a role for PP2A in centrosome maturation was identified in a genome-wide RNAi screen. The current results indicate that PP2A and the regulatory subunit Tws are required for centriole duplication by dephosphorylating and stabilizing Plk4. Without PP2A^Tws, Plk4 cannot be stabilized, and centrioles fail to duplicate. PP2A is also required for centriole assembly in C. elegans embryos but functions downstream in the centriole assembly process (Kitagawa, 2011; Song, 2011). Although the catalytic and structural PP2A subunits are abundant, regulatory subunits are needed for intracellular targeting and recognition of a myriad of substrates. Tws overexpression is sufficient to stabilize Plk4 in a dose-dependent manner, causing centriole amplification and multipolar spindle formation. Like Plk4, Tws protein levels are low during interphase but rise and peak during mitosis. Accordingly, the results suggest that PP2A^Tws stabilizes mitotic Plk4 by counteracting Plk4 autophosphorylation, enabling cells to switch Plk4 activity (and thus centriole duplication) on and off. This mechanism is inherently highly sensitive to the presence of Tws, a rate-limiting component. Moreover, this is likely a conserved mechanism because overexpression of human Tws also stabilizes fly Plk4 in S2 cells. Clearly, an important goal for future studies is to establish whether the regulation of Tws levels and cell cycle control are linked. In addition, the results suggest that up-regulation of Tws could be a means to amplify centrioles in multiciliated cells and that increased Tws activity could be a condition found in cancerous cells (Brownlee, 2011).

Centrosome amplification is a hallmark of cancer and is also observed upon expression of DNA tumor virus proteins, which include SV40 ST, human papillomavirus E7, human T cell leukemia virus type-1 Tax, hepatitis B virus oncoprotein X, and human adenovirus E1A. However, mechanisms for centrosome amplification by viral oncoproteins are not known. SV40 ST has been found to directly bind the highly conserved Drosophila catalytic and structural PP2A subunits and to induce centrosome overduplication in cultured fly cells (Kotadia, 2008). Notably, ST is a well-established PP2A inhibitor and is known to bind structural PP2A subunits, forcing endogenous PP2A regulatory subunits to be displaced and inhibiting PP2A activity. However, the current results demonstrate that ST expression does not inhibit all PP2A activities but, instead, stimulates PP2A stabilization of Plk4. This represents the first evidence that ST mimics the function of a PP2A regulatory subunit in cells. It will be important to determine whether ST targets additional PP2A substrates during tumorigenesis and whether other tumorigenic viruses (e.g., human papillomavirus and hepatitis B) known to promote centrosome amplification exploit this same mechanism. Intriguingly, human papillomavirus E7 oncoprotein binds PP2A catalytic and structural subunits and prevents PP2A from dephosphorylating Akt. Although a previous study has suggested that PP2A may function as a tumor suppressor, these findings indicate that unregulated PP2A activity leads to centriole amplification and chromosomal instability and should therefore be considered as a potential oncogenic factor (Brownlee, 2011).

Essential roles of the Tap42-regulated protein phosphatase 2A (PP2A) family in wing imaginal disc development of Drosophila melanogaster

Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but understanding of the in vivo function and regulation of these enzymes is limited. This study investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42^RNAi-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described in this study identifies crucial roles for Tap42 phosphatase complexes in governing imaginal disc and fly development (Wang, 2012).

Understanding about the in vivo function of α4/Tap42, especially in development, is limited in part because global knockout of this gene in mice and flies leads to early embryonic death (see Cygnar, 2005 and Kong, 2004). Cellular studies have also revealed that depletion of α4/Tap42 causes death in embryonic stem cells, mouse embryonic fibroblasts, adipocytes, hepatocytes, B and T cells of the spleen and thymus, and Drosophila S2 cells (Bielinski, 2007; Kong, 2004; Yamashita, 2006). Although studies of a conditional (Cre-LoxP) α4 knockout in mouse hepatocytes and a mosaic assay of Tap42 in Drosophila wing disc have provided insights into the cellular biology of α4 and Tap42 (Cygnar, 2005; Kong, 2004), the impact of these gene products on the development of tissues and host have not yet been described. This report utilized Tap42-targeted RNAi and the Gal4/UAS system to investigate the biological effects of silencing Tap42 expression in specific Drosophila tissues. Suppressing the Tap42 gene using two tissue-specific drivers (pnr-Gal4 and ap-Gal4) led to a pleiotropic fly phenotype, which included major deformities in the adult thorax and wings as well as decreased survival rates. The experimental platform described in this study has allowed exploration of the role of Tap42 and Tap42-regulated phosphatases in the control of cellular signaling, tissue development, and Drosophila viability (Wang, 2012).

Analyses of Tap42^RNAi wing discs revealed significant alterations in multiple signal transduction pathways including JNK, DPP, and HH. Marked increases in p-JNK signals were found in ap-Gal4>Tap42^RNAi wing discs. This observation, together with previous studies showing increased c-Jun phosphorylation in α4-null mouse embryonic fibroblasts (Kong, 2004) and activated JNK in Tap42-depleted clones of fly wing discs (Cygnar, 2005), indicate that α4/Tap42 likely plays a negative role in regulation of JNK signaling. Silencing the Tap42 gene in the ap gene domain also changed DPP and HH signaling in the wing discs. Although ap-Gal4-mediated silencing of Tap42 had a profound effect on JNK, DPP, and HH signaling, these pathways were unaffected in pnr-Gal4>Tap42^RNAi wing discs, thus demonstrating that the thorax cleft phenotype seen in the pnr-Gal4>Tap42^RNAi flies is not due to alterations in these pathways. Collectively, these findings indicate that Tap42 plays a crucial role in the modulation of JNK, DPP, and HH signaling, but the effects of Tap42 on these pathways appear to play a minimal role in normal thorax development (Wang, 2012).

The HH pathway is one of the major guiding signals for imaginal disc development. Recent investigations have revealed that the phosphorylation state of Ci and Smo, two components of the HH signaling pathway, are controlled by Drosophila PP2A (Mts) and PP4 (Jia, 2009). Additional studies implicate a role for specific Mts complexes in the control of HH signaling, whereby holoenzyme forms of Mts containing the Wdb and Tws regulatory B subunits act at the level of Smo and Ci, respectively (Su, 2011). Together, these findings point to key roles for Mts and PP4 in HH signaling and suggest that a common subunit of these phosphatases, namely Tap42, may also be involved in HH signaling. Indeed, the current data clearly show that Tap42 plays an important regulatory role in this pathway as silencing of Tap42 within the wing discs leads to an elimination of both Smo and Ci expression. Although the precise role(s) of Tap42 in the control of HH signaling remains unclear, it likely involves Tap42-dependent regulation of one or more phosphatase catalytic subunits (e.g., Mts, PP4, and possibly PPV) or specific holoenzymes forms of these phosphatases (e.g., Wdb/Mts, Tws/Mts). The pleiotropic effects of Tap42^RNAi on JNK, DPP, and HH signaling could be due to loss of Tap42's regulation of phosphatase activity, cellular levels, holoenzyme assembly, or subcellular localization (Wang, 2012).

Depletion of α4 in mouse embryonic fibroblasts caused an increase in phosphorylation of a variety of established PP2A substrates, which was attributed to a 'generalized defect in PP2A activity.' Instead of the expected unidirectional increase in protein phosphorylation, the current findings demonstrate a dual role for Tap42 in the control of JNK activation as hyperphosphorylation and hypophosphorylation of JNK were observed in the dorsal and ventral sides of the Tap42^RNAi wing disc, respectively, relative to control wing discs. Silencing of Tap42 in the ap domain also impacted DPP in a bi-directional fashion; these flies exhibited significantly decreased DPP expression in the scutellum but augmented expression around the wing blade. Consistent with previous studies showing that PP2A functions at different levels within the Ras1 and HH pathways, the current data indicate that Tap42-regulated phosphatases likely target multiple substrates within the JNK and DPP pathways in different regions of wing discs (Wang, 2012).

Close examination of the PE cells in the wing disc revealed that Tap42 expression occurs in only a fraction of these cells. It is noteworthy that the majority of Tap42 localized in rows of cells delineating the PE/DP (peripodial epithelium/disc proper) boundary. These cells are commonly referred to as 'medial edge' cells, which represent a subpopulation of PE cells that play a crucial role in thorax closure during metamorphosis. Interestingly, α4-PP2A complexes appear to play a major role in the control of cell spreading, migration, and cytoskeletal architecture, presumably via their ability to modulate the activity of the small G-protein Rac. Yeast Tap42 has also been implicated in the cell cycle-dependent and polarized distribution of actin via a Rho GTPase-dependent mechanism. Therefore, it is hypothesized that the wing disc structural deformities and thorax cleft phenotype of Tap42^RNAi flies are a result of unregulated phosphatases leading to defective spreading and migration of the medial edge cells during metamorphosis. The thorax cleft phenotype provides an opportunity to delineate the precise roles of Tap42-phosphatase complexes in processes controlling thoracic closure (e.g., cell spreading and migration) (Wang, 2012).

α4/Tap42 appears to function as an essential anti-apoptotic factor as cells lacking this common regulatory subunit of PP2A family members undergo rapid death. These studies implicate a role for α4/Tap42-dependent regulation of PP2A-like enzymes, and presumably the phosphorylation state of multiple pro- and anti-apoptotic proteins, in the maintenance of cell survival. The current findings reveal that silencing Tap42 in wing discs triggers apoptosis, thus providing supportive in vivo evidence that depletion of Tap42 (α4) leads to deregulated phosphatase action, which switches these enzymes from pro-survival to pro-apoptotic mediators. Because JNK activation is a hallmark feature of apoptosis, the overlap of apoptotic cells and hyperphosphorylated JNK indicates that the Tap42^RNAi-induced apoptosis may be dependent on JNK activation (Wang, 2012).

Since α4 is required for maintaining the normal function of PP2A, PP4, and PP6, it is suspected that misregulation of these phosphatases could be responsible for the pleiotrophic phenotypes observed in Tap42^RNAi flies. Consistent with this idea, introduction of the mts^XE2258 heterozygous allele into ap-Gal4>UAS-Tap42^RNAi flies partially rescued the thorax and wing defects, and significantly improved fly survival rates. The partial rescue by mts^XE2258 suggests that the defects seen in the Tap42^RNAi flies are due, in part, to unregulated Mts activity, possibly as a result of increased Mts levels or enzymatic activity. Indeed, previous studies have demonstrated an accumulation of Mts in Tap42-depleted clones of the fly wing disc. Thus, mts^XE2258 appears to function as a mild mutant that partially restores misregulated Mts function following depletion of Tap42. However, given the biochemical findings showing that Tap42 also interacts with PP4 and PPV, additional studies will be needed to determine the relative contribution of these phosphatases to the Tap42^RNAi-induced defects (Wang, 2012).

The phenotypes observed in flies expressing Tap42^RNAi could also be attributed to loss of a phosphatase-independent function(s) of Tap42 that controls normal fly development. However, introduction of a phosphatase binding-defective mutant of Tap42 (Tap42^ED) into the Tap42^RNAi background failed to rescue the phenotypes and lethality associated with Tap42 depletion. In contrast to Tap42^ED, introduction of Tap42^WT fully rescued the phenotypes and lethality of the Tap42^RNAi flies. These findings indicate that the Tap42^RNAi-induced phenotypes are entirely due to the impaired interactions between Tap42 and PP2A family members, and provide compelling support for the hypothesis that Tap42-dependent regulation of the functions of these enzymes is crucial for normal wing disc development and Drosophila viability (Wang, 2012).

Although understanding the exact molecular mechanisms underlying Tap42's regulation of PP2A family members is still incomplete, these studies clearly demonstrate that Tap42-phosphatase interactions play crucial roles in the control of multiple signaling pathways governing cell growth and survival. The experimental platform described in this report will undoubtedly serve as a valuable system to further explore the in vivo function and regulation of Tap42-phosphatase complexes. Furthermore, given the remarkable phenotypes seen in the Tap42^RNAi flies (e.g., thorax cleft and deformed wings), it is anticipated that this model system will drive future studies (e.g., phenotype-based suppressor/enhancer screens) aimed at identifying direct targets of Tap42-regulated phosphatases, as well as additional pathways under the control of these phosphatase complexes (Wang, 2012).

Histone Chaperone NAP1 Mediates Sister Chromatid Resolution by Counteracting Protein Phosphatase 2A

Chromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. This study found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed that NAP1 and cohesin co-localize at multiple genomic loci. Proteomic and biochemical analysis established that NAP1 associates with the full cohesin complex, but it also forms a separate complex with the cohesin subunit stromalin (SA). NAP1 binding to cohesin is cell-cycle regulated and increases during G2/M phase. This causes the dissociation of protein phosphatase 2A (PP2A) from cohesin, increased phosphorylation of SA and cohesin removal in early mitosis. PP2A depletion led to a loss of centromeric cohesion. The distinct mitotic phenotypes caused by the loss of either PP2A or NAP1, were both rescued by their concomitant depletion. In is concluded that the balanced antagonism between NAP1 and PP2A controls cohesin dissociation during mitosis (Moshkin, 2013).

As reflected by their name, a major activity of histone chaperones is to prevent illicit liaisons and guide newly synthesized histones to sites of chromatin assembly. This study describes a mitotic function for the canonical histone chaperone NAP1 that is unrelated to nucleosome assembly. NAP1 was found to bind cohesin and block dephosphorylation of SA by PP2A, thereby promoting cohesin dissociation from the chromosome arms. Consequently, chromosomal binding of cohesin during mitosis is controlled by the balance between the opposing activities of NAP1 and PP2A (Moshkin, 2013).

NAP1 is part of a large assemblage including the full cohesin complex and PP2A. In addition, NAP1 and SA form a subcomplex, which lacks the other cohesin subunits and PP2A. An attractive scenario is that the NAP1-SA module or NAP1 alone competes with PP2A-bound SA within the full cohesion complex. PP2A displacement by NAP1 allows stable phosphorylation of cohesin and its dissociation during early mitosis. NAP1 might also act as a direct inhibitor of PP2A catalytic activity, because a mammalian NAP1 homolog, SET, has been identified as a potent PP2A inhibitor, which promotes sister chromatid segregation during mouse oocyte miosis (Qi, 2013; Chambon, 2012). In addition, NAP1 might help cohesin phosphorylation by tethering Polo kinase to cohesin. In fact, a potential association between NAP1 and Polo kinase was detected. However, the dramatic chromosome condensation defects after Polo kinase depletion precluded a functional evaluation of a possible role of NAP1 in its function. Nevertheless, although additional NAP1 activities cannot be excluded, functional experiments established that blockage of PP2A suffices to explain the crucial role of NAP1 during sister chromatid resolution (Moshkin, 2013).

NAP1 not only regulates the chromosomal distribution of cohesin and PP2A, but also that of MeiS332, a fly homolog of Sgo. The function of MeiS332 and PP2A appears to be largely conserved from mammals to flies because they bind each other and depletion of either factor causes a loss of centromeric cohesion. Either knockdown of NAP1 or over-expression of PP2A caused spreading of MeiS332 onto the arms of mitotic chromosomes, accompanying the loss of sister chromatid resolution. Thus, the balanced antagonism between NAP1 and PP2A controls chromosomal association of both cohesin and MeiS332 during mitosis (Moshkin, 2013).

One level of regulation involves changes in NAP1's subcellular localization and chromatin binding through the cell cycle. At prophase there is a strong increase in the level of nuclear NAP1, but by metaphase, NAP1 and cohesin have dissociated from the chromosomes. Thus, the dynamic behavior of NAP1 correlates well with its function in promoting cohesin release at early mitosis. Regulation of NAP1 localization may involve cyclin B-cdc2/cdk1 kinase complexes. Previously it was found that yeast and vertebrate NAP1 are phosphorylated by cyclin B-cdc2 and that yeast cyclin B requires NAP1 for its full range of mitotic functions (Moshkin, 2013).

It is suggested that histone chaperones are at the hubs of specialized protein networks that perform a wide variety of tasks in chromosome biology. Through association with distinct partners, NAP1 is able to perform different functions. By acting as a histone chaperone, NAP1 mediates chromatin assembly. Through recruitment of the histone H3 deacetylase and H3K4 demethylase complex RLAF, NAP1 controls gene-selective silencing at developmental loci. Finally, by binding cohesin and blocking SA dephosphorylation by PP2A, NAP1 mediates sister chromatid resolution during mitosis. These results emphasize the surprisingly diverse- and specific regulatory functions of histone chaperones in chromosome biology (Moshkin, 2013).

Contribution of Orb2A stability in regulated amyloid-like oligomerization of Drosophila Orb2

How learned experiences persist as memory for a long time is an important question. In Drosophila the persistence of memory is dependent upon amyloid-like oligomers of the Orb2 protein. However, it is not clear how the conversion of Orb2 to the amyloid-like oligomeric state is regulated. The Orb2 has two protein isoforms, and the rare Orb2A isoform is critical for oligomerization of the ubiquitous Orb2B isoform. This study reports the discovery of a protein network comprised of protein phosphatase 2A (PP2A), Transducer of Erb-B2 (Tob), and Lim Kinase (LimK) that controls the abundance of Orb2A. PP2A maintains Orb2A in an unphosphorylated and unstable state, whereas Tob-LimK phosphorylates and stabilizes Orb2A. Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain. Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation. These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories (White-Grindley, 2014).

Previous work suggested that conversion of neuronal CPEB to an amyloid-like oligomeric state provides a molecular mechanism for the persistence of memory. However, it is not known how Orb2 oligomerization is regulated so that it will occur in a neuron/synapse-specific and activity-dependent manner. This study reports that factors that influence Orb2A stability and thereby abundance regulate Orb2 oligomerization (White-Grindley, 2014)

Tob, a previously known regulator of SMAD-dependent transcription and CPEB-mediated translation, associates with both forms of Orb2, but increases the half-life of only Orb2A. Stimulation with tyramine or activation of mushroom body neurons enhances the association of Tob with Orb2, and overexpression of Tob enhances Orb2 oligomerization. Both Orb2 and Tob are phosphoproteins. Phosphorylation destabilizes Orb2-associated Tob, whereas it stabilizes Orb2A. Tob promotes Orb2 phosphorylation by recruiting LimK, and PP2A controls the phosphorylation status of Orb2A and Orb2B (White-Grindley, 2014).

PP2A, an autocatalytic phosphatase, is known to act as a bidirectional switch in activity-dependent changes in synaptic activity. PP2A activity is down-regulated upon induction of long-term potentiation of hippocampal CA1 synapses (LTP) and up-regulated during long-term depression (LTD). Similarly, Lim Kinase, which is synthesized locally at the synapse in response to synaptic activation, is also critical for long-term changes in synaptic activity and synaptic growth (White-Grindley, 2014).

Based on these observations a model is proposed for activity-dependent and synapse-specific regulation of amyloid-like oligomerization of Orb2. It is postulated that in the basal state synaptic PP2A keeps the available Orb2A in an unphosphorylated and thereby unstable state. Neuronal stimulation results in synthesis of Orb2A by a yet unknown mechanism. The Tob protein that is constitutively present at the synapse binds to and stabilizes the unphosphorylated Orb2A and recruits the activated LimK to the Tob-Orb2 complex, allowing Orb2 phosphorylation. Concomitant decreases in PP2A activity and phosphorylation by other kinases enhances and increases Orb2A half-life. The increase in Orb2A level as well as phosphorylation may induce conformational change in Orb2A, which allows Orb2A to act as a seed. Alternatively, accumulation and oligomerization of Orb2A may create an environment that is conducive to overall Orb2 oligomerization. In the absence of an in vitro Orb2A-Orb2B oligomerization assay, it is not possible to distinguish between these two possibilities (White-Grindley, 2014).

For Tob, initial Orb2 association stabilizes Tob. However, association with Orb2 as well as suppression of PP2A activity leads to additional phosphorylation, which results in dissociation of Tob from the Orb2-Tob complex and destabilization. The destabilization of Orb2-associated Tob provides a temporal restriction to the Orb2 oligomerization process. The coincident inactivation of PP2A and activation of LimK may also provide a mechanism for stimulus specificity and synaptic restriction (White-Grindley, 2014).

Orb2A and Orb2B are phosphorylated at multiple sites, including serine/threonine and presumably tyrosine residues. These phosphorylation events are likely mediated by multiple kinases because overexpression of LimK did not affect Orb2 phosphorylation to the extent observed with the inhibition or activation of PP2A, raising several interesting questions. In what order do these phosphorylations occur? What function do they serve individually and in combination? What kinases are involved? Moreover, similar to mammalian CPEB family members, in addition to changing stability, phosphorylation may also influence the function of Orb2A and Orb2B (White-Grindley, 2014).

Does Tob regulate Orb2 function? In mammals Tob has been shown to recruit Caf1 to CPEB3 target mRNA, resulting in deadenylation, and CPEB3 is known to act as a translation repressor when ectopically expressed. This study found Drosophila Tob also interacts with Pop2/Caf1 and Orb2A and Orb2B can repress translation of some mRNA. Orb2 has also been identified as a modifier of Fragile-X Mental Retardation Protein (FMRP)-dependent translation, and Fragile-X is believed to act in translation repression (Cziko, 2009). Therefore, the Tob-Orb2 association may contribute to Orb2-dependent translation repression, and the degradation of Orb2-associated Tob may relieve translation repression. Additionally, if the oligomeric Orb2 has an altered affinity for either mRNA or other translation regulators, Tob can affect Orb2 function by inducing oligomerization. However, the relationship between Tob phosphorylation and its function is unclear at this point (White-Grindley, 2014).

Does involvement of Tob both in transcription and translation serve a specific purpose in the nervous system? Tob inhibits BMP-mediated activation of the Smad-family transcription activators (Smad 1/5/8) by promoting association of inhibitory Smads (Smad 6/7) with the activated receptor. In Drosophila BMP induces synaptic growth via activation of the Smad-family of transcriptional activators, and subsequent stabilization of these newly formed synapses via activation of LimK. These studies suggest Tob and LimK also regulate Orb2-dependent translation, raising the possibility Tob may coordinate transcriptional activation in the cell body to translational regulation in the synapse (White-Grindley, 2014).

Feedback control of chromosome separation by a midzone Aurora B gradient

Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids before nuclear envelope reassembly (NER). However, how these two processes are coordinated remains unknown. This study, carried out in Drosophila S2 cells, identified a conserved feedback control mechanism that delays chromosome decondensation and NER in response to incomplete chromosome separation during anaphase. A midzone-associated Aurora B gradient was found to monitor chromosome position along the division axis and to prevent premature chromosome decondensation by retaining Condensin I. PP1/PP2A phosphatases counteracted this gradient and promoted chromosome decondensation and NER. Thus, an Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome decondensation and NER until effective separation of sister chromatids is achieved. This allows the correction and reintegration of lagging chromosomes in the main nuclei before completion of NER (Afonso, 2014).

DEVELOPMENTAL BIOLOGY

Embryonic to pupal period

There is a maternal contribution of transcripts to the embryo. Transcripts for both isoforms are expressed simultaneously but in variable amounts in all tisues examined. The level of maternal transcripts remains very high throughout the syncytial stage of embryogenesis and then, around cellularization, decreases rapidly to below the detection. At full germband extension (stage 10) zygotic transcripts can be detected in the neuroblasts and the migrating gonads. This expression is more clearly seen after germband retraction. In the later stages of embryogenesis, the expression can be detected not only in the nervous system and the gonads, but also in the hindgut, the anal pads and the Malpighian tubules. There is a uniform distribution of both types of transcripts in discs and in the testes of late pupae, and lower transcript levels in the larval brain, where they appear restricted to regions of cell proliferation in optic lobes (Mayer-Jaekel, 1993).

Adult

In ovarioles, there is a high level of expression in nurse cells of the developing egg chamber, and transcripts are detectable starting from around stage 6 in oogenesis. Upon oocyte maturation, the transcripts are transported into the egg cell, and high transcript levels can be detected in the unfertilized egg.

EFFECTS OF MUTATION

Mutations in the twins locus causes mitotic abnormalities during early embryogenesis and late larval development in an allele-specific manner. Homozygotes have small brains and die during late pupal stages. These individuals probably survive to late developmental stages due to the presence of a maternally provided wild-type product. There is a range of mitotic abnormalities during metaphase and anaphase in third-instar larval neuroblasts. The abnormal metaphase figures are characterized by excessive chromosome condensation, low level of polyploidy, and in some cases, the presence of irregular chromatid condenstion. These abnormal phenotypes are probably not due to the absence of functional spindles, since anaphases can be easily found in these brains. However, most anaphases appear abnormal. They are characterized by the presence of stretched chromatids, which extend all the way between the poles, and/or lagging chromatids, which are left in the mid-zone between the two poles. Some anaphases also show a variable degree of chromosome condensation. This mutation causes an increase in the mitotic index. The data suggest that the twins mutation causes a delay in the initiation of anaphase, which results in the large number of metaphase figures with condensed chromosomes (Gomes, 1993).

Mutation of twins causes a pattern duplication in Drosophila imaginal discs. Inactivation of twins induces the formation of extra wing blade anlagen in the posterior compartment. The duplication is mirror symmetrical, and the line of symmetry does not correspond to any of the known compartment borders. The wing duplication in twins is associated with partial losses of engrailed expression outside the wing pouches. It is thought that the proximal domain of the posterior compartment is missing, with the missing region corresponding to ventral structures of the notum (Uemura, 1993).

Partial loss-of-function mutations in twins alter cell fate lineage in peripheral nervous system mechanoreceptor development. Hypomorphic twins mutations do not plock division of the sensory organ precursor, but most likely lresult in production of accessory (shaft and socket) cells at the expense of neural and glial cells. Almost all duplicated sockets are physically connected or fused to one another. No identifiable mitotic arrest of sensory organ precursors is found. The other two subunits of phosphatase are expressed at normal levels in twins mutants. A similar transformation is seen in musashi and numb, and the opposite transformation is seen in tramtrack mutants. Homozygous twins alleles produce a slightly rough eye phenotype, suggesting a transformation of non-neuronal cone cells to R7 photoreceptor cells (Shiomi, 1994). (Note: also see below, Wassarman, 1996).

Genetic evidence suggests that protein phosphatase regulates Ras mediated photoreceptor development in Drosophila. Using transgenic flies expressing constitutively activated Ras1 or Raf proteins that function independently of upstream signaling events, it has been shown that a reduction in the dose of the gene encoding the catalytic subunit of PP2A stimulates signaling from Ras1 but impairs signaling from Raf. The dominant Ras1 transgene results in a transformation of non-neuronal cone cells to R7 photoreceptor cells producing a visibly rough eye. Mutants in the catalytic subunit enhance the rough eye phenotype, causing a 48% increase in the number of supernumary R7 cells. Combining mutant PP2A catalytic subunit with constitutively activated ras results in a 30% decrease in the number of R7 cells per ommatidium. Germ-line clones of PP2A catalytic subunit block oogenesis suggesting a role for PP2A in oogenesis. What are the substrates for PP2A in the Ras1 pathway? Phosphorylation of Raf at different sites can either activate or inhibit its kinase activity, making it a possible substrate for negative or positive regulation by PP2A. Ksr kinase is also a potential target for negative regulation by PP2A; it appears to function between Ras1 and Raf and contains four consensus phosphorylation sites for MAPK (see Drosophila rolled), but it is not known whether phosphorylation of Ksr modulates its activity. PP2A has been shown to dephosphorylate and inactivate both MEK and MAPK in vitro, but PP2A alleles do not interact with MEK or MAPK (Wassarman, 1996).

Progression through mitosis requires the ubiquitin-mediated proteolysis of several regulatory proteins. A large multisubunit complex known as the anaphase-promoting complex or cyclosome (APC/C) plays a key role as an E3 ubiquitin-protein ligase in this process. The APC/C adds chains of ubiquitin to substrate proteins, targeting them for proteolysis by the 26S proteasome. The gene makos (mks) encodes the Drosophila counterpart of the Cdc27 subunit of the anaphase promoting complex (APC/C). Neuroblasts from third-larval-instar mks mutants arrest mitosis in a metaphase-like state but show some separation of sister chromatids. In contrast to metaphase-checkpoint-arrested cells, such mutant neuroblasts contain elevated levels not only of cyclin B but also of cyclin A. Mutations in mks enhance the reduced ability of hypomorphic polo mutant alleles to recruit and/or maintain the centrosomal antigens gamma-tubulin and CP190 at the spindle poles. Absence of the MPM2 epitope from the spindle poles in such double mutants suggests Polo kinase is not fully activated at this location. Thus, it appears that spindle pole functions of Polo kinase require the degradation of early mitotic targets of the APC/C, such as cyclin A, or other specific proteins. The metaphase-like arrest of mks mutants cannot be overcome by mutations in the spindle integrity checkpoint gene bub1, confirming this surveillance pathway has to operate through the APC/C. However, mutations in the twins/aar gene, which encodes the 55kDa regulatory subunit of PP2A, do suppress the mks metaphase arrest and so permit an alternative means of initiating anaphase. Thus the APC/C might normally be required to inactivate wild-type twins/aar gene product (Deak, 2003).

The metaphase-like arrest of mks cells cannot be overcome by the bub1 mutation. This is consistent with known functions of the APC/C downstream of the spindle integrity checkpoint. However, the ability of mutants in the aar/twins gene to overcome the metaphase arrest of mks is suggestive of an alternate mechanism for regulating the transition. In fact, the aar/twins mutant appears to be totally epistatic to (functioning downstram of) mks. Thus, the mks aar/twins double mutant shows a similar proportion of anaphase figures to the aar/twins mutant alone, and this is higher than the frequency of anaphases seen in wild-type cells. These observations cast some light on the possible multiple functions of the regulatory subunit of PP2A encoded by aar/twins in regulating anaphase and mitotic exit. It suggests that APC/C function might normally be required to inactivate the wild-type 55 kDa PP2A subunit that, in turn, negatively regulates sister chromatid separation. Thus, in the absence of aar/twins function, this aspect of APC/C involvement would not be required for anaphase, thus accounting for the epistasis of aar/twins to mks. The mutant aar/twins phenotype that then develops is akin to that observed following the expression of non-degradable forms of cyclin B, in which mitosis proceeds into anaphase. This outcome would be reinforced by a failure to exit mitosis as a result of the reduced ability of aar/twins mutants to dephosphorylate substrates of Cdk1 (Deak, 2003).

The anaphases in aar/twins and in the double mutant are highly abnormal, indicating that the checkpoint pathway that monitors chromosome alignment at metaphase and works through regulation of the APC/C is being circumvented. Consequently, there are many bridging and lagging chromatids in both aar/twins and mks aar/twins anaphase figures. This phenotype bears a striking resemblance to that seen in mutants of the CDC55 gene of budding yeast that encodes the orthologous regulatory subunit of PP2A. Cells with a cdc55 mutation have also been shown to leave mitosis without B-type cyclin destruction, in this case apparently owing to inhibitory tyrosine phosphorylation. However, it is also postulated in budding yeast that Cdc55p function is required for the kinetochore/spindle checkpoint. Such cdc55 mutants are sensitive to nocodazole and, in contrast to the situation for Drosophila cells, cdc55 mutations do not overcome the arrest imposed by mutation in an APC/C protein, in this case Cdc23p. Nevertheless, the abnormal morphology of cdc55 mutants and their conditional lethality is suppressed by a cdc28F19 mutation that encodes a variant kinase not susceptible to inhibitory phosphorylation. By contrast, nocodazole sensitivity cannot be suppressed by cdc28F19. This suggests that, in yeast, Cdc55p might have a checkpoint role that is independent of Cdc28/Cdk1 and a second role in regulating Cdc28 phosphorylation (Deak, 2003).

At the present time, it is not possible to account for how the APC/C might regulate the function of the 55kDa PP2A subunit although one possibility is through its direct proteolysis. It appears that this regulatory subunit of PP2A must participate in regulating the metaphase-anaphase transition, in controlling the activity of Cdk1 and in dephosphorylating Cdk1 substrates. It therefore remains a question of considerable future interest to determine exactly how these activities are coordinated (Deak, 2003).

Twins regulates Armadillo levels in response to Wg/Wnt signal

Protein Phosphatase 2A (PP2A) has a heterotrimeric-subunit structure, consisting of a core dimer of ~36 kDa catalytic and ~65 kDa scaffold subunits complexed to a third variable regulatory subunit. Several studies have implicated PP2A in Wg/Wnt signaling. However, reports on the precise nature of the PP2A role in Wg/Wnt pathway in different organisms are conflicting. twins (tws), which codes for the B/PR55 regulatory subunit of PP2A in Drosophila, is shown to be a positive regulator of Wg/Wnt signaling. In tws^- wing discs both short- and long-range targets of Wingless morphogen are downregulated. Analyses of tws^- mitotic clones suggest that requirement of Tws in Wingless pathway is cell-autonomous. Epistatic genetic studies indicate that Tws functions downstream of Dishevelled and upstream of Sgg and Armadillo. These results suggest that Tws is required for the stabilization of Armadillo/ß-catenin in response to Wg/Wnt signaling. Interestingly, overexpression of an otherwise normal Tws protein induces dominant-negative phenotypes. The conflicting reports on the role of PP2A in Wg/Wnt signaling could be due to the dominant-negative effect caused by the overexpression of one of the subunits (Bajpai, 2004).

Results of these studies show that Twins is involved in modifying Wg signaling. Partial to complete downregulation of short- (Ct and Sca) and long-range (Dll and Vg) targets of Wg pathway is observed in tws^- background. The downregulation of Wg signaling in wing discs is reflected in adult phenotypes, such as serrated wing margin in mitotic clones of tws. Loss-of-Wg phenotypes (induced by the overexpression of DN-TCF/pan or Sgg or Cad^intra) are enhanced in tws heterozygous mutant background. In addition, mutation in tws suppresses the phenotypes induced by Dsh, a positive component of Wg signaling. Finally, some of the phenotypes induced by the overexpression of Tws are characteristic of gain-of-Wg phenotypes. These results suggest that Tws functions as a positive regulator of Wg signaling (Bajpai, 2004).

Overexpression of otherwise normal Tws protein induces dominant-negative phenotypes. The dominant-negative phenotype is unlikely to be neomorphic or antimorphic, since UAS-Tws rescues tws alleles (at the levels of both Wingless-dependent and independent developmental events) and also induces gain-of-Wg phenotypes. The dominant-negative phenotype is probably due to imbalance in the relative amounts of the three subunits in the heterotrimeric complex, proper formation of which is obligatory for PP2A function. Thus, the conflicting reports on the role of PP2A in Wnt signaling could be due to the dominant negative effect caused by the overexpression of one of the subunits (Bajpai, 2004).

In tws mutant background, cytoplasmic Arm levels are downregulated. Even overexpressed Arm is degraded in tws^- background. Furthermore, loss of tws had no effect on the degradation-resistant form of Arm, which suggests that Tws functions upstream of Arm to mediate Wg signaling. These results could not be confirmed directly by Western blotting, since only a very small fraction (such as DV cells) of wing disc shows changes in Arm levels in response to Wg signaling. Nevertheless, results presented in this report suggest that stabilization of the cytoplasmic form of Arm by Wg signaling is dependent on Tws function (Bajpai, 2004).

A dominant-negative form of Sgg/GSK-3ß is able to rescue tws^- phenotype at the level of Dll expression. However, overexpression of Dsh failed to rescue Dll expression in tws^- discs, suggesting that Tws functions downstream of Dsh and upstream of Sgg to stabilize cytoplasmic Arm in response to Wg signaling. Preliminary results presented here suggest that function of Tws in Wg pathway is inactivation of Sgg. Normally, overexpressed APC sequesters Arm only in those cells in which Sgg activity is downregulated. In other cells, APC participates in Arm-degradation machinery. In tws^- wing discs, overexpressed APC fails to sequester Arm in DV cells, suggesting that loss of tws results in upregulation of Sgg activity. However, it has been reported that PR/B56epsilon functions upstream of Dsh to regulate Wnt signaling in Xenopus embryos. The PR/B56epsilon homolog in Drosophila is widerborst (with 80% identity at the protein level), which is involved in the determination of planar cell polarity. widerborst is also known to be functional upstream of Dsh, but not in the canonical Wg/Wnt pathway. Although Tws homologs in other organisms have not been well characterized, the current studies are consistent with a role for PP2A in dephosphorylation of Axin (Bajpai, 2004).

The next question regards the substrate of PP2A function in the Wg pathway. In mammalian cells, Axin is dephosphorylated in response to Wnt signaling. Furthermore, dephosphorylated Axin binds ß-catenin less efficiently than the phosphorylated form. Thus, dephosphorylation of Axin would free ß-catenin from the degradation machinery. Thus, Tws may function by inhibiting the activity of Axin, which acts a scaffold protein to bring Sgg and Arm to close proximity. Further biochemical work is in progress to determine phosphorylated status of Arm in tws^- background and to determine if Tws directly binds to Sgg or Axin or both (Bajpai, 2004).

Multiple protein phosphatases are required for mitosis in Drosophila

Approximately one-third of the Drosophila kinome has been ascribed some cell-cycle function. However, little is known about which of its 117 protein phosphatases (PPs) or subunits have counteracting roles. This study investigated mitotic roles of PPs through systematic RNAi. It was found that G2-M progression requires Puckered, the JNK MAP-kinase inhibitory phosphatase and PP2C in addition to string (Cdc25). Strong mitotic arrest and chromosome congression failure occurs after Pp1-87B downregulation. Chromosome alignment and segregation defects also occurs after knockdown of PP1-Flapwing, not previously thought to have a mitotic role. Reduction of several nonreceptor tyrosine phosphatases produced spindle and chromosome behavior defects, and for corkscrew, premature chromatid separation. RNAi of the dual-specificity phosphatase, Myotubularin, or the related Sbf 'antiphosphatase' resulted in aberrant mitotic chromosome behavior. Finally, for PP2A, knockdown of the catalytic or A subunits led to bipolar monoastral spindles, knockdown of the Twins B subunit led to bridged and lagging chromosomes, and knockdown of the B' Widerborst subunit led to scattering of all mitotic chromosomes. Widerborst is associated with MEI-S332 (Shugoshin) and is required for its kinetochore localization. This study has identified cell-cycle roles for 22 of 117 Drosophila PPs. Involvement of several PPs in G2 suggests multiple points for its regulation. Major mitotic roles are played by PP1 with tyrosine PPs and Myotubularin-related PPs having significant roles in regulating chromosome behavior. Finally, depending upon its regulatory subunits, PP2A regulates spindle bipolarity, kinetochore function, and progression into anaphase. Discovery of several novel cell-cycle PPs identifies a need for further studies of protein dephosphorylation (Chen, 2007).

P2A is a heterotrimeric serine/threonine phosphatase composed of invariant catalytic (C) and structural (A) subunits together with one member of a family of B regulatory subunits thought to direct the AC core to different substrates. The Drosophila gene for the catalytic subunit of type 2A protein serine/threonine phosphatase (PP2A) is known as microtubule star (mts) because mutant embryos show multiple individual centrosomes with disorganized astral arrays of microtubules. In agreement with this mutant phenotype, it was found that S2 cells depleted for Mts (PP2A-C) displayed aberrant elongated arrays of microtubules with a high proportion (5- to 10-fold increase over the control) of bipolar monoastral spindles or monopolar spindles emanating from a single centrosomal mass. This phenotype is also consistent with the observations in Xenopus egg extracts where mitotic microtubules grow longer and bipolar spindles can not be assembled after inhibition of PP2A by low concentrations of okadaic acid (OA). It is speculated that the monopolar spindle phenotype after mts dsRNA treatment is a consequence of the spindle collapse rather than a failure in centrosome duplication or separation because most of the RNAi-treated cells showed well-separated centrosomes during prophase. In support of this view, spindle bipolarity can be rescued by restoration of microtubule dynamics in OA-treated Xenopus egg extracts (Chen, 2007).

In Drosophila, as in many other eukaryotes, mitosis-specific phosphorylation of histone H3 requires Aurora B activity, but the identity of the opposing phosphatase remains unclear. Because P-H3 (Ser 10) levels were used for monitoring the mitotic index in this analysis, it is possible that a high mitotic index observed after RNAi for PPs may also reflect a defect in dephosphorylating P-H3 in the absence of PPs upon mitotic exit. The phosphorylation state of this histone was therefore studied after RNAi for PPs that displayed a significant increase in the mitotic index in the screen. The immunostaining of control cells showed that P-H3 signals began to decrease at early telophase and then disappeared completely at late telophase. After RNAi knockdown of Mts (PP2A-C) or Pp1-87B, however, the majority of mitotic cells were arrested at prometaphase, but late telophase figures could occassionally be found showing an abnormal accumulation of P-H3 on decondensed chromosomes. To better assess the effect of depletion of these two PPs on P-H3 dephosphorylation, the spindle-assembly checkpoint was inactivated by simultaneously knocking down BubR1. It was found that this rescued the prometaphase arrest of cells simultaneously depleted for Mts or Pp1-87B; this allowed a study of telophase cells. P-H3 was present in the majority of such telophase cells compared to control cells, indicating that both PPs are required for P-H3 dephosphorylation. These results are in accordance with previous studies showing that reduction of PP1 activity can partially suppress defects in the mitotic histone H3 phosphorylation in yeast and C. elegans (Chen, 2007).

Downregulation of Pp2A-29B, the structural A subunit, revealed almost identical aberrant phenotypes to those observed after mts (PP2A-C) RNAi. Consistently, knockdown of Pp2A-29B (PP2A-A) led to a reduction of the protein level of Mts (PP2A-C) (Chen, 2007).

The Drosophila genome contains 4 B-type PP2A regulatory subunits, twins/tws/aar (B sub-type), widerborst/wdb (B' sub-type), Pp2A-B' (B' sub-type), and Pp2A-B" (B" sub-type), but mitotic defects have so far only been reported for mutants of tws. Consistent with the phenotype of tws mutants, it was observed that RNAi for this gene led to an increased proportion of anaphase figures showing lagging chromosomes and chromosome bridges (Chen, 2007).

In metazoans, the B' regulatory subunits of PP2A have evolved into two related subclasses with conserved central regions and diverged amino and carboxy termini. The protein encoded by widerborst (wdb) is more closely related to the human α and ɛ subunits (79%-80% identity) than to the β, γ, or δ subunits (69%-75% identity). Whereas RNAi for tws led to lagging chromosomes, wdb RNAi led to dramatic scattering of chromosomes throughout the spindle. Whether this dramatic effect of wdb RNAi on chromosome segregation reflected any particular subcellular localization of this regulatory subunit was examined. To this end, a GFP-tagged Wdb was expressed in S2 cells. During interphase and prophase, Wdb::GFP partially colocalized with the centromeric marker CID (CENP-A). After spindle formation, Wdb::GFP was found adjacent and external to the centromeres. Although less pronounced, this distribution remained during chromosome segregation at anaphase. Because MEI-S332 (Drosophila Shugoshin) is a dynamic centromeric marker, its distribution was examined in wdb RNAi cells. In control cells, MEI-S332 localized in a band between each pair of the centromeres at metaphase. After downregulation of wdb, however, greatly reduced MEI-S332 staining was found on the metaphase chromosome. In contrast, depletion of MEI-S332 by RNAi did not affect the normal localization of the Wdb B' PP2A subunit. Thus, it is concluded that the Wdb B' subunit is required for correct localization of MEI-S332 but not vice versa. Whether the two proteins existed in the same complex was examined. To address this, a Protein A (PrA)-tagged form of MEI-S332 was expressed in S2 cells to purify potential protein complexes and identify its components by mass spectrometry. The catalytic C (Mts), the structural A (PP2A-29B), and the regulatory B' (Wdb) and B (Tws) subunits of PP2A were identified associated with MEI-S332. Three recent studies also identified PP2A complexed to the B' subunit bound to Shugoshin (Sgo) in human and yeast cells, where they are thought to protect centromeric cohesin subunits from phosphorylation that would promote premature sister-chromatid separation. As with the archetypal family member, Drosophila MEI-S332, the Shugoshins function primarily to protect sister chromatids from separation in the first meiotic division but are also present in mitotic divisions. Consistent with these observations in Drosophila S2 cells, it has been found that depletion of PP2A in human cells led to premature dissociation of Shugoshin 1 (Sgo1) from the kinetochore and loss of mitotic centromere cohesion. The finding of Shugoshin complexed to PP2A/B' in yeast and human, and now in Drosophila, points to a highly evolutionally conserved role for this particular PP2A heterotrimer in regulating sister-chromatid cohesion. Interestingly, Tws B regulatory subunit was also recovered associated with MEI-S332. How this subunit of PP2A might function with MEI-S332 should be the subject of future investigations (Chen, 2007).

Only a moderatedly elevated mitotic index (by approximately 10%) was observed after downregulation of the second Drosophila B' regulatory subunit (Pp2A-B'/B56-1). However, when this second B' subunit was simultaneously knocked down with Wdb, this led to similar phenotypes seen in Mts (PP2A-C) or Pp2A-29B (PP2A-A)-depleted cells. Western-blot analysis showed that the Mts (PP2A-C) level decreased after simultaneous knockdown of both B' subunits, suggesting that this phenotype could be partially due to the loss of PP2A catalytic subunit, although the possibility that the two B' subunits share partially redundant mitotic functions cannot be excluded (Chen, 2007).

Cell-cycle kinases represent a large family of enzymes governing the cell division cycle. It is therefore not surprising that a considerable number of counteracting cell-cycle phosphatases (19% of the genes for tested) were identified in the current study. In addition to finding all the well-known PPs required for cell-cycle progression in Drosophila (Mts, Tws, String, Pp4-19C, and Pp1-87B), the Drosophila counterparts of some eight PPs implicated in cell-cycle functions were identified from studies on other organisms together with six PPs for which novel cell-cycle roles were ascribe. These results were validated by confirming the observed phenotypes with a second nonoverlapping dsRNA. In two cases (flw and csw), their mitotic roles were confirmed through the analysis of phenotypes in mutant larval neuroblasts. The RNAi phenotypes of catalytic subunits were evaluated by observing similar phenotypes after downregulation of the corresponding regulatory subunits (e.g., Pp4-19C and PPP4R2r, Mts/PP2A-C and Pp2A-29B/PP2A-A, and simultaneous RNAi of the two PP2A-B' regulatory subunits). Although a recent large-scale RNAi screen based solely on flow cytometry in Drosophila S2 cells identified many regulators of the cell cycle, cell size, and cell death, this study showed a very low degree of overlap with the cureent analysis (only six), reflecting the need for more sensitive small-scale screens that can examine the functional requirements of assayed proteins in greater detail. These results have provided novel insights into the cell-cycle functions of the Drosophila PPs, and it is likely that, in many cases, these functions have been conserved in other metazoans including humans. This study should guide future work aimed at elucidating the significance and mechanisms of the balanced activities of PKs and PPs in regulating the cell division cycle. The challenge ahead will be to match up the functions of the PPs that were identified with their corresponding counteracting PKs and to identify their common key substrates (Chen, 2007).

Suppression of Scant identifies Endos as a substrate of Greatwall Kinase and a negative regulator of Protein Phosphatase 2A in mitosis

Protein phosphatase 2A (PP2A) plays a major role in dephosphorylating the targets of the major mitotic kinase Cdk1 at mitotic exit, yet how it is regulated in mitotic progression is poorly understood. This study shows that mutations in either the catalytic or regulatory twins/B55 subunit of PP2A act as enhancers of gwl^Scant, a gain-of-function allele of the Greatwall kinase gene that leads to embryonic lethality in Drosophila when the maternal dosage of the mitotic kinase Polo is reduced. It was also shown that heterozygous mutant endos alleles suppress heterozygous gwl^Scant; many more embryos survive. Furthermore, heterozygous PP2A mutations make females heterozygous for the strong mutation polo¹¹ partially sterile, even in the absence of gwl^Scant. Heterozygosity for an endos mutation suppresses this PP2A/polo¹¹ sterility. Homozygous mutation or knockdown of endos leads to phenotypes suggestive of defects in maintaining the mitotic state. In accord with the genetic interactions shown by the gwl^Scant dominant mutant, the mitotic defects of Endos knockdown in cultured cells can be suppressed by knockdown of either the catalytic or the Twins/B55 regulatory subunits of PP2A but not by the other three regulatory B subunits of Drosophila PP2A. Greatwall phosphorylates Endos at a single site, Ser68, and this is essential for Endos function. Together these interactions suggest that Greatwall and Endos act to promote the inactivation of PP2A-Twins/B55 in Drosophila. The involvement of Polo kinase in such a regulatory loop is discussed (Rangone, 2011).

This study identified endos mutations as heterozygous suppressors of the dominant mutant phenotype of polo¹ gwl^Scant. This suggests that Greatwall and Endos promote the same mitotic pathway. In accord with this it was found that the consequences of loss of gwl and of endos function in mitosis are very similar. This study found that larval neuroblasts from homozygous endos mutants show poorly condensed chromosomes and anaphase bridging, a phenotype very similar to recessive gwl mutants. In cultured Drosophila cells, depletion of endos interferes with proper mitotic exit and allows cells to accumulate that have elongated spindles but have not undertaken chromatid separation or Cyclin B destruction. This is similar to the removal of Gwl from CSF Xenopus extracts; there, an unusual mitotic exit occurs in which cyclins remained undegraded but Cyclin-dependent kinase 1 (Cdk1) is inactivated by phosphorylation at Thr14 and Tyr15 (Rangone, 2011).

Three lines of genetic evidence indicate that Greatwall and Endos are required to down-regulate the function of B55/Twins-bound PP2A. Lowering the dosage of either the catalytic C subunit or the B55/Twins regulatory subunit of PP2A enhances the maternal dominant effect of polo¹ gwl^Scant and this is suppressed by lowering the dosage of endos. Secondly, opposing roles for Endos and PP2A in regulating Polo kinase function are seen in the absence of the gwl^Scant mutation; the low fertility of twins/polo trans-heterozygous females is also dramatically suppressed by one mutant copy of endos. Thirdly, the Endos depletion phenotype in cultured cells is suppressed by simultaneous depletion of either the catalytic C subunit, the structural A subunit, or the B55/Twins regulatory subunit of PP2A but notably not by co-depletion of the three other regulatory B subunits. Together these interactions suggest that Greatwall activates Endos leading to the inhibition of PP2A-B55/Twins. This is in accord with recent studies in Xenopus showing that inhibition or depletion of PP2A-B55 from mitotic extracts rescues the inability of Gwl-depleted extracts to enter M phase and also with two recent biochemical studies that show that the Xenopus counterpart of Gwl kinase can phosphophorylate two related members of the cAMP-regulated phosphoprotein family, Ensa (the Endos counterpart) or Arpp19, to make these molecules highly effective inhibitors of PP2A. Endos is the unique cAMP-regulated phosphoprotein family member in Drosophila. Indeed, such is the degree of conservation that Drosophila Gwl kinase phosphorylates Endos only at Serine 68, a site essential for Endos function; this is the exact counterpart of the Serine 67 site in Xenopus. Studies in Drosophila, Xenopus and human cells indicate that PP2A is a major protein phosphatase acting to dephosphorylate Cdk1 substrates. Thus gwl or endos reduced-function mutants should have increased activity of PP2A and therefore accumulate dephosphorylated Cdk1 substrates. Failure of Cdk1 substrates to become maximally phosphorylated in spite of high levels of Cyclin B accumulation would account for the prolonged prometaphase-like state and the eventual development of elongated spindles without having appeared to activate the anaphase-promoting complex in these mutantsThis leads to a model in which Greatwall kinase is active in mitosis in order to convert Endos into an inhibitor of PP2A-Twins/B55, which is then inactived upon mitotic exit to permit the dephosphorylation of Cdk1 substrates by this phosphatase (Rangone, 2011).

The above simple model is, however, confounded by genetic interactions suggesting that the gain-of-function mutation gwl^Scant negatively regulates the function of the mitotic kinase Polo or one of its downstream targets. Such evidence comes largely from the search for suppressors of polo¹¹ gwl^Scant that identified mutations in two broad categories: (1) those that decrease the effect of Gwl or its downstream targets as exemplified by endos mutations and reversion of gwl^Scant to recessive mutant alleles; (2) those that increase the activity of Polo kinase such as the polo⁺ duplications that were obtained. Moreover, the degree of sterility (adult progeny per female) and frequency of embryonic centrosome loss co-vary with strength of polo allele. polo¹, a hypomorphic allele with sufficient residual Polo function to be homozygous viable, is slightly fertile heterozygous with Scant and its embryos are only moderately defective, whereas polo¹¹, a lethal amorphic mutation, is completely sterile heterozygous with Scant and its embryos are much more defective. Furthermore, over-expressing Map205 (a known binding partner of Polo which sequesters the kinase on microtubules) in ovaries of polo¹¹/+ mothers mimics Scant regarding the centrosome detachment phenotype, and more defective nuclei are seen when the transgene carries a mutation preventing Polo release (Rangone, 2011).

Together these results suggest that the specific defect in Scant polo-derived embryos, detachment of centrosomes from the nuclear envelope, is a consequence of the reduction of the level of functional Polo below a critical threshold. Indeed this is the only phenotype that could be attributed to the Scant allele of gwl and its sensitivity to the gene dosage of polo suggests that this function requires the highest level of Polo kinase activity in comparison to all of Polo's other roles. It is important to note that centrosome detachment is an interphase phenotype. It occurs after the centrosomes have separated, which in wild type is during telophase in anticipation of the next round of mitosis in the rapidly alternating S and M phases of the syncytial Drosophila embryo. In the normal mitotic cycle, Greatwall kinase would not be active at this stage. Thus the functional complex of PP2A and its B55/Twins regulatory subunit seems to be required to positively regulate Polo activity or a process controlled by Polo between the exit from one mitotic cycle and entry into the next. This accounts for the finding that mutations in the PP2A subunit genes, mts and twins, enhance sterility when transheterozygous with polo¹¹, and that this sterility is in turn relieved by heterozygous endos mutations. Although it is possible that PP2A removes an inhibitory phosphorylation from Polo, this seems unlikely because no such phosphorylation has been identified to date. Thus the alternative is favored, that PP2A acts to stimulate a process promoted by Polo and a dephosphorylated partner. Indeed it is known that Polo interacts with phosphorylated partners after mitotic entry and with dephosphorylated partners from late anaphase onwards (Rangone, 2011).

PP2A-twins is antagonized by greatwall and collaborates with polo for cell cycle progression and centrosome attachment to nuclei in drosophila embryos

Cell division and development are regulated by networks of kinases and phosphatases. In early Drosophila embryogenesis, 13 rapid nuclear divisions take place in a syncytium, requiring fine coordination between cell cycle regulators. The Polo kinase is a conserved, crucial regulator of M-phase. An antagonism exists between Polo and Greatwall (Gwl), another mitotic kinase, in Drosophila embryos (Archambault, 2007). However, the nature of the pathways linking them remained elusive. A comprehensive screen was conducted for additional genes functioning with polo and gwl. A strong interdependence was uncovered between Polo and Protein Phosphatase 2A (PP2A) with its B-type subunit Twins (Tws). Reducing the maternal contribution of Polo and PP2A-Tws together is embryonic lethal. Polo and PP2A-Tws were found to collaborate to ensure centrosome attachment to nuclei. While a reduction in Polo activity leads to centrosome detachments observable mostly around prophase, a reduction in PP2A-Tws activity leads to centrosome detachments at mitotic exit, and a reduction in both Polo and PP2A-Tws enhances the frequency of detachments at all stages. Moreover, Gwl was shown to antagonize PP2A-Tws function in both meiosis and mitosis. This study highlights how proper coordination of mitotic entry and exit is required during embryonic cell cycles and defines important roles for Polo and the Gwl-PP2A-Tws pathway in this process (Wang, 2011).

These results shed new light on cell cycle regulation and syncytial embryogenesis. High Polo activity is needed to promote the normal cohesion between centrosomes and nuclei, and this is mostly observable around the time of mitotic entry. Interestingly, transiently detached centrosomes can be recaptured by the assembling spindle and nuclear division can then be completed. This centrosome recapture is probably essential for successful development of the syncytial embryo. A systematic genetic screen unveiled a very strong and specific functional link between Polo and a specific form of PP2A associated with its B-type subunit Tws. PP2A-Tws activity is required for centrosome cohesion with nuclei, although in late M-phase, around the time of mitotic exit. This is consistent with a recent study where centrosome defects were observed in late M-phase when the small T antigen of SV40, which binds PP2A, was expressed in Drosophila embryos (Kotadia, 2008}. PP2A-B55δ (ortholog of Twins) has been recently implicated in promoting mitotic exit in vertebrates, by inactivating Cdc25C and by directly dephosphorylating Cdk1 mitotic substrates (Castilho, 2009; Forester, 2007). The closely related isoform PP2A-B55α has been shown to promote the timely reassembly of the nuclear envelope at mitotic exit. Thus, the failure to reattach centrosomes to nuclei during mitotic exit in PP2A-Tws compromised embryos could be due to problems or a delay in nuclear envelope resealing (Wang, 2011).

The results indicate that the proper regulation of the events of mitotic entry and exit by Polo and PP2A-Tws is crucial. This may be particularly true in the syncytial embryo due to the rapidity of the cycles, where one mitosis is almost immediately followed by another, and because of the obligatory cohesion between centrosomes and nuclei for their migration towards the cortex of the syncytium. Combining partial decreases in the activities of Polo and Tws strongly enhances the frequency of centrosome detachments observed. This suggests that when centrosomes fail to attach properly for too long between mitotic exit and the next mitotic entry, they become permanently detached from nuclei, leading to failures in mitotic divisions (Wang, 2011).

The differences in timing between the detachments observed in polo and tws hypomorphic situations led to a proposal that the two enzymes act in parallel pathways, of which the disruption can lead to a failure in centrosome-nucleus cohesion. This is also supported by the prominent roles of Polo in regulating centrosome maturation and mitotic entry (Archambault, 2009), and the specific requirements of PP2A-Tws/B55 at mitotic exit. However, it cannot be excluded that Polo, Gwl and PP2A-Tws could function on a common substrate, or even in the same linear pathway, where the different players of the pathway could become more or less influential at different times of the cell cycle. In has been proposed that PP2A promotes full expression of Polo in larval neuroblasts and in S2 cells (Wang, 2009). It has also been shown that depletion of Tws by RNAi leads to centrosome maturation defects in S2 cells (Dobbelaere, 2008), which could be explained by a reduction in Polo levels. However, no significant difference has been detected in Polo levels in embryos from gwl^Scant/+ or tws/+ females, compared to wild-type controls by Western blotting. Deeper genetic and molecular dissection of those pathways should lead to a clearer understanding of the regulation of centrosome and nuclear dynamics during mitotic entry and exit (Wang, 2011).

These results add strong support to an emerging model for a pathway that controls entry into and exit from mitosis and meiosis in animal cells. It is increasingly clear that a form of PP2A associated with a B-type regulatory subunit plays a crucial and conserved role in competing with Cdk1. In Xenopus egg extract, PP2A-B55δ activity is high in interphase and low in M phase. PP2A-B55δ must be down-regulated to allow mitotic entry, and conversely, it appears to promote mitotic exit both by inactivating Cdc25C and by dephosphorylating Cdk1 substrates. In human cells, depletion in B55α delays the events of mitotic exit, including nuclear envelope reassembly. Already some years ago, mutations in Drosophila tws were found to lead to a mitotic arrest in larval neuroblasts, and extracts from tws mutants were shown to have a reduced ability to dephosphorylate Cdk substrates. Mutations in mts resulted in an accumulation of nuclei in mitosis in the embryo. The budding yeast now appears to be a particular case, as its strong reliance on the Cdc14 phosphatase to antagonize Cdk1 may reflect the need for insertion of the anaphase spindle through the bud neck prior to mitotic exit, a constraint that does not exist in animal cells. Nevertheless, additional phosphatases to PP2A, including PP1 are likely to play conserved roles in promoting mitotic and meiotic exit, and this remains to be dissected (Wang, 2011 and references therein).

Identification of PP2A genes as functional interactors of polo and gwl is the result of an unbiased genetic screen. It was found that an elevation in Gwl function combined with a reduction in PP2A-Tws activity leads to a block in M phase, either in metaphase of meiosis I or in the early mitotic cycles. However, positioning of Gwl as an antagonist of PP2A-Tws was facilitated by reports that appeared subsequent to the screen, proposing that the main role of Gwl in promoting M-phase was to lead to the inactivation of PP2A-B55δ in Xenopus egg extracts. Results consistent with this idea were also obtained in mammalian cells (Wang, 2011 and references therein).

More recently, two seminal biochemical studies using Xenopus egg extracts showed that the antagonism of PP2A-B55δ by Gwl is mediated by α-endosulfine/Ensa and Arpp19, two small, related proteins which, when phosphorylated by Gwl at a conserved serine residue, become able to bind and inhibit PP2A-B55δ (Gharbi-Ayachi, 2010; Mochida, 2010). By this mechanism, Gwl activation at mitotic entry leads to the inhibition of PP2A-B55γ, which results in an accumulation of the phosphorylated forms of Cdk1 substrates. Depletion of human Arpp19 also perturbs mitotic progression in Hela cells (Gharbi-Ayachi, 2010), suggesting a conserved role among vertebrates (Wang, 2011).

In an independent study, the group of David Glover has recently identified mutations in Drosophila endosulfine (endos) as potent suppressors of the embryonic lethality that occurs when gwl^Scant (the gain-of-function allele) is combined with a reduction in polo function, in a maternal effect (Rangone, 2011). endos is the single fly ortholog of Xenopus α-endosulfine and Arpp19. That the identification of endos by Rangone came from another unbiased genetic screen testifies of the specificity and conservation of the Gwl-Endos-PP2A pathway in animal cells. The authors went as far as showing that the critical phosphorylation site of Gwl in Endos is conserved between frogs and flies, and is critical for the function of Endos in antagonizing PP2A-Tws in cultured cells. These findings are consistent with a previous report showing that mutations in endos lead to a failure of oocytes to progress into meiosis until metaphase I (Von Stetina, 2008). Moreover, loss of Gwl specifically in the female germline also leads to meiotic failure, although in that case oocytes do reach metaphase I but exit the arrest aberrantly (Archambault, 2007). Although the meaning of those phenotypic differences is not yet understood, Gwl and Endos are both required for meiotic progression in Drosophila. Conversely, this study shows that excessive Gwl activity relative to PP2A-Tws prevents exit from the metaphase I arrest, suggesting that the inhibition of PP2A-Tws by Gwl and Endos must be relieved to allow completion of meiosis. Moreover, Rangone (2011) shows that the Endos pathway also regulates the mitotic cell cycle in the early embryo, in larval neuroblasts and in cultured cells (Wang, 2011).

Together, the systematic and unbiased identifications of mutations in PP2A-Tws subunit genes as enhancers (this paper), and of mutations in endos as suppressors (Rangone, 2011) of gwl^Scant provide strong evidence for a pathway connecting these genes to control M phase in flies. These studies provide a convincing genetic and functional validation of the recent biochemical results from Xenopus extracts, and show that the Gwl-Endos-PP2A-Tws/B55 pathway is conserved and plays a key role in regulating both meiosis and mitosis in a living animal (Wang, 2011).

Bypassing the Greatwall-Endosulfine pathway: plasticity of a pivotal cell-cycle regulatory module in Drosophila melanogaster and Caenorhabditis elegans

In vertebrates, mitotic and meiotic M phase is facilitated by the kinase Greatwall (Gwl), which phosphorylates a conserved sequence in the effector Endosulfine (Endos). Phosphorylated Endos inactivates the phosphatase PP2A/B55 to stabilize M-phase-specific phosphorylations added to many proteins by cyclin-dependent kinases (CDKs). This module functions essentially identically in Drosophila and is necessary for proper mitotic and meiotic cell division in a wide variety of tissues. Despite the importance and evolutionary conservation of this pathway between insects and vertebrates, it can be bypassed in at least two situations. First, heterozygosity for loss-of-function mutations of twins, which encodes the Drosophila B55 protein, suppresses the effects of endos or gwl mutations. Several types of cell division occur normally in twins heterozygotes in the complete absence of Endos or the near absence of Gwl. Second, this module is nonessential in the nematode Caenorhaditis elegans. The worm genome does not contain an obvious ortholog of gwl, although it encodes a single Endos protein with a surprisingly well-conserved Gwl target site. Deletion of this site from worm Endos has no obvious effects on cell divisions involved in viability or reproduction under normal laboratory conditions. In contrast to these situations, removal of one copy of twins does not completely bypass the requirement for endos or gwl for Drosophila female fertility, although reducing twins dosage reverses the meiotic maturation defects of hypomorphic gwl mutants. These results have interesting implications for the function and evolution of the mechanisms modulating removal of CDK-directed phosphorylations (Kim, 2012).

EVOLUTIONARY HOMOLOGS

Protein phosphatase 2A (PP2A) appears to be involved in the regulation of many cellular processes. Control mechanisms that lead to the activation (and deactivation) of the various holoenzymes to initiate appropriate dephosphorylation events remain obscure. The core components of all PP2A holoenzymes are the catalytic (PP2Ac) and 63-65-kD regulatory (PR65) subunits. Monospecific and affinity-purified antibodies against both PP2Ac and PR65 show that these proteins are ubiquitously localized in the cytoplasm and the nucleus in nontransformed fibroblasts. The core subunits of PP2A are twofold more concentrated in the nucleus than in the cytoplasm. Detailed analysis of synchronized cells reveals striking changes in the nuclear to cytoplasmic ratio of PP2Ac-specific immunoreactivity, albeit the total amounts of PP2Ac and PR65 in each compartment do not alter significantly during the cell cycle. These results imply that differential methylation of PP2Ac occurs at the G0/G1 and G1/S boundaries. Specifically, a demethylated form of PP2Ac is found in the cytoplasm of G1 cells, and in the nucleus of S and G2 cells. In addition, nuclear PP2A holoenzymes appear to undergo conformational changes at the G0/G1 and G1/S boundaries. During mitosis PP2A is lost from the nuclear compartment; unlike protein phosphatase 1, PP2A shows no specific association with the condensed chromatin (Turowski, 1995).

Differential association of regulatory B subunits with a core heterodimer, composed of a catalytic (C) and a structural (A) subunit, is an important mechanism that regulates protein phosphatase 2A (PP2A). Three novel cDNAs related to the B' subunit of bovine cardiac PP2A have been isolated and characterized. Two human (B'alpha1 and B'alpha2) and a mouse (B'alpha3) cDNA encode for alternatively spliced variants of the B subunit. The deduced primary sequences of these clones contain 12 of 15 peptides derived from the purified bovine B' subunit. Differences between the deduced sequences of the B alpha splice variants and the cardiac peptide sequences suggest the existence of multiple isoforms of the B' subunit. Comparison of the protein and nucleotide sequences of the cloned cDNAs show that all three forms of B'alpha diverge at a common splice site near the 3'-end of the coding regions. Northern blot and reverse transcription-polymerase chain reaction analyses reveals that the B'alpha transcripts (4.3-4.4 kb) are widely expressed and very abundant in heart and skeletal muscle. The expressed human and mouse B'alpha proteins readily associate with the PP2A core enzyme in both in vitro and in vivo complex formation assays. Epitope-tagged B'alpha has been localized in both the cytosol and nuclei of transiently transfected cells. The efficiency of binding of all three expressed proteins to a glutathione S-transferase-A subunit fusion protein is greatly enhanced by the addition of the C subunit. Expression of the B'alpha subunits in insect Sf9 cells results in formation of AC.B'alpha heterotrimers with the endogenous insect A and C subunits. These results show that the B' subunit, which is the predominant regulatory subunit in cardiac PP2A, is a novel protein whose sequence is unrelated to other PP2A regulatory subunits. The nuclear localization of expressed B'alpha suggests that some variants of the B' subunit are involved in the nuclear functions of PP2A (Tehrani, 1998).

The phosphoprotein phosphatase 2A (PP2A) catalytic subunit contains a methyl ester on its C-terminus, which in mammalian cells is added by a specific carboxyl methyltransferase and removed by a specific carboxyl methylesterase. Genes in yeast have been identified that show significant homology to human carboxyl methyltransferase and methylesterase. The methyltransferase homolog is not present in C. elegans or D. melanogaster. Extracts of wild-type yeast cells contain carboxyl methyltransferase activity, while extracts of strains deleted for one of the methyltransferase genes, PPM1, lack all activity. Mutation of PPM1 partially disrupts the PP2A holoenzyme in vivo and ppm1 mutations exhibit synthetic lethality with mutations in genes encoding the B or B' regulatory subunit. Inactivation of PPM1 or overexpression of PPE1, the yeast gene homologous to bovine methylesterase, yields phenotypes similar to those observed after inactivation of either regulatory subunit. These phenotypes can be reversed by overexpression of the B regulatory subunit. These results demonstrate that Ppm1 is the sole PP2A methyltransferase in yeast and that its activity is required for the integrity of the PP2A holoenzyme (Wu, 2000).

In contrast to its effect on heterotrimer formation, loss of PP2A methyltransferase activity does not diminish the affinity of the C subunit for another regulatory partner in the cell, Tap42. The retention of interaction of the unmethylated C subunit with Tap42 is consistent with the rapamycin resistance of ppm1 strains, since resistance to rapamycin depends on retaining association between Tap42 and the C subunit. This difference in the effect of carboxyl methylation on affinity of the C subunit for B regulatory subunits compared with Tap42/alpha4 raises the possibility that methylation could provide a mechanism for modulating the distribution of the C subunit among these different regulatory elements. These results indicate that methylation of the PP2A C subunits is in dynamic equilibrium through the competing reactions catalyzed by Ppm1 and Ppe1. Accordingly, inhibition of PP2A methyltransferase or activation of PPME would tend to diminish PP2A activity while strengthening the Tor-mediated pathway (Tor kinase mediates phosphorylation of Tap42). Thus, PPMT or PPME could serve as a locus through which internal or external signals impinge on cellular proliferation (Wu, 2000).

Phosphoprotein phosphatase 2A (PP2A) is a major phosphoserine/threonine protein phosphatase in all eukaryotes. It has been isolated as a heterotrimeric holoenzyme composed of a 65 kDa A subunit, which serves as a scaffold for the association of the 36 kDa catalytic C subunit, and a variety of B subunits that control phosphatase specificity. The C subunit is reversibly methyl esterified by specific methyltransferase and methylesterase enzymes at a completely conserved C-terminal leucine residue. Methylation plays an essential role in promoting PP2A holoenzyme assembly and demethylation has an opposing effect. Changes in methylation indirectly regulate PP2A phosphatase activity by controlling the binding of regulatory B subunits to AC dimers. Since PP2A has been implicated in the regulation of cell proliferation, it seems likely that changes in methylation function in cell cycle regulation. Results with mammalian fibroblasts in tissue culture indicate that methylation levels may depend on the cell cycle, with different patterns of regulation in the nucleus and cytoplasm. Cytoplasmic PP2A appears to become demethylated at the G0/G1 boundary and remethylated as cells entered S phase, at which point nuclear PP2A is demethylated (Tolstykh, 2000).

Okadaic acid (OA) enhances the resumption of meiosis in mouse oocytes, indicating that serine/threonine protein phosphatase-1 (PP1) and/or PP2A is involved. However, specific identification of PP1 and/or PP2A in mouse oocytes has not been reported. Fully grown germinal vesicle-intact (GVI) mouse oocytes contain mRNA corresponding to two isotypes of PP1: PP1alpha and PP1gamma. Also present is the transcript for PP2A. At the protein level only PP1alpha and PP2A are recognized in fully grown GVI oocytes, using Western blot analysis. Neither of the PP1gamma spliced variant proteins, PP1gamma1 and PP1gamma2, is detectable. Immunohistochemical analysis of ovarian tissue from gonadotropin-stimulated adult mice results in subcellular localization of both PP1alpha and PP2A, but not PP1gamma, in oocytes from all stages of folliculogenesis. In primordial oocytes, PP1alpha and PP2A are present in the cytoplasm. In more advanced stages of oogenesis, PP1alpha, although still present in the cytoplasm, is highly concentrated in the nucleus, whereas PP2A is predominantly cytoplasmic with a distinct reduction in the nuclear area. Both PP1alpha and PP2A are immunodetectable in oocytes during the prepubertal period. Eleven-day-old mouse oocytes, considered OA-insensitive and germinal vesicle breakdown (GVB)-incompetent, display both PP1alpha and PP2A predominantly in the cytoplasm. By 15 days of age mouse oocytes, which are beginning to acquire OA sensitivity and GVB competence, show a relocation of PP1alpha into the nucleoplasm while PP2A remains predominantly cytoplasmic. This is the first specific identification of PP1alpha and PP2A in mouse oocytes. The differential localization of PP1alpha and PP2A, in addition to the relocation of PP1alpha during the acquisition of meiotic competence, suggests that these PPs have distinct regulatory roles during the resumption of meiosis (Smith, 1998).

Structure of PP2A

The PR65/A subunit of protein phosphatase 2A serves as a scaffolding molecule to coordinate the assembly of the catalytic subunit and a variable regulatory B subunit, generating functionally diverse heterotrimers. Mutations of the beta isoform of PR65 are associated with lung and colon tumors. The crystal structure of the PR65/Aalpha subunit, at 2.3 A resolution, reveals the conformation of its 15 tandemly repeated HEAT sequences, degenerate motifs of approximately 39 amino acids present in a variety of proteins, including huntingtin and importin beta. Individual motifs are composed of a pair of antiparallel alpha helices that assemble in a mainly linear, repetitive fashion to form an elongated molecule characterized by a double layer of alpha helices. Left-handed rotations at three interrepeat interfaces generate a novel left-hand superhelical conformation. The protein interaction interface is formed from the intrarepeat turns that are aligned to form a continuous ridge (Groves, 1999).

Alpha4 is a ubiquitin-binding protein that regulates protein serine/threonine phosphatase 2A ubiquitination

Multiple regulatory mechanisms control the activity of the protein serine/threonine phosphatase 2A catalytic subunit (PP2Ac), including post-translational modifications and its association with regulatory subunits and interacting proteins. Alpha4 is a PP2Ac-interacting protein that is hypothesized to play a role in PP2Ac ubiquitination via its interaction with the E3 ubiquitin ligase Mid1, which targets phosphatase 2A for degradation. This report shows that murine alpha4 serves as a necessary adaptor protein that provides a binding platform for both PP2Ac and Mid1. Aa novel ubiquitin-interacting motif (UIM) was identified within alpha4 (amino acid residues 46-60), and the interaction between alpha4 and ubiquitin was analyze using NMR. Consistent with other UIM-containing proteins, alpha4 is monoubiquitinated. Interestingly, deletion of the UIM within alpha4 enhances its association with polyubiquitinated proteins. Lastly, it was demonstrated that addition of wild-type alpha4 but not an alpha4 UIM deletion mutant suppresses PP2Ac polyubiquitination. Thus, the polyubiquitination of PP2Ac is inhibited by the UIM within alpha4. These findings reveal direct regulation of PP2Ac polyubiquitination by a novel UIM within the adaptor protein alpha4 (McConnell, 2010).

PP2A mutation

Protein phosphatase 2A (PP2A) is a multimeric enzyme, containing a catalytic subunit complexed with two regulatory subunits. The catalytic subunit PP2A C is encoded by two distinct and unlinked genes, termed Calpha and Cbeta. The specific function of these two catalytic subunits is unknown. To address the possible redundancy between PP2A and related phosphatases as well as between Calpha and Cbeta, the Calpha subunit gene was deleted by homologous recombination. Homozygous null mutant mice are embryonically lethal, demonstrating that the Calpha subunit gene is an essential gene. Because PP2A exerts a range of cellular functions, including cell cycle regulation and cell fate determination, it was surprising to find that these embryos develop normally until postimplantation, around embryonic day 5.5/6.0. While no Calpha protein is expressed, comparable expression levels of PP2A C are found at a time when the embryo is degenerating. Despite a 97% amino acid identity, Cbeta cannot completely compensate for the absence of Calpha. Degenerated embryos can be recovered even at embryonic day 13.5, indicating that although embryonic tissue is still capable of proliferating, normal differentiation is significantly impaired. While the primary germ layers (ectoderm and endoderm) are formed, mesoderm is not formed in degenerating embryos (Gotz, 1998).

The PPP2R1B gene, which encodes the beta isoform of the A subunit of the serine/threonine protein phosphatase 2A (PP2A), has been identified as a putative human tumor suppressor gene. Sequencing of the PPP2R1B gene, located on human chromosome 11q22-24, reveals somatic alterations in 15% (5 out of 33) of primary lung tumors, 6% (4 out of 70) of lung tumor-derived cell lines, and 15% (2 out of 13) of primary colon tumors. One deletion mutation generated a truncated PP2A-Abeta protein that is unable to bind to the catalytic subunit of the PP2A holoenzyme. The PP2R1B gene product may suppress tumor development through its role in cell cycle regulation and cellular growth control (Wang, 1998).

Lipids have been implicated in signal transduction and in several stages of membrane trafficking, but these two functions have not been functionally linked. In yeast, sphingoid base synthesis is required for the internalization step of endocytosis and organization of the actin cytoskeleton. Inactivation of a protein phosphatase 2A (PP2A) or overexpression of one of two kinases, Yck2p or Pkc1p, can specifically suppress the sphingoid base synthesis requirement for endocytosis. The two kinases have an overlapping function because only a mutant with impaired function of both kinases is defective in endocytosis. An ultimate target of sphingoid base synthesis may be the actin cytoskeleton, because overexpression of the kinases and inactivation of PP2A substantially corrected the actin defect due to the absence of sphingoid base. These results suggest that sphingoid base controls protein phosphorylation, perhaps by activating a signal transduction pathway that is required for endocytosis and proper actin cytoskeleton organization in yeast (Friant, 2000).

PP2A in yeast

Tor proteins, homologous to DNA-dependent protein kinases, participate in a signal transduction pathway in yeast that regulate protein synthesis and cell wall expansion in response to nutrient availability. The anti-inflammatory drug rapamycin inhibits yeast cell growth by inhibiting Tor protein signaling. This leads to diminished association of a protein, Tap42, with two different protein phosphatase catalytic subunits; one encoded redundantly by PPH21 and PPH22, and one encoded by SIT4. Inactivation of either Cdc55 or Tpd3, which regulate Pph21/22 activity, results in rapamycin resistance and this resistance correlates with an increased association of Tap42 with Pph21/22. Tor-dependent phosphorylation of Tap42 is shown both in vivo and in vitro and this phosphorylation is rapamycin sensitive. Inactivation of Cdc55 or Tpd3 enhances in vivo phosphorylation of Tap42. It is concluded that Tor phosphorylates Tap42 and that phosphorylated Tap42 effectively competes with Cdc55/Tpd3 for binding to the phosphatase 2A catalytic subunit. Furthermore, Cdc55 and Tpd3 promote dephosphorylation of Tap42. Thus, Tor stimulates growth-promoting association of Tap42 with Pph21/22 and Sit4, while Cdc55 and Tpd3 inhibit this association both by direct competition and by dephosphorylation of Tap42. These results establish Tap42 as a target of Tor and add further refinement to the Tor signaling pathway (Jiang, 1999).

Protein phosphatase 2A (PP2A) is an essential intracellular serine/threonine phosphatase containing a catalytic subunit that possesses the potential to dephosphorylate promiscuously tyrosine-phosphorylated substrates in vitro. How PP2A acquires its intracellular specificity and activity for serine/threonine-phosphorylated substrates is unknown. A novel and phylogenetically conserved mechanism is reported to generate active phospho-serine/threonine-specific PP2A in vivo. Phosphotyrosyl phosphatase activator (PTPA), a protein of so far unknown intracellular function, is required for the biogenesis of active and specific PP2A. Deletion of the yeast PTPA homologs generates a PP2A catalytic subunit with a conformation different from the wild-type enzyme, as indicated by its altered substrate specificity, reduced protein stability, and metal dependence. Complementation and RNA-interference experiments show that PTPA fulfills an essential function conserved from yeast to man (Fellner, 2003).

Homologue segregation during the first meiotic division requires the proper spatial regulation of sister chromatid cohesion and its dissolution along chromosome arms, but its protection at centromeric regions. This protection requires the conserved MEI-S332/Sgo1 proteins that localize to centromeric regions and also recruit the PP2A phosphatase by binding its regulatory subunit, Rts1. Centromeric Rts1/PP2A then locally prevents cohesion dissolution possibly by dephosphorylating the protein complex cohesin. This study shows that Aurora B kinase in Saccharomyces cerevisiae (Ipl1) is also essential for the protection of meiotic centromeric cohesion. Coupled with a previous study in Drosophila, this meiotic function of Aurora B kinase appears to be conserved among eukaryotes. Furthermore, Sgo1 recruits Ipl1 to centromeric regions. In the absence of Ipl1, Rts1 can initially bind to centromeric regions but disappears from these regions after anaphase I onset. It is suggested that centromeric Ipl1 ensures the continued centromeric presence of active Rts1/PP2A, which in turn locally protects cohesin and cohesion (Yu, 2007).

In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. This study identified the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. The protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function (Chan, 2009).

Tension-dependent removal of pericentromeric shugoshin is an indicator of sister chromosome biorientation

During mitosis and meiosis, sister chromatid cohesion resists the pulling forces of microtubules, enabling the generation of tension at kinetochores upon chromosome biorientation. How tension is read to signal the bioriented state remains unclear. Shugoshins form a pericentromeric platform that integrates multiple functions to ensure proper chromosome biorientation. This study shows that budding yeast shugoshin Sgo1 (see Drosophila Mei-S332) dissociates from the pericentromere reversibly in response to tension. The antagonistic activities of the kinetochore-associated Bub1 kinase and the Sgo1-bound phosphatase protein phosphatase 2A (PP2A)-Rts1 underlie a tension-dependent circuitry that enables Sgo1 removal upon sister kinetochore biorientation. Sgo1 dissociation from the pericentromere triggers dissociation of condensin and Aurora B (see Drosophila Aurora B) from the centromere, thereby stabilizing the bioriented state. Conversely, forcing sister kinetochores to be under tension during meiosis I leads to premature Sgo1 removal and precocious loss of pericentromeric cohesion. Overall, this study shows that the pivotal role of shugoshin is to build a platform at the pericentromere that attracts activities that respond to the absence of tension between sister kinetochores. Disassembly of this platform in response to intersister kinetochore tension signals the bioriented state. Therefore, tension sensing by shugoshin is a central mechanism by which the bioriented state is read (Nerusheva, 2014).

PP2A regulates vulval induction in C. elegans

Protein phosphatase 2A (PP2A) can both positively and negatively influence the Ras/Raf/MEK/ERK signaling pathway, but its relevant substrates are largely unknown. In C. elegans, the PR55/B regulatory subunit of PP2A, encoded by sur-6, positively regulates Ras-mediated vulval induction and acts at a step between Ras and Raf. The catalytic subunit (C) of PP2A, encoded by let-92, also positively regulates vulval induction. Therefore SUR-6/PR55 and LET-92/PP2A-C probably act together to dephosphorylate a Ras pathway substrate. PP2A has been proposed to activate the Raf kinase by removing inhibitory Ser259 phosphates from Raf-1 or from equivalent Akt phosphorylation sites in other Raf family members. However, mutant forms of C. elegans LIN-45 RAF that lack these sites still require sur-6. Therefore, SUR-6 must influence Raf activity via a different mechanism. SUR-6 and KSR (kinase suppressor of Ras) function at a similar step in Raf activation but genetic analysis suggests that KSR activity is intact in sur-6 mutants. The kinase PAR-1 has been identified as a negative regulator of vulval induction; it is shown to act in opposition to SUR-6 and KSR-1. In addition to their roles in Ras signaling, SUR-6/PR55 and LET-92/PP2A-C cooperate to control mitotic progression during early embryogenesis (Kao, 2004).

In other systems, PR55/B and PP2A have been found to have both positive and negative effects on Ras signaling. For example, in Drosophila a positive role for PR55/PP2A is supported by findings that mutations in tws/PR55 suppress the lethality caused by activated Sevenless receptor and activated Ras, and mutations in the PP2A catalytic subunit enhance photoreceptor defects caused by a hypomorphic Draf allele. However, a negative role for PR55/PP2A is supported by findings that RNAi against tws/PR55 elevates the level of phospho-ERK in cultured S2 cells, and mutations in the PP2A catalytic subunit enhance photoreceptor defects caused by activated Ras. Thus, in Drosophila the role of PR55/PP2A appears complex, and PP2A may act on multiple substrates within the Ras pathway. Similarly, in mammalian cells PP2A has been suggested to positively regulate Ras signaling by removing inhibitory phosphates from Raf and to negatively regulate Ras signaling by removing activating phosphates from MEK or ERK. By contrast, no evidence was found for a negative role of SUR-6/PR55 or LET-92/PP2A in C. elegans, despite having tested sur-6 and let-92 mutations in numerous genetic backgrounds. Therefore either PP2A lacks a negative role in C. elegans, or its negative role is masked by its stronger positive role (Kao, 2004 and references therein).

The sur-6 maternal effect lethal phenotype reveals that in addition to Ras signaling, sur-6 is required for mitotic progression. sur-6(sv30) and sur-6(RNAi) embryos display a variety of mitotic defects such as ectopic and aberrant cytokinesis, the collapse and re-elaboration of well-extended anaphase spindles, abnormally shaped spindles and chromatin bridges during anaphase. Similar mitotic defects have been observed in Drosophila tws/PR55 mutants. Premature sister chromatid separation and cytokinesis defects have also been observed in S. cerevisiae cdc55/PR55 mutants. Thus, the mitotic role of PR55 appears to be evolutionarily conserved. The early C. elegans embryo is a particularly tractable system for further study of this poorly understood mitotic role of PR55 (Kao, 2004 and references therein).

A PP2A regulatory subunit regulates C. elegans insulin/IGF-1 signaling by modulating AKT-1 phosphorylation

The C. elegans insulin/IGF-1 signaling (IIS) cascade plays a central role in regulating life span, dauer, metabolism, and stress. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine-specific protein kinases. An RNAi screen for serine/threonine protein phosphatases that counterbalance the effect of the kinases in the IIS pathway identified pptr-1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr-1 affects IIS pathway-associated phenotypes including life span, dauer, stress resistance, and fat storage. PPTR-1 functions by regulating worm AKT-1 phosphorylation at Thr 350. With striking conservation, mammalian B56beta regulates Akt phosphorylation at Thr 308 in 3T3-L1 adipocytes. In C. elegans, this ultimately leads to changes in subcellular localization and transcriptional activity of the forkhead transcription factor DAF-16. This study reveals a conserved role for the B56 regulatory subunit in regulating insulin signaling through AKT dephosphorylation, thereby having widespread implications in cancer and diabetes research (Padmanabhan, 2009).

Protein phosphatase 2A cooperates with the autophagy-related kinase UNC-51 to regulate axon guidance in Caenorhabditis elegans

UNC-51 is a serine/threonine protein kinase conserved from yeast to humans. The yeast homolog Atg1 regulates autophagy (catabolic membrane trafficking) required for surviving starvation. In C. elegans, UNC-51 regulates the axon guidance of many neurons by a different mechanism than it and its homologs use for autophagy. UNC-51 regulates the subcellular localization (trafficking) of UNC-5, a receptor for the axon guidance molecule UNC-6/Netrin; however, the molecular details of the role for UNC-51 are largely unknown. This study reports that UNC-51 physically interacts with LET-92, the catalytic subunit of serine/threonine protein phosphatase 2A (PP2A-C), which plays important roles in many cellular functions. A low allelic dose of LET-92 partially suppresses axon guidance defects of weak, but not severe, unc-51 mutants, and a low allelic dose of PP2A regulatory subunits A (PAA-1/PP2A-A) and B (SUR-6/PP2A-B) partially enhance the weak unc-51 mutants. It was also found that LET-92 can work cell-non-autonomously on axon guidance in neurons, and that LET-92 colocalizes with UNC-51 in neurons. In addition, PP2A dephosphorylates phosphoproteins that have been phosphorylated by UNC-51. These results suggest that, by forming a complex, PP2A cooperates with UNC-51 to regulate axon guidance by regulating phosphorylation. This is the first report of a serine/threonine protein phosphatase functioning in axon guidance in vivo (Ogura, 2010).

par-1, atypical pkc and PP2A/B55 sur-6 are implicated in the regulation of exocyst-mediated membrane trafficking in Caenorhabditis elegans

The exocyst is a conserved protein complex that is involved in tethering secretory vesicles to the plasma membrane and regulating cell polarity. Despite a large body of work little is known how exocyst function is controlled. To identify regulators for exocyst function, a targeted RNAi screen was performed in Caenorhabditis elegans to uncover kinases and phosphatases that genetically interact with the exocyst. Six kinase and seven phosphatase genes were found that display enhanced phenotypes when combined with hypomorphic alleles of exoc-7 (exo70), exoc-8 (exo84), or an exoc-7;exoc-8 double mutant. In line with its reported role in exocytotic membrane trafficking, a defective exoc-8 caused accumulation of exocytotic SNARE proteins in both intestinal and neuronal cells in C. elegans. Down-regulation of the PP2A phosphatase regulatory subunit sur-6/B55 gene resulted in accumulation of exocytic SNARE proteins SNB-1 and SNAP-29 in wild-type and in exoc-8 mutant animals. In contrast, RNAi of the kinase par-1 caused reduced intracellular GFP signal for the same proteins. Double RNAi experiments for par-1, pkc-3 and sur-6/B55 in C. elegans suggest a possible cooperation and involvement in post-embryo lethality, developmental timing, as well as SNARE protein trafficking. Functional analysis of the homologous kinases and phosphatases in Drosophila median neurosecretory cells showed that aPKC kinase and phosphatase PP2A regulate exocyst-dependent insulin-like peptide secretion. Collectively, these results characterize kinases and phosphatases implicated in the regulation of exocyst function, and suggest the possibility for interplay between the par-1 and pkc-3 kinases and the PP2A phosphatase regulatory subunit sur-6 in this process (Jiu, 2013).

PP2A and receptor function

Leptin exerts its weight-reducing effects by binding to its receptor and activating signal transduction in hypothalamic neurons and other cell types. To identify the components of the leptin signal transduction pathway, an approach was developed in which bacterially expressed phosphorylated fragments of Ob receptor b (Ob-Rb) were used as affinity agents. Leptin binding to the Ob-Rb form of the leptin receptor leads to tyrosyl phosphorylation of the cytoplasmic domain of its receptor. Two of the three cytoplasmic tyrosines of Ob-Rb, at positions 985 and 1138, are phosphorylated after leptin treatment. Affinity chromatography using a tyrosine-phosphorylated fragment spanning Tyr 985 of Ob-Rb was used to identify proteins that bind to this site. The SH2 domain containing protein tyrosine phosphatase 2 (SHP-2) was isolated from bovine and mouse hypothalamus by using this method. After cotransfection into 293T cells of Ob-Rb, Janus kinase 2 (JAK2), and SHP-2, leptin treatment results in direct binding of SHP-2 to the phosphorylated Tyr 985. The bound SHP-2 is itself tyrosine phosphorylated after leptin treatment. SHP-2 is not phosphorylated after leptin treatment when a Y to F 985 receptor mutant is cotransfected. In the absence of SHP-2 phosphorylation, the level of JAK2 phosphorylation is increased. Tyrosyl phosphorylation of the leptin receptor and signal transducer and activator of transcription 3 (STAT3) are not affected by phosphorylation of SHP-2. These data suggest that activation of SHP-2 by the leptin receptor results in a decreased phosphorylation of JAK2 and may act to attenuate leptin signal transduction. The data also suggest that the dephosphorylation of JAK2 is a direct action of SHP-2. Thus, a point mutation that ablates SHP-2 phosphatase activity also ablates its effects on the state of JAK2 phosphorylation. Although SHP-2 does have intrinsic phosphatase activity, it also could lead to dephosphorylation of JAK2 indirectly by functioning as an adapter protein. For example, binding of SHP-2 to the activated platelet-derived growth factor receptor leads to its own phosphorylation at position Tyr 584, which in turn leads to binding of Grb2. Grb2 then activates ras and the mitogen-activated protein kinase signaling pathway. Previous studies have shown that leptin can activate mitogen-activated protein kinase. Indeed the available data are consistent with the possibility that SHP-2 could both decrease JAK2 phosphorylation and stimulate signaling via the mitogen-activated protein kinase or other pathways (Li, 1999).

PP2A and DNA replication

Protein phosphatase 2A (PP2A) is an abundant, multifunctional serine/threonine-specific phosphatase that stimulates simian virus 40 DNA replication. The question as to whether chromosomal DNA replication also depends on PP2A was addressed by using a cell-free replication system derived from Xenopus laevis eggs. Immunodepletion of PP2A from Xenopus egg extract results in strong inhibition of DNA replication. PP2A is required for the initiation of replication but not for the elongation of previously engaged replication forks. Therefore, the initiation of chromosomal DNA replication depends not only on phosphorylation by protein kinases but also on dephosphorylation by PP2A (Lin, 1998).

PP2A, Pin1 and proline isomerization

The reversible protein phosphorylation on serine or threonine residues that precede proline (pSer/Thr-Pro) is a key signaling mechanism for the control of various cellular processes, including cell division. The pSer/Thr-Pro moiety in peptides exists in the two completely distinct cis and trans conformations whose conversion is catalyzed specifically by the essential prolyl isomerase Pin1 (Drosophila homolog dodo). Previous results suggest that Pin1 might regulate the conformation and dephosphorylation of its substrates. However, it is not known whether phosphorylation-dependent prolyl isomerization occurs in a native protein and/or affects dephosphorylation of pSer/Thr-Pro motifs. The major Pro-directed phosphatase PP2A is conformation-specific and effectively dephosphorylates only the trans pSer/Thr-Pro isomer. Furthermore, Pin1 catalyzes prolyl isomerization of specific pSer/Thr-Pro motifs both in Cdc25C and tau to facilitate their dephosphorylation by PP2A. Moreover, Pin1 and PP2A show reciprocal genetic interactions, and prolyl isomerase activity of Pin1 is essential for cell division in vivo. Thus, phosphorylation-specific prolyl isomerization catalyzed by Pin1 is a novel mechanism essential for regulating dephosphorylation of certain pSer/Thr-Pro motifs (Zhou, 2000).

PP2A, mitosis and cell cycle arrest

A temperature-sensitive S. cerevisiae type 2A phosphatase (PP2A) mutant, pph21-102 arrests predominantly with small or aberrant buds, with abnormal actin cytoskeleton and chitin deposition. The involvement of PP2A in bud growth may be due to the role of PP2A in actin distribution during the cell cycle. Moreover, after a shift to the non-permissive temperature, the pph21-102 cells are blocked in G2 and have low activity of Clb2-Cdc28 kinase. Expression of Clb2 from the S.cerevisiae ADH promoter in pph21-102 cells is able to partially bypass the G2 arrest in the first cell cycle, but is not able to stimulate passage through a second mitosis. These cells have higher total amounts of Clb2-Cdc28 kinase activity, but the Clb2-normalized specific activity is lower in the pph21-102 cells compared with wild-type cells. Unlike wild-type strains, a PP2A-deficient strain is sensitive to the loss of MIH1, which is a homolog of the S. pombe mitotic inducer cdc25+. Furthermore, the cdc28F19 mutation cures the synthetic defects of a PP2A-deficient strain containing a deletion of MIH1. These results suggest that PP2A is required during G2 for the activation of Clb-Cdc28 kinase complexes for progression into mitosis (Lin, 1995).

The giant, unicellular alga Acetabularia is a well known experimental model for the study of actin-dependent intracellular organelle motility. In the cyst stage, however, which is equivalent to the gametophytic stage, organelles are immobile, even though an actin cytoskeleton is present. To test the hypothesis that organelle motility could be under the control of posttranslational modification by protein phosphorylation, cysts were treated with submicromolar concentrations of okadaic acid or calyculin A, both potent inhibitors of serine/threonine protein phosphatases (ser/thr-PPases). The effects were dramatic: Instead of linear actin bundles typical for control cysts, circular arrays of actin bundles form in the cortical cyst cytoplasm. Concomitant with the formation of these actin rings, the cytoplasmic layers beneath the rings begin to slowly rotate in a continuous and uniform counter-clockwise fashion. This effect suggests that protein phosphorylation acts on the actin cytoskeleton at two levels: (1) it changes the assembly properties of the actin filament system to the extent that novel cytoskeletal configurations are formed and (2) it raises the activity of putative motor proteins involved in the rotational movements to levels sufficiently high to support motility at a stage when organelle motility does not normally occur. PP2A is strongly expressed at this developmental stage whereas PP1 is not detectable, suggesting that PP2A is the likely target of the protein phosphatase inhibitors (Menzel, 1995).

Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28-cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. The relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis has been investigated. Budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55 delta cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids. It is concluded that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. The role of PP2A and Cdc28 phosphorylation in cell-cycle control is discussed; it is possibile that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint (Minshull, 1996).

Saccharomyces cerevisiae, like most eukaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle. Cdc55p (regulatory B subunit of protein phosphatase 2A [PP2A]) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast. ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity. In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) are nocodazole sensitive and exhibit the phenotypes of previously identified kinetochore/spindle checkpoint mutants. Inactivating CDC55 does not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrest normally at elevated temperatures. CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants show normal sensitivity to gamma radiation and hydroxyurea. The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 are suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p. In contrast, the nocodazole sensitivity of cdc55::LEU2 is not suppressed by cdc28F19. Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p. Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis (Wang, 1997).

PPAR gamma is an adipose-selective nuclear hormone receptor that plays a key role in the control of adipocyte differentiation. Previous studies have indicated that activation of ectopically expressed PPAR gamma induces differentiation when cells have ceased growth because of confluence. Ligand activation of PPAR gamma is sufficient to induce growth arrest in fibroblasts and SV40 large T-antigen transformed, adipogenic HIB1B cells. Cell cycle withdrawal is accompanied by a decrease in the DNA-binding and transcriptional activity of the E2F/DP complex (See Drosophila E2F), which is attributable to an increase in the phosphorylation of these proteins, especially DP-1. This effect is a consequence of decreased expression of the catalytic subunit of the serine-threonine phosphatase PP2A. These data suggest an important role for PP2A in the control of E2F/DP activity and a new mode of cell cycle control in differentiation (Altiok, 1997).

MPF, a protein kinase complex consisting of cyclin and p34cdc2 subunits, promotes the G2 to M phase transition in eukaryotic cells. The pathway of activation and inactivation of MPF is not well understood, although there is strong evidence that removal of phosphate from a tyrosine residue on p34cdc2 is part of the activation process. INH was originally identified as an activity that could inhibit the posttranslational activation of a latent form of MPF, called pre-MPF, in immature (G2 phase-arrested) Xenopus oocytes. INH is a form of protein phosphatase 2A. Both INH and the catalytic subunit of protein phosphatase 2A can directly inactivate an isolated p34cdc2-cyclin complex. Both cyclin and p34cdc2 become dephosphorylated; the rate of inactivation closely parallels the removal of phosphate from a specific site on p34cdc2. It is proposed that INH opposes MPF activation by reversing this critical phosphorylation (Lee, 1991).

INH, a type 2A protein phosphatase (PP2A), negatively regulates entry into M phase and the cyclin B-dependent activation of cdc2 in Xenopus extracts. INH appears to be central to the mechanism of the trigger for mitotic initiation, as it prevents the premature activation of cdc2. INH is a conventional form of PP2A with a B alpha regulatory subunit. Although PP2A inhibits the switch in tyrosine kinase and tyrosine phosphatase activities accompanying mitosis, this switch is a consequence of the inhibition of some other rate-limiting event. In the preactivation phase, PP2A inhibits the pathway leading to T161 phosphorylation, suggesting that this activity may be one of the rate-limiting events for transition. However, these results also suggest that the accumulation of active cdc2/cyclin complexes during the lag is only one of the events required for triggering entry into mitosis (Lee, 1994).

A WD-40 repeat protein, TRIP-1, associates with the type II transforming growth factor beta (TGF-beta) receptor. Another WD-40 repeat protein, the Balpha subunit of protein phosphatase 2A, associates with the cytoplasmic domain of type I TGF-beta receptors. This association depends on the kinase activity of the type I receptor; it is increased by coexpression of the type II receptor (which is known to phosphorylate and activate the type I receptor) and allows the type I receptor to phosphorylate Balpha. Furthermore, Balpha enhances the growth inhibition activity of TGF-beta in a receptor-dependent manner. Because Balpha has been characterized as a regulator of phosphatase 2A activity, these observations suggest possible functional interactions between the TGF-beta receptor complex and the regulation of protein phosphatase 2A (Griswold-Prenner, 1998).

The effect of Balpha on the growth inhibition response of TGF-beta complements the role of Smads as effectors of TGF-beta receptor signaling. Smads function as transcriptional activators that induce the expression of various genes. Since the transcription of several genes is induced by Smads, Smads may induce growth inhibition by inducing transcription of the cdk inhibitors p15 and p21 in response to TGF-beta. Overexpression of Balpha induces growth inhibition to a level comparable to that of overexpression of Smads and, like the Smads, the effect of Balpha on growth inhibition depends on receptor activity. Furthermore, the antiproliferative effect of Balpha does not depend on Smad4, suggesting that TGF-beta receptor activation may induce two parallel pathways that lead to the antiproliferative response, one propagated by Smad proteins and the other one propagated through Balpha. Although the mechanism of the receptor-dependent growth inhibition by Balpha is not known, one possibility is that it acts through the ability of PP2A to regulate MAP kinase activity, especially since PP2A is a major enzyme involved in dephosphorylating MAP kinase. Therefore, altered PP2A activity following TGF-beta receptor activation might contribute to growth inhibition by deactivating this growth stimulatory pathway, theregy complementing the direct induction of growth inhibition by Smads. Moreover, a possible regulation of PP2A activity by TGF-beta may also directly affect the cell cycle, which would be consistent with the observed role of PP2A in cell cycle control (Griswold-Prenner, 1998 and references).

The activation of Cdc2 kinase induces the entry into M-phase of all eukaryotic cells. A cell-free system prepared from prophase-arrested Xenopus oocytes has been developed to analyze the mechanism initiating the all-or-none activation of Cdc2 kinase. Inhibition of phosphatase 2A, the major okadaic acid-sensitive Ser/Thr phosphatase in these extracts, provokes Cdc2 kinase amplification and concomitant hyperphosphorylation of Cdc25 phosphatase, with a lag of about 1 h. Polo-like kinase (Plx1 kinase) is activated slightly after Cdc2. All these events are totally inhibited by the cdk inhibitor p21(Cip1), demonstrating that Plx1 kinase activation depends on Cdc2 kinase activity. Addition of a threshold level of recombinant Cdc25 induces a linear activation of Cdc2 and Plx1 kinases and a partial phosphorylation of Cdc25. It is proposed that the Cdc2 positive feedback loop involves two successive phosphorylation steps of Cdc25 phosphatase: the first one is catalyzed by Cdc2 kinase and/or Plx1 kinase but it does not modify Cdc25 enzymatic activity; the second one requires a new kinase counteracted by phosphatase 2A. Under the conditions of this assay, Cdc2 amplification and MAP kinase activation are two independent events (Kara, 1998).

The initiation of anaphase and exit from mitosis depend on the anaphase-promoting complex (APC), which mediates the ubiquitin-dependent proteolysis of anaphase-inhibiting proteins and mitotic cyclins. An investigation was carried out using Xenopus egg extracts to see if protein phosphatases are required for mitotic APC activation. In Xenopus egg extracts, APC activation occurs normally in the presence of protein phosphatase 1 inhibitors, suggesting that the anaphase defects caused by protein phosphatase 1 mutation in several organisms are not due to a failure to activate the APC. Contrary to this, the initiation of mitotic cyclin B proteolysis is prevented by inhibitors of protein phosphatase 2A, such as okadaic acid. Okadaic acid induces an activity that inhibits cyclin B ubiquitination. This activity is referred to as inhibitor of mitotic proteolysis because it also prevents the degradation of other APC substrates. A similar activity exists in extracts of Xenopus eggs that are arrested at the second meiotic metaphase by the cytostatic factor activity of the protein kinase mos. In Xenopus eggs, the initiation of anaphase II may therefore be prevented by an inhibitor of APC-dependent ubiquitination (Vorlaufer, 1998).

Efficient translation of the mRNA encoding the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A (PP2A) is prevented by an out of frame upstream AUG and a stable stem-loop structure (delta G = -55.9 kcal/mol) in the 5'-untranslated region (5'-UTR). Deletion of the 5'-UTR allows efficient translation of the PR65 alpha message in vitro and overexpression in COS-1 cells. Insertion of the 5'-UTR into the beta-galactosidase leader sequence dramatically inhibits translation of the beta-galactosidase message in vitro and in vivo, confirming that this sequence functions as a potent translation regulatory sequence. Cells transfected or microinjected with a PR65 alpha expression vector lacking the 5'-UTR, express high levels of PR65 alpha, accumulating in both nucleus and cytoplasm. PR65 alpha overexpressing rat embryo fibroblasts (REF-52 cells) become multinucleated. These data suggest that PP2A participates in the regulation of both mitosis and cytokinesis (Wera, 1995).

Protein phosphatase 2A (PP2A) is present on microtubules in neuronal and nonneuronal cells. Interphase and mitotic spindle microtubules, as well as centrosomes, were all labeled with antibodies against individual PP2A subunits, showing that the AB alpha C holoenzyme is associated with microtubules. PP2A can be reversibly bound to microtubules in vitro; approximately 75% of the PP2A in cytosolic extracts can interact with microtubules. The activity of microtubule-associated PP2A is differentially regulated during the cell cycle. Enzymatic activity is high during S phase and intermediate during G1, while the activity in G2 and M is 20-fold lower than during S phase. The amount of microtubule-bound PP2A remains constant throughout the cell cycle, implying that cell cycle regulation of its enzymatic activity involves factors other than microtubules. These results raise the possibility that PP2A regulates cell cycle-dependent microtubule functions, such as karyokinesis and membrane transport (Sontag, 1995).

Assembly of a mitotic spindle requires the accurate regulation of microtubule dynamics; this is accomplished, at least in part, by phosphorylation-dephosphorylation reactions. The role of serine-threonine phosphatases in the control of microtubule dynamics has been investigated using specific inhibitors in Xenopus egg extracts. Type 2A phosphatases are required to maintain the short steady-state length of microtubules in mitosis by regulating the level of microtubule catastrophes, in part by controlling the the microtubule-destabilizing activity and phosphorylation of Op18/stathmin. Type 1 phosphatases are only required for control of microtubule dynamics during the transitions into and out of mitosis. Thus, although both type 2A and type 1 phosphatases are involved in the regulation of microtubule dynamics, they have distinct, non-overlapping roles (Tournebize, 1997).

Most cancer cells have increased levels of telomerase activity implicated in cell immortalization. Activation of telomerase, a ribonucleoprotein complex, catalyzes the elongation of the ends of mammalian chromosomal DNA (telomeres), the length of which regulates cell proliferation. Currently, how telomerase is regulated in cancer is not yet established. The present study shows that telomerase activity is regulated by protein phosphorylation in human breast cancer cells. Incubation of cell nuclear telomerase extracts with protein phosphatase 2A (PP2A) abolishes the telomerase activity; in contrast, cytoplasmic telomerase activity is unaffected, and protein phosphatases 1 and 2B are ineffective. Inhibition of telomerase activity by PP2A is both concentration- and time-dependent and is prevented by the protein phosphatase inhibitor okadaic acid. In addition, nuclear telomerase inhibited by PP2A is reactivated by endogenous protein kinase(s) in the presence of ATP, but not in the presence of ATPgammaS. Telomerase activity in cultured human breast cancer PMC42 cells is stimulated by okadaic acid, consistent with a role for PP2A in the regulation of telomerase activity in intact cells. These findings suggest that protein phosphorylation reversibly regulates the function of telomerase and that PP2A is a telomerase inhibitory factor in the nucleus of human breast cancer cells (Li, 1997).

The auto-catalytic activation of the cyclin-dependent kinase Cdc2 or MPF (M-phase promoting factor) is an irreversible process responsible for the entry into M phase. In Xenopus oocyte, a positive feed-back loop between Cdc2 kinase and its activating phosphatase Cdc25 allows the abrupt activation of MPF and the entry into the first meiotic division. The Cdc2/Cdc25 feed-back loop was studied using cell-free systems derived from Xenopus prophase-arrested oocyte. The findings support the following two-step model for MPF amplification: during the first step, Cdc25 acquires a basal catalytic activity resulting in a linear activation of Cdc2 kinase. In turn, Cdc2 partially phosphorylates Cdc25 but no amplification takes place; under this condition Plx1 kinase and its activating kinase Plkk1 are activated. However, their activity is not required for the partial phosphorylation of Cdc25. This first step occurs independent of PP2A or Suc1/Cks-dependent Cdc25/Cdc2 association. On the contrary, the second step involves the full phosphorylation and activation of Cdc25 and the initiation of the amplification loop. It depends both on PP2A inhibition and Plx1 kinase activity. Suc1-dependent Cdc25/Cdc2 interaction is required for this process (Karaiskou, 1999).

On TGF-beta binding, the TGF-beta receptor directly phosphorylates and activates the transcription factors Smad2/3, leading to G₁ arrest. Evidence is presented for a second, parallel, TGF-beta-dependent pathway for cell cycle arrest, achieved via inhibition of p70^s6k. TGF-beta induces association of its receptor with protein phosphatase-2A (PP2A)-Balpha. Concomitantly, three PP2A-subunits, Balpha, Abeta, and Calpha, associate with p70^s6k, leading to its dephosphorylation and inactivation. Although either pathway is sufficient to induce G₁ arrest, abrogation of both, the inhibition of p70^s6k, and transcription through Smad proteins is required for release of epithelial cells from TGF-beta-induced G₁ arrest. TGF-beta thereby modulates the translational and posttranscriptional control of cell cycle progression (Petritsch, 2000).

On receptor activation, PP2A-Balpha specifically binds the activated TbetaRI and is catalytically activated by TGF-beta. PP2A-Balpha then recruits PP2A-Abeta and PP2A-C to bind and dephosphorylate p70^s6k. Complexes containing at the same time PP2A, TbetaRI and p70^s6k, could not be detected, indicating that on activation the phosphatase is released from the receptor to bind to the target molecule. Immunolocalization of the endogenous proteins supports this model. p70^s6k activity controls the translational upregulation of proteins important for G₁/S progression and is itself essential for cell cycle progression. Most of the transcripts isolated to date represent ribosomal proteins and elongation factors of protein synthesis. TGF-beta-induced inactivation of p70^s6k leads to the translational regulation of a group of cell cycle regulators for G₁ progression. It remains unclear, however, if the repression of those cell cycle regulators result from global repression of protein translation or represent a class of specifically translationally repressed mRNAs. It is conceivable that the regulation of crucial components of the cell cycle machinery is mediated at the transcriptional, translational, and posttranslational levels (Petritsch, 2000).

Expression of the regulatory subunit PP2A-Balpha itself appears to be a prerequisite for the PP2A-mediated inhibition of p70^s6k by TGF-beta. Cells with nondetectable PP2A-Balpha expression remain solely responsive to TGF-beta-mediated transcriptional responses. p70^s6k is not inhibited by TGF-beta in these cells; this reflects the differential sensitivity of epithelial cells and mesenchymal cells to growth inhibitory effects of TGF-beta. The chromosomal localization of PP2A-Balpha has not been investigated; PP2A-Abeta, however, has been mapped to a human tumor suppressor locus on 11q22-24 and appears to be mutated in a subset of human lung tumors. It is tempting to speculate that mutations of the regulatory subunits of PP2A in human tumors abolish the regulation of p70^s6k to TGF-beta and confer a selective advantage to growing tumors (Petritsch, 2000).

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates. PP2A is required for chromosome segregation, but PP2A's substrates in this process remain unknown. To identify PP2A substrates, a two-hybrid screen was carried out with the regulatory B/PR55 subunit. A human homolog of C. elegans HCP6, a protein distantly related to the condensin subunit hCAP-D2 was isolated, and this homolog was named hHCP-6. Both C. elegans HCP-6 and condensin are required for chromosome organization and segregation. HCP-6 binding partners are unknown, whereas condensin is composed of the structural maintenance of chromosomes proteins SMC2 and SMC4 and of three non-SMC subunits. hHCP-6 becomes phosphorylated during mitosis and its dephosphorylation by PP2A in vitro depends on B/PR55, suggesting that hHCP-6 is a B/PR55-specific substrate of PP2A. Unlike condensin, hHCP-6 is localized in the nucleus in interphase, but similar to condensin, hHCP-6 associates with chromosomes during mitosis. hHCP-6 is part of a complex that contains SMC2, SMC4, kleisin-ß, and the previously uncharacterized HEAT repeat protein FLJ20311. hHCP-6 is therefore part of a condensin-related complex that associates with chromosomes in mitosis and may be regulated by PP2A (Yeong, 2003).

The success of cell division relies on the activation of its master regulator Cdc2-cyclin B, and many other kinases controlling cellular organization, such as Aurora-A. Most of these kinase activities are regulated by phosphorylation. Despite numerous studies showing that okadaic acid-sensitive phosphatases regulate both Cdc2 and Aurora-A activation, their identity has not yet been established in Xenopus oocytes and the importance of their regulation has not been evaluated. Using an oocyte cell-free system, it has been demonstrated that PP2A depletion is sufficient to lead to Cdc2 activation, whereas Aurora-A activation depends on Cdc2 activity. The activity level of PP1 does not affect Cdc2 kinase activation promoted by PP2A removal. PP1 inhibition is also not sufficient to lead to Aurora-A activation in the absence of active Cdc2. It is therefore conclude that in Xenopus oocytes, PP2A is the key phosphatase that negatively regulates Cdc2 activation. Once this negative regulator is removed, endogenous kinases are able to turn on the activator Cdc2 system without any additional stimulation. In contrast, Aurora-A activation is indirectly controlled by Cdc2 activity independently of either PP2A or PP1. This strongly suggests that in Xenopus oocytes, Aurora-A activation is mainly controlled by the specific stimulation of kinases under the control of Cdc2 and not by downregulation of phosphatase (Maton, 2005).

DNA-responsive checkpoints prevent cell-cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. Ser287 phosphorylation is a major locus of G2/M checkpoint control, although several checkpoint-independent kinases can phosphorylate this site. Mitotic entry requires 14-3-3 removal and Ser287 dephosphorylation. DNA-responsive checkpoints also activate PP2A/B56Δ phosphatase complexes to dephosphorylate Cdc25 at a site distinct from Ser287 (T138), the phosphorylation of which is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. These data suggest that creation of a 14-3-3 'sink,' consisting of phosphorylated 14-3-3 binding intermediate filament proteins, including keratins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56Δ as a central checkpoint effector and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis (Margolis, 2006).

A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest

Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. This study reports the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 binds to the core APC, and this binding is disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 is antagonized by PP2A, which can bind to Emi2 and promote Emi2-APC interactions. It is concluded that constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest (Wu, 2007)

Phosphorylation of mammalian Sgo2 by Aurora B recruits PP2A and MCAK to centromeres

Shugoshin (Sgo) is a conserved centromeric protein. Mammalian Sgo1 collaborates with protein phosphatase 2A (PP2A) to protect mitotic cohesin from the prophase dissociation pathway. Although another shugoshin-like protein, Sgo2, is required for the centromeric protection of cohesion in germ cells, its precise molecular function remains largely elusive. This study demonstrates that hSgo2 plays a dual role in chromosome congression and centromeric protection of cohesion in HeLa cells, while the latter function is exposed only in perturbed mitosis. These functions partly overlap with those of Aurora B, a kinase setting faithful chromosome segregation. Accordingly, phosphorylation of hSgo2 by Aurora B was identified at the N-terminal coiled-coil region and the middle region, and these phosphorylations were shown to separately promote binding of hSgo2 to PP2A and MCAK, factors required for centromeric protection and chromosome congression, respectively. Furthermore, these phosphorylations are essential for localizing PP2A and MCAK to centromeres. This mechanism seems applicable to germ cells as well. Thus, this study identifies Sgo2 as a hitherto unknown crucial cellular substrate of Aurora B in mammalian cells (Tanno, 2010).

Formation of stable attachments between kinetochores and microtubules depends on the B56-PP2A phosphatase

Error-free chromosome segregation depends on the precise regulation of phosphorylation to stabilize kinetochore-microtubule attachments (K-fibres) on sister chromatids that have attached to opposite spindle poles (bi-oriented). In many instances, phosphorylation correlates with K-fibre destabilization. Consistent with this, multiple kinases, including Aurora B and Plk1, are enriched at kinetochores of mal-oriented chromosomes when compared with bi-oriented chromosomes, which have stable attachments. Paradoxically, however, these kinases also target to prometaphase chromosomes that have not yet established spindle attachments and it is therefore unclear how kinetochore-microtubule interactions can be stabilized when kinase levels are high. This study shows, using human retinal pigment epithelial cells and HeLa cells, that the generation of stable K-fibres depends on the B56-PP2A phosphatase, which is enriched at centromeres/kinetochores of unattached chromosomes. When B56-PP2A is depleted, K-fibres are destabilized and chromosomes fail to align at the spindle equator. Strikingly, B56-PP2A depletion increases the level of phosphorylation of Aurora B and Plk1 kinetochore substrates as well as Plk1 recruitment to kinetochores. Consistent with increased substrate phosphorylation, it was found that chemical inhibition of Aurora or Plk1 restores K-fibres in B56-PP2A-depleted cells. These findings reveal that PP2A, an essential tumour suppressor, tunes the balance of phosphorylation to promote chromosome-spindle interactions during cell division (Foley, 2011).

PP2A, Mdm2, Cyclin G and p53

A fuller understanding of the function of cyclin G, a commonly induced p53 target, has remained elusive. Cyclin G forms a quaternary complex in vivo and in vitro with enzymatically active phosphatase 2A (PP2A) holoenzymes containing B' subunits. Interestingly, cyclin G also binds in vivo and in vitro to Mdm2 and markedly stimulates the ability of PP2A to dephosphorylate Mdm2 at T216. Consistent with these data, cyclin G null cells have both Mdm2 that is hyperphosphorylated at T216 and markedly higher levels of p53 protein when compared to wild-type cells. Cyclin G expression also results in reduced phosphorylation of human Hdm2 at S166. Thus, these data suggest that cyclin G recruits PP2A in order to modulate the phosphorylation of Mdm2 and thereby to regulate both Mdm2 and p53 (Okamoto, 2002).

Eukaryotic proteins are frequently regulated through their state of phosphorylation. Although protein kinases frequently recognize sequence motifs to target them to their sites in substrates, there is often less specificity in the sequence requirements of the major cellular phosphatases. Therefore, other mechanisms are needed for direction of phosphatases to their substrates, and these results suggest that cylin G serves such a role. In fact, PP2A is likely to be extensively regulated. Individual PP2A complexes have been shown to differ in some cases in their roles, localization, and substrate specificity. Thus, the apparently exclusive association of cyclin G with the B' subfamily is tantalizing. Cyclin G is of course not the only protein that has been shown to be able to interact with PP2A. Among the proteins shown to associate with PP2A and regulate its activity are the small t antigens encoded by SV40 and polyomavirus, the adenovirus E4orf4 protein, casein kinase II, Hox II, PKR, and several others. In some cases, the interaction results in negative regulation of PP2A activity. Although the effect of recruitment of cyclin G on the specific activity of PP2A is not known, cyclin G clearly does not block PP2A enzymatic activity, supporting the possibility that cyclin G serves to recruit PP2A to specific substrates (Okamoto, 2002).

The discovery that cyclin G binds to Mdm2 provided the impetus for testing whether Mdm2 might serve as such a substrate. The data strongly support the conclusion that at least two phosphorylation sites (Mdm2 T216 and Hdm2 S166) are substrates of cyclin G-directed PP2A. Since Mdm2 can associate with a host of cellular proteins, a future challenge will be to determine whether such interactions are regulated by phosphorylation, and if so, which of these are regulated by the cyclin G-PP2A complex. It is, of course, also possible that the cyclin G-PP2A interaction is relevant to other potential substrates, and therefore, the identification of cellular proteins that can interact with cyclin G may prove to be very interesting (Okamoto, 2002).

Mouse cells lacking cyclin G contain both Mdm2 that is hyperphosphorylated at T216 and higher p53 levels when compared to wild-type cells. These two observations are very likely interrelated. Although it is still not fully understood how modification of p53 affects its functions in vivo, phosphorylation of p53 at some N-terminal residues (that are modified in cells in response to DNA damage) decreases the ability of p53 to bind to Mdm2 in vitro. Modification of Mdm2 also impacts on its interactions with p53; phosphorylation of human Hdm2 by DNA PK (at S17 within the N terminus) and phosphorylation of murine Mdm2 by cyclin A/CDK2 (at T216 within the acidic domain) block and attenuate, respectively, the ability of either protein to bind to p53. Moreover, phosphorylation of human Mdm2 (Hdm2) at S395, a process that can be accomplished by ATM kinase in vitro, counteracts Mdm2's ability to target p53 for degradation in vivo. It can thus be speculated that in general, activated p53 and deactivated Mdm2 are the more phosphorylated forms of each protein. The data imply that the function of cyclin G is to serve as a negative regulator of p53 by activating Mdm2 through dephosphorylation. When seen in this context, it becomes less surprising that many previous studies have indicated that cyclin G expression is associated with growth promotion rather than arrest. Most exciting is the evidence that cyclin G null mice have fewer and smaller carcinogen-induced liver tumors, consistent with the hypothesis that cyclin G serves to negatively regulate the tumor suppressor function of p53 (Okamoto, 2002).

Structure and function of the PP2A-shugoshin interaction

Accurate chromosome segregation during mitosis and meiosis depends on shugoshin proteins that prevent precocious dissociation of cohesin from centromeres. Shugoshins associate with PP2A, which is thought to dephosphorylate cohesin and thereby prevent cleavage by separase during meiosis I. A crystal structure of a complex between a fragment of human Sgo1 and an AB'C PP2A holoenzyme reveals that Sgo1 forms a homodimeric parallel coiled coil that docks simultaneously onto PP2A's C and B' subunits. Sgo1 homodimerization is a prerequisite for PP2A binding. While hSgo1 interacts only with the AB'C holoenzymes, its relative, Sgo2, interacts with all PP2A forms and may thus lead to dephosphorylation of distinct substrates. Mutant shugoshin proteins defective in the binding of PP2A cannot protect centromeric cohesin from separase during meiosis I or support the spindle assembly checkpoint in yeast. Finally, evidence is provided that PP2A's recruitment to chromosomes may be sufficient to protect cohesin from separase in mammalian oocytes (Xu, 2009).

Sgol2 provides a regulatory platform that coordinates essential cell cycle processes during meiosis I in oocytes: Sgol2's ability to protect cohesin depends on its interaction with PP2A

Accurate chromosome segregation depends on coordination between cohesion resolution and kinetochore-microtubule interactions (K-fibers), a process regulated by the spindle assembly checkpoint (SAC). How these diverse processes are coordinated remains unclear. This study shows that in mammalian oocytes Shugoshin-like protein 2 (Sgol2; Drosophila homolog Mei-S332) in addition to protecting cohesin, plays an important role in turning off the SAC, in promoting the congression and bi-orientation of bivalents on meiosis I spindles, in facilitating formation of K-fibers and in limiting bivalent stretching. Sgol2's ability to protect cohesin depends on its interaction with PP2A, as is its ability to silence the SAC, with the latter being mediated by direct binding to Mad2. In contrast, its effect on bivalent stretching and K-fiber formation is independent of PP2A and mediated by recruitment of MCAK and inhibition of Aurora C kinase activity respectively. By virtue of its multiple interactions, Sgol2 links many of the processes essential for faithful chromosome segregation (Rattani, 2013).

The production of haploid gametes from diploid germ cells depends on two rounds of chromosome segregation (meiosis I and II) without an intervening round of DNA replication. Defects during the first or second meiotic division in oocytes lead to formation of aneuploid eggs, which in humans occurs with a frequency between 10 and 30% and is a major cause of fetal miscarriage. Understanding the causes of meiotic chromosome missegregation will require clarifying not only the forces and regulatory mechanisms governing meiotic chromosome segregation but also how these are coordinated (Rattani, 2013).

At the heart of this process are two opposing forces. The pulling forces produced by kinetochore-microtubules attachments (K-fibers) and resisting forces generated by sister chromatid cohesion, which counteracts K-fiber forces if and when kinetochores attach to microtubules with different polarities. During meiosis I, cohesion along chromosome arms holds bivalent chromosomes together following the creation of chiasmata produced by reciprocal recombination between homologous non-sister chromatids. This cohesion must persist during the attachment of maternal and paternal kinetochores to microtubules from opposite poles (bi-orientation) and resist the resulting spindle forces. The degree of traction exerted by meiotic spindles must create sufficient tension to facilitate bi-orientation. During the bi-orientation process, the initial attachment of kinetochores to the surface of the microtubule lattice (lateral attachment) is converted to attachments to their plus ends (end on attachment), creating K-fibers. Because this process is intrinsically error prone, inappropriate attachments, for example those that connect maternal and paternal kinetochores to the same pole, must be disrupted through phosphorylation of kinetochore proteins by Aurora B/C kinases. However, because these kinases disrupt K-fibers, they must subsequently be down-regulated once bivalents bi-orient correctly (Rattani, 2013).

The first meiotic division is eventually triggered by activation of a gigantic ubiquitin protein ligase called the anaphase-promoting complex or cyclosome (APC/C) whose destruction of securin and cyclin B activates a thiol protease called separase that cleaves the kleisin subunit of the cohesin complex holding sister chromatids together. This process converts chromosomes from bivalents to dyads. It is delayed until all bivalents have bi-oriented by the production, at kinetochores that have not yet come under tension, of a potent inhibitor of the APC/C called the mitotic checkpoint complex (MCC) whose Mad2 subunit binds tightly to the APC/C's Cdc20 co-activator protein. This regulatory mechanism, called the spindle assembly checkpoint (SAC), must be turned off before APC/C^Cdc20 can direct destruction of securin and cyclin B and thereby activate separase. Another pre-condition for cleavage, at least in yeast, is phosphorylation of cohesin's kleisin subunit by a pair of protein kinases, namely CK1δ/ε and DDK. During the first meiotic division, cohesin is phosphorylated along chromosome arms but not at centromeres, which ensures that only cohesion along arms is destroyed by separase at the onset of anaphase I. The consequent persistence of cohesion at centromeres promotes bi-orientation of dyads during meiosis II (Rattani, 2013).

Centromeric cohesin avoids phosphorylation and therefore cleavage during the first meiotic division because separase activation is preceded by the recruitment to centromeres of orthologues of the Drosophila MEI-S332 protein, called shugoshins. Members of this family contain a conserved C-terminal basic region and an N-terminal homodimeric parallel coiled coil, which provides a docking site for protein phosphatase 2A's (PP2A) C and B' subunits (Xu, 2009). In budding yeast, mutant proteins defective specifically in PP2A binding fail to confer protection of centromeric cohesion during meiosis I (Rattani, 2013).

Mammals have two members of the shugoshin family: Shugoshin-like protein 1 (Sgol1), and Shugoshin-like protein 2 (Sgol2). The former prevents centromeric cohesin from a process called the 'prophase pathway' that removes cohesin from chromosomes by a non-proteolytic mechanism soon after cells enter mitosis (McGuinness, 2005; Liu, 2013). Sgol2, on the other hand, protects centromeric cohesin from separase at the first meiotic division (Lee, 2008; Llano, 2008). Sgol2's coiled coil domain binds PP2A in vitro (Xu, 2009) but whether this is vital for protecting centromeric cohesion is not known. Sgol2 also interacts with MCAK (Huang, 2007), a microtubule depolymerizing kinesin, implicated in correcting inappropriate kinetochore-microtubule interactions (error correction), and with Mad2 an essential component of the MCC (Orth, 2011). Shugoshins, though not explicitly Sgol2, have also been implicated in recruiting to kinetochores the Aurora B kinase, necessary both for the SAC and for error correction (Tsukahara, 2010; Yamagishi, 2010). Based on its interactions, Sgol2 has been linked to cohesion protection, the spindle assembly checkpoint, and error correction pathways. However, the physiological significance of these multiple interactions remain unclear (Rattani, 2013).

This study shows that Sgol2 defective in PP2A binding fails to protect centromeric cohesin, as found for Sgo1 in yeast (Xu, 2009). However, if this were the sole function of Sgol2, then chromosome behavior during meiosis I should be unaffected. Surprisingly it was found that Sgol2 deficiency caused striking changes in chromosome and microtubule dynamics. It delayed bi-orientation of bivalents on meiosis I spindles, caused increased bivalent stretching, and greatly increased Aurora B/C kinase activity at kinetochores, which was accompanied by an increase in lateral and a decrease in end on kinetochore-microtubules attachments. Lastly, Sgol2 deficiency delayed considerably APC/C^Cdc20 activation, suggesting that it was required to shut off the SAC. Sgol2 helps to turn off the SAC by binding directly both PP2A and the MCC protein Mad2, it moderates chromosome stretching by recruiting to kinetochores the kinesin MCAK, and likely promotes formation of K-fibers by down-regulating the activity of Aurora B/C kinase specifically at kinetochores. Meiotic chromosome segregation is a highly complex process dependent on an array of different biochemical processes. These findings imply that through its multi-domain structure, Sgol2 has an important if not unique role in coordinating many of the key processes within this array (Rattani, 2013).

Cyclin B-Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase-independent mechanism>

Entry into M phase is governed by cyclin B-Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B-Cdk1-dependent inhibition of the protein phosphatase 2A (PP2A)-B55, which antagonizes cyclin B-Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B-Cdk1 to phosphorylate and activate alpha-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B-Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. This study shows that cyclin B-Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B-Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles (Okumura, 2014).

PP2A and the cytoskeleton

Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. Retrotransposon insertion produces an N-terminally truncated form (Deltagamma1) of the B56gamma1 regulatory subunit isoform of protein phosphatase (PP) 2A in BL6 cells, but not in F10 cells. An interaction of paxillin is found with PP2A C and B56gamma subunits by co-immunoprecipitation. B56gamma1 co-localizes with paxillin at focal adhesions, suggesting a role for this isoform in targeting PP2A to paxillin. In this regard, Deltagamma1 behaves similarly to B56gamma1. However, the Deltagamma1-containing PP2A heterotrimer is insufficient for the dephosphorylation of paxillin. Transfection with Deltagamma1 enhances paxillin phosphorylation on serine residues and recruitment into focal adhesions, and cell spreading with an actin network. In addition, Deltagamma1 renders F10 cells as highly metastatic as BL6 cells. These results suggest that mutations in PP2A regulatory subunits may cause malignant progression (Ito, 2000).

PP2A and organelle movement

Melanophores, cells specialized for regulated organelle transport, were used to study signaling pathways involved in the regulation of transport. Immortalized Xenopus melanophores were transfected with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogs of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. alpha-Melanocyte-stimulating hormone (MSH), a stimulus that increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. It is concluded that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A (Reilein, 1998).

PP2A regulates transcription factor phosphorylation

The bHLH factors HAND1 and HAND2 are required for heart, vascular, neuronal, limb, and extraembryonic development. Unlike most bHLH proteins, HAND factors exhibit promiscuous dimerization properties. Phosphorylation/dephosphorylation via PKA, PKC, and a specific heterotrimeric protein phosphatase 2A (PP2A) modulates HAND function. The PP2A targeting-subunit B56delta specifically interacts with HAND1 and -2, but not other bHLH proteins. PKA and PKC phosphorylate HAND proteins in vivo, and only B56delta-containing PP2A complexes reduce levels of HAND1 phosphorylation. During RCHOI trophoblast stem cell differentiation, B56delta expression is downregulated and HAND1 phosphorylation increases. Mutations in phosphorylated residues result in altered HAND1 dimerization and biological function. Taken together, these results suggest that site-specific phosphorylation regulates HAND factor functional specificity (Firulli, 2003).

Acquisition of epidermal barrier function, that serves to prevent water loss, occurs late in mouse gestation. Several days before birth a wave of barrier acquisition sweeps across murine fetal skin, converging on dorsal and ventral midlines. The molecular pathways active during epidermal barrier formation were investigated. Akt signaling increased as the barrier wave crossed epidermis and Jun was transiently dephosphorylated. Inhibitor experiments on embryonic explants showed that the dephosphorylation of Jun was dependent on both Akt and protein phosphatase 2A (Pp2a). Inhibition of Pp2a and Akt signaling also caused defects in epidermal barrier formation. These data are compatible with a model for developmental barrier acquisition mediated by Pp2a regulation of Jun dephosphorylation, downstream of Akt signaling. Support for this model was provided by siRNA-mediated knockdown of Ppp2r2a (Pr55alpha or B55alpha), a regulatory subunit of Pp2a expressed in an Akt-dependent manner in epidermis during barrier formation. Ppp2r2a reduction caused significant increase in Jun phosphorylation and interfered with the acquisition of barrier function, with barrier acquisition being restored by inhibition of Jun phosphorylation. These data provide strong evidence that Ppp2r2a is a regulatory subunit of Pp2a that targets this phosphatase to Jun, and that Pp2a action is necessary for barrier formation. This study therefore describes a novel Akt-dependent Pp2a activity that acts at least partly through Jun to affect initial barrier formation during late embryonic epidermal development (O'Shaughnessy, 2009).

PP2A and differentiation

A regulatory B subunit of protein phosphatase 2A (PP2A) positively regulates an RTK-Ras-MAP kinase signaling cascade during Caenorhabditis elegans vulval induction. Although reduction of sur-6 PP2A-B function causes few vulval induction defects in an otherwise wild-type background, sur-6 PP2A-B mutations suppress the Multivulva phenotype of an activated ras mutation and enhance the Vulvaless phenotype of mutations in lin-45 raf, sur-8, or mpk-1. Double mutant analysis suggests that sur-6 PP2A-B acts downstream or in parallel to ras, but likely upstream of raf, and functions with ksr-1 in a common pathway to positively regulate Ras signaling (Sieburth, 1999).

KSR proteins positively regulate Ras signaling in C. elegans and Drosophila, as well as in Xenopus oocytes and certain mammalian cells. Murine KSR associates with several proteins in vivo, including Raf, MEK, and MAP kinase, and has been proposed to function as a scaffold protein involved in signal propagation through the Raf/MEK/MAP kinase cascade. Murine KSR is a phosphoprotein; although the role of phosphorylation in KSR regulation is unclear. Thus, KSR-1 is a potential target for regulation by PP2A during vulval induction. Alternatively, KSR-1 may act to regulate PP2A-B function. Another potential sur-6 PP2A-B-dependent PP2A target is LIN-45 Raf. The mechanism of Raf activation is still poorly understood, but there is evidence for both inhibitory and activating phosphates on Raf. Whereas in vitro studies suggest that PP2A can dephosphorylate Raf, it is probably not the major phosphatase to remove activating phosphates. However, a role for PP2A in removing inhibitory phosphates has not been ruled out. The placement of a B regulatory subunit of PP2A as a positive regulator of the Ras pathway, and the unexpected finding that it acts together with KSR-1, should lead to a better understanding of PP2A regulation and its physiological substrates (Sieburth, 1999).

Neurofilament-L (NF-L) mRNA and protein levels in mouse P19 embryonal carcinoma cells are enhanced by retinoic acid-induced differentiation. Okadaic acid (OA) treatment represses this differentiation-dependent expression and neurite outgrowth. OA treatment does not affect NF-L gene transcription level, but it does reduce the stability of NF-L mRNAs. The expression and activity of PP2A and PP2B increases in accordance with the enhanced NF-L gene expression. The presence of OA during the course of neural differentiation reduces PP2A activity. These results demonstrate that the OA treatment inhibits the differentiation-dependent increase in NF-L gene expression by destabilizing its mRNAs and suggest that PP2A plays key roles in the differentiation-dependent enhanced expression of the NF-L gene (Sasahara, 1996).

Three families of cellular B subunits have been identified to date: B55, B56 and PR72/130. Five genes encode human B56 isoforms. Most code for phosphoproteins. There are distinct patterns of intracellular targeting by different B56 isoforms. Specifically, B56alpha, B56beta and B56epsilon complexed with A and C subunits localize to the cytoplasm, whereas B56delta, B56gamma1 and B56gamma3 are concentrated in the nucleus. Two isoforms are expressed in adult brain. mRNA of the brain isoforms increases when neuroblastoma cell lines are induced to differentiate by retinoic acid treatment (McCright, 1996).

Angiotensin receptors elicit a stimulation of PP2A. Ang II elicits significant increases in MAP kinase (ERK) activity in cultured neurons, mediated via Ang II type 1 (AT1) receptors. This stimulatory effect of Ang II on MAPK activity is potentiated by blockade of AT2 receptors. An AT2 receptor agonist causes a significant decrease in neural MAPK activity, which is abolished by the PP2A inhibitor okadaic acid. This indicates that AT1 and AT2 receptors have opposite actions an MAPK activity in neonatal neurons. Since MAP kinases are involved in the regulation of growth/differentiation and apoptosis, these data may provide an intracellular basis for the modulatory effects of Ang II receptors on these processes (Huang, 1996).

The BR beta regulatory subunit of rat PP2A is specifically expressed in rat brain and testis, the lengths of mRNAs in these two organs being different. In the testis, the BR beta mRNA is first detected 40 days after birth, increasing gradually thereafter, and is expressed specifically in elongated spermatids, while mRNA of the alpha-isotype (BR alpha) is expressed equally in all spermatogenic cells. After meiosis, round spermatids change morphologically to elongated spermatids. BR beta may regulate the activity of the PP2A catalytic subunit in spermatids, and be involved in spermatogenic maturation, especially spermatid elongation (Hatano, 1993).

Hox11 codes for a homeobox protein that controls genesis of the spleen. Hox 11 is also oncogenic, having been isolated from a chromosomal breakpoint in human T-cell leukemia. Transgenic mice that redirect Hox11 expression to the thymus demonstrate cell-cycle aberrations and progression to malignancy. In order to understand the cell cycle disruptions caused by HOX11 protein, the protein was tested for interaction partners. HOX11 directly interacts with the PP2A catalytic subunit and the related PP1C. The physical interaction domain of HOX11 is outside the homeodomain. PP2A can regulate the cell cycle of Xenopus oocytes by maintaining G2 meiotic arrest preventing activation of maturation-promoting factor. Microinjection of HOX11 into Xenopus oocytes arrested at the G2 phase of the cell cycle promotes progression to M phase. The interaction of HOX11 with PP2a suggests a mechanism by which a homeobox protein can alter the cell cycle (Kawabe, 1997).

Protein phosphatase type-2A (PP2A) is a highly conserved serine/threonine phosphatase known to play a key role in cell proliferation and differentiation in vitro, but the role of PP2A in mammalian embryogenesis remains unexplored. No particular information exists as to the tissue or cell specific expression of PP2A or the relevance of PP2A expression to mammalian development in vivo. To examine expression of PP2A during mammalian lung development, fetal rats were studied from day 14 of gestation (the lung bud is formed on day 12 of gestation) to parturition. Western analysis with a specific PP2A catalytic subunit antibody identifies a single 36 kDa protein, with protein levels two-fold higher in the 17 and 19 day embryonic lung, as compared to the adult. Both mRNA and protein for PP2A are localized equally to the epithelial lining of the embryonic lung airway and the surrounding mesenchyme in the 14 day embryonic lung. With maturation of the lung, PP2A becomes highly expressed in respiratory epithelium. The highest level of expression is in the earliest developing airways with columnar epithelium (the pseudoglandular stage, 15-18 days of gestation). There is a decrease in expression with the transformation to cuboidal epithelium by day 20 of gestation. This is most noticeable in the developing bronchial epithelium of the 19 and 20 day gestation lungs, where only an occasional cell continues to express PP2A. Mesenchymal hybridization is most obvious in early endothelial cells of forming vascular channels at 17-19 days of gestation. PP2A respiratory epithelial expression mimics the centrifugal development of the respiratory tree where the highest expression is in the peripheral columnar epithelium (15-18 days gestation) with only an occasional central bronchiolar cell continuing to express PP2A at 19 and 20 days gestation. Endothelial hybridization decreases with muscularization of large pulmonary arteries with low levels of expression detected in bronchial and vascular smooth muscle. In the newborn lung PP2A expression is decreased, but detectable in alveolar epithelium and vascular endothelium. In summary: (1) PP2A mRNA and protein exhibit cell specific expression during rat lung development; (2) PP2A is highly expressed in the respiratory epithelium of the fetal rat lung and is temporally related to the maturation of the bronchial epithelium, and (3) the PP2A subunit is highly expressed in early vascular endothelium, but not smooth muscle of the rat lung (Xue, 1998).

PP2A and signal transduction

A Sonic hedgehog (see Drosophila Hedgehog) response element was identified in the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) promoter (see Drosophila Seven-up). The Shh response element binds to a factor distinct from Gli, a gene known to mediate Shh signaling. Although this binding activity is specifically stimulated by Shh-N (amino-terminal signaling domain), it can also be unmasked with protein phosphatase treatment in the mouse cell line P19, and induction by Shh-N can be blocked by phosphatase inhibitors. Thus, Shh-N signaling may result in dephosphorylation of a target factor that is required for activation of COUP-TFII-, Islet1-, and Gli response element-dependent gene expression. This finding identifies another step in the Shh-N signaling pathway. The phosphatase that mediates this dephosphorylation in response to Shh-N treatment is PP2A or is like PP2A. This particular response is channeled through a protein with DNA binding activity apparently unrelated to that of the Ci/Gli family. A similar protein phosphatase activity is also required in the Ci/Gli-mediate branch of the Drosophila Hh signaling pathway (Krishnan, unpublished result). Thus, activation of specific protein phosphatase activity appears to be a general feature of Hh signal transduction (Krishnan, 1997).

Timely deactivation of kinase cascades is crucial to the normal control of cell signaling and is partly accomplished by protein phosphatase 2A (PP2A). The catalytic (alpha) subunit of the serine-threonine kinase casein kinase 2 (CK2) binds to PP2A in vitro and in mitogen-starved cells; binding requires the integrity of a sequence motif common to CK2alpha and SV40 small t antigen. Overexpression of CK2alpha results in deactivation of mitogen-activated protein kinase kinase (MEK) and suppression of cell growth. Moreover, CK2alpha inhibits the transforming activity of oncogenic Ras, but not that of constitutively activated MEK. Thus, CK2alpha may regulate the deactivation of the mitogen-activated protein kinase pathway (Heriche, 1997).

Inhibition of protein phosphatase 2A (PP2A) by the expression of SV40 small t stimulates the mitogenic MAP kinase cascade. SV40 small t can substitute for either tumor necrosis factor-alpha (TNF-alpha) or serum, and stimulate atypical protein kinase C zeta (PKC zeta) activity, resulting in MEK activation, cell proliferation and NF-kappaB-dependent gene transcriptional activation in CV-1 and NIH 3T3 cells. These effects are abrogated by co-expression of kinase-deficient PKC zeta and inhibition of phosphatidylinositol 3-kinase p85alpha-p110 by wortmannin, LY294002 and a dominant-negative mutant of p85alpha. In contrast, expression of kinase-inactive ERK2 inhibits small t-dependent cell growth but is unable to abolish small t-induced NF-kappaB transactivation. These results provide the first in vivo evidence for a critical regulatory role of PP2A in bifunctional PKC zeta signaling pathways controlled by phosphatidylinositol 3-kinase. Constitutive activation of PKC zeta and NF-kappaB following inhibition of PP2A supports new mechanisms by which SV40 small t promotes cell growth and transformation. By establishing PP2A as a key player in the response of cells to growth factors and stress signals like TNF-alpha, these findings could explain why PP2A is a primary target for the alteration of cellular behavior, as utilized during SV40 infection (Sontag, 1997).

Dysregulation of Wnt-beta-catenin signaling disrupts axis formation in vertebrate embryos and underlies multiple human malignancies. The adenomatous polyposis coli (APC) protein, axin, and glycogen synthase kinase 3beta form a Wnt-regulated signaling complex that mediates the phosphorylation-dependent degradation of beta-catenin. A protein phosphatase 2A (PP2A) regulatory subunit, B56, interacts with APC in the yeast two-hybrid system. Expression of B56 reduces the abundance of beta-catenin and inhibits transcription of beta-catenin target genes in mammalian cells and Xenopus embryo explants. The B56-dependent decrease in beta-catenin is blocked by oncogenic mutations in beta-catenin or APC, and by proteasome inhibitors. B56 may direct PP2A to dephosphorylate specific components of the APC-dependent signaling complex and thereby inhibit Wnt signaling. Loss of PP2A function may provide an additional route to activation of Wnt signaling and oncogenesis. Consistent with this, mutations in the gene encoding the beta isoform of the PP2A subunit have been identified in colon and lung cancers (Seeling, 1999).

Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and beta-catenin. Axin has been shown to have distinct binding sites for GSK-3 and beta-catenin and to promote the phosphorylation of beta-catenin and its consequent degradation. This provides an explanation for the ability of Axin to inhibit signaling through beta-catenin. In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of beta-catenin. The results are reported of a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no binding sites for other proteins have previously been identified. Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A through a domain between amino acids 632 and 836. This interaction was confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells. These results suggest that protein phosphatase 2A might interact with the Axin.APC.GSK-3.beta-catenin complex, where it could modulate the effect of GSK-3 on beta-catenin or other proteins in the complex. A region of Axin was identified that may allow it to form dimers or multimers. Through two-hybrid and co-immunoprecipitation studies, it was demonstrated that the C-terminal 100 amino acids of Axin can bind to this same region (Hsu, 1999).

Wnt signaling increases ß-catenin abundance and transcription of Wnt-responsive genes. The B56 regulatory subunit of protein phosphatase 2A (PP2A) inhibits Wnt signaling. Okadaic acid (a phosphatase inhibitor) increases, while B56 expression reduces, ß-catenin abundance; B56 also reduces transcription of Wnt-responsive genes. Okadaic acid is a tumor promoter, and the structural A subunit of PP2A is mutated in multiple cancers. Taken together, the evidence suggests that PP2A is a tumor suppressor. However, other studies suggest that PP2A activates Wnt signaling. The B56, A and catalytic C subunits of PP2A each have ventralizing activity in Xenopus embryos. B56 is epistatically positioned downstream of GSK3ß and axin but upstream of ß-catenin, and axin co-immunoprecipitates B56 A and C subunits suggesting that PP2A:B56 is in the ß-catenin degradation complex. PP2A appears to be essential for ß-catenin degradation, since ß-catenin degradation is reconstituted in phosphatase-depleted Xenopus egg extracts by PP2A, but not PP1. These results support the hypothesis that PP2A:B56 directly inhibits Wnt signaling and plays a role in development and carcinogenesis (Li, 2001).

The FKBP12-rapamycin-associated protein (FRAP; also called RAFT1/mTOR) regulates translation initiation and entry into the cell cycle. Depriving cells of amino acids or treating them with the small molecule rapamycin inhibits FRAP and results in rapid dephosphorylation and inactivation of the translational regulators 4E-BP1(eukaryotic initiation factor 4E-binding protein 1) and p70(s6k) (the 70-kDa S6 kinase: see Drosophila RPS6-p70-protein kinase). Data published recently have led to the view that FRAP acts as a traditional mitogen-activated kinase, directly phosphorylating 4E-BP1 and p70(s6k) in response to mitogenic stimuli. FRAP controls 4E-BP1 and p70(s6k) phosphorylation indirectly by restraining a phosphatase. A calyculin A-sensitive phosphatase is required for the rapamycin- or amino acid deprivation-induced dephosphorylation of p70(s6k), and treatment of Jurkat I cells with rapamycin increases the activity of the protein phosphatase 2A (PP2A) toward 4E-BP1. PP2A is shown to associate with p70(s6k) but not with a mutated p70(s6k) that is resistant to rapamycin- and amino acid deprivation-mediated dephosphorylation. FRAP also is shown to phosphorylate PP2A in vitro, consistent with a model in which phosphorylation of PP2A by FRAP prevents the dephosphorylation of 4E-BP1 and p70(s6k), whereas amino acid deprivation or rapamycin treatment inhibits FRAP's ability to restrain the phosphatase (Peterson, 1999).

Protein phosphatase 2A (PP2A) plays central roles in development, cell growth and transformation. Inactivation of the murine gene encoding the PP2A catalytic subunit Calpha by gene targeting generates a lethal embryonic phenotype. No mesoderm is formed in Calpha minus embryos. During normal early embryonic development Calpha is predominantly present at the plasma membrane whereas the highly homologous isoform Cbeta is localized to the cytoplasm and nuclei, suggesting the inability of Cbeta to compensate for vital functions of Calpha in Calpha minusembryos. In addition, PP2A is found in a complex containing the PP2A substrates E-cadherin and beta-catenin. In Calpha minus embryos, E-cadherin and beta-catenin are redistributed from the plasma membrane to the cytosol. Cytosolic concentrations of beta-catenin are low. These results suggest that Calpha is required for stabilization of E-cadherin/ beta-catenin complexes at the plasma membrane (Gotz, 2000).

These data suggest that PP2A is involved in wnt signaling. One likely explanation is that PP2A containing the Calpha subunit binds to and stabilizes the E-cadherin/beta-catenin complex within the plasma membrane. PP2A forms a complex with beta-catenin and E-cadherin. It is likely that PP2A regulates the phosphorylation status of E-cadherin and beta-catenin at the plasma membrane. In the cytosol, the activity of PP2A may not be high enough to offset GSK3 beta-mediated phosphorylation of beta-catenin. Wnt signaling increases cytoplasmic concentrations of PP2A by releasing it from the plasma membrane into the cytosol. Increased concentrations of PP2A effectively offset the GSK3 beta-mediated phosphorylation of beta-catenin thus preventing its degradation, and promoting its translocation to the nucleus with subsequent activation of wnt target genes. In the Calpha knockout situation both E-cadherin and beta-catenin are no longer stabilized in the plasma membrane. It is likely that both are phosphorylated and translocated to the cytoplasm. These data show that cytoplasmic E-cadherin is stable, and suggest that the associated beta-catenin is targeted for degradation resulting in reduced beta-catenin levels. It is proposed that in the absence of PP2A Calpha, wnt signaling releases beta-catenin from the axin/APC/GSK3 beta complex; beta-catenin associates with the highly abundant cytoplasmic E-cadherin before it reaches the nucleus, where it is phosphorylated and becomes degraded. Consequently, no target genes of wnt signaling, including Brachyury or goosecoid, are transcribed and embryonic development halts (Gotz, 2000).

This model is consistent with the finding that Calpha is mainly plasma membrane-associated in the inner cell mass, but this association becomes lost during differentiation. This model would also explain why the wnt target gene T-brachyury is negatively regulated by E-cadherin: T-brachyury mRNA levels are high in E-cad minus embryonic stem cells (ES) cells, but absent in wild-type ES cells. Basal cytoplasmic levels of non-phosphorylated beta-catenin may be quenched by cytoplasmic or membrane-bound E-cadherin, preventing transcription of T-brachyury in the absence of a wnt signal. In E-cad minus cells, the quencher is missing, allowing low but significant amounts of non-phosphorylated beta-catenin to be translocated into the nucleus, and to activate transcription of T-brachyury. This is consistent with the structural organization of beta-catenin itself; most of its protein-interacting motifs overlap so that interaction with one partner can block binding of another at the same time. Using recombinant proteins, it has been found that E-cadherin and lymphocyte-enhancer factor-1 (LEF-1), which targets beta-catenin to the nucleus, form mutually exclusive complexes with beta-catenin. This model is also consistent with the known association of PP2A with axin. Axin is a negative regulator of embryonic axis formation in vertebrates (Gotz, 2000).

The Wnt/ß-catenin pathway plays important roles during embryonic development and growth control. The B56 regulatory subunit of protein phosphatase 2A (PP2A) has been implicated as a regulator of this pathway. Loss-of-function analysis of PP2A:B56epsilon during early Xenopus embryogenesis has been examined. PP2A:B56epsilon is required for Wnt/ß-catenin signaling upstream of Dishevelled and downstream of the Wnt ligand. Maternal PP2A:B56epsilon function is required for dorsal development, and PP2A:B56epsilon function is required later for the expression of the Wnt target gene engrailed, for subsequent midbrain-hindbrain boundary formation, and for closure of the neural tube. These data demonstrate a positive role for PP2A:B56epsilon in the Wnt pathway (Yang, 2003).

Loss-of-function analysis in Drosophila and zebrafish suggests that PP2A:B56 family members play roles in the PCP pathway upstream of dsh. Although Frizzled and Dsh function in both the PCP pathway and the canonical Wnt/ß-catenin pathway, the requirement for PP2A:B56epsilon upstream of Dsh in Wnt/ß-catenin signaling has not been shown previously. The loss-of-function analysis of PP2A:B56epsilon presented here directly supports the hypothesis that PP2A:B56epsilon is required for Wnt/ß-catenin signaling. PP2A:B56epsilon is required for the expression of multiple Wnt/ß-catenin target genes, including Xnr5, Xnr6 during pre-MBT stages; Xnr3, siamois, noggin, goosecoid, cerberus and Xdkk1, during the gastrula stage, and en2 during neurulation. Furthermore, PP2A:B56epsilon is required for ß-catenin accumulation in cells that respond to endogenous Wnt signaling, such as dorsal blastomeres in blastula-stage embryos and in the neural ectoderm of early neurulae. In contrast, the constitutive turnover of ß-catenin in ventral blastomeres and in oocytes is not sensitive to PP2A:B56epsilon depletion. The requirement for PP2A:B56epsilon in canonical Wnt signaling is further supported by epistatic analysis. Dorsal gene expression is rescued by dsh and downstream Wnt signaling components but not by Wnt8b. Based on these observations, it is concluded that PP2A:B56epsilon is required for Wnt/ß-catenin signaling downstream of the Wnt ligand and upstream of dsh (Yang, 2003).

In contrast to observations with depletion of PP2A:B56epsilon, overexpression of PP2A:B56 family members inhibits Wnt/ß-catenin signaling. Other B56 family members, such as B56alpha, may have different functions in the Wnt/ß-catenin pathway. For example, overexpression of PP2A:B56epsilon in dorsal blastomeres leads to anterior truncation of embryos without altering dorsal gene expression, a phenotype that is distinct from the strong ventralization caused by PP2A:B56alpha overexpression. These observations are consistent with the suggestion that the two isoforms have different functions in the Wnt pathway. However, overexpressed PP2A:B56epsilon also inhibits secondary dorsal axis induction by positive modulators of Wnt/ß-catenin pathway. To explain this, PP2A:B56epsilon could regulate multiple steps in Wnt signaling, and loss-of-function at an upstream step may obscure an inhibitory role at a downstream step in the pathway. Alternatively, overexpression of PP2A:B56 or other subunits that interact with the PP2A complex could sequester essential components of the phosphatase complex and interfere with its normal function. It also remains unclear whether PP2A:B56epsilon regulates Wnt signaling through modulation of PP2A activity. Further experiments are needed in order to understand the mechanism by which PP2A:B56epsilon regulates the Wnt/ß-catenin pathway (Yang, 2003).

Kinase Suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to facilitate signal transmission through the MAPK cascade. Although recruitment of KSR1 from the cytosol to the plasma membrane is required for its scaffolding function, the precise mechanism(s) regulating the translocation of KSR1 have not been fully elucidated. Using mass spectrometry to analyze the KSR1-scaffolding complex, the serine/threonine protein phosphatase PP2A has been identified as a KSR1-associated protein; PP2A is a critical regulator of KSR1 activity. The enzymatic core subunits of PP2A (PR65A and catalytic C) constitutively associate with the N-terminal domain of KSR1, whereas binding of the regulatory PR55B subunit is induced by growth factor treatment. Specific inhibition of PP2A activity prevents the growth factor-induced dephosphorylation event involved in the membrane recruitment of KSR1 and blocks the activation of KSR1-associated MEK and ERK. Moreover, PP2A activity is required for activation of the Raf-1 kinase and that both Raf and KSR1 must be dephosphorylated by PP2A on critical regulatory 14-3-3 binding sites for KSR1 to promote MAPK pathway activation. These findings identify KSR1 as novel substrate of PP2A and demonstrate the inducible dephosphorylation of KSR1 in response to Ras pathway activation. Further, these results elucidate a common regulatory mechanism for KSR1 and Raf-1 whereby their localization and activity are modulated by the PP2A-mediated dephosphorylation of critical 14-3-3 binding sites (Ory, 2003).

PP2A, Receptors, and Memory

Calcineurin is a calcium-sensitive serine/threonine phosphatase that is present at high levels in the hippocampus and enriched at synapses. Once activated, calcineurin can act both directly and indirectly on protein substrates, including CREB. (1) It can dephosphorylate target proteins directly and thereby regulate specific cellular functions. (2) It can modulate an even larger variety of substrates indirectly by its ability to dephosphorylate inhibitor 1 (I-1). I-1, when phosphorylated, inhibits the function of protein phosphatase 1 (PP1). Dephosphorylation of I-1 by calcineurin activates PP1 and leads to the dephosphorylation of a large and independent set of target proteins. One interesting feature of the regulatory actions of calcineurin comes from its interactions with the cAMP cascade. Calcineurin inhibits the action of I-1 by dephosphorylating the site on I-1 phosphorylated by the cAMP-dependent kinase, PKA (see Drosophila PKA). Indeed, calcineurin and PKA antagonistically regulate the function of several proteins, including NMDA and GluR6 glutamate receptors (Winder, 1998 and references).

The interactions of Protein kinase and calcineurin are of particular interest in the context of LTP. Based on the requirement for macromolecular synthesis, LTP can be divided into at least two components: an early component (E-LTP) and a late component (L-LTP). Delivery to the Schaffer collateral-CA1 pyramidal cell (SC-CA1) synapse of a single 100 Hz train lasting 1s elicits E-LTP, a relatively short-lived and weak enhancement of synaptic transmission that does not require protein and RNA synthesis and is not dependent on PKA. By contrast, administration of three or four trains of 100 Hz elicits L-LTP, a more robust and stable form of LTP lasting many hours that is dependent on the activation of PKA as well as the synthesis of both RNA and protein. Recent experiments with inhibitors of phosphatases suggest that one role of PKA in LTP in area CA1 may be to suppress the actions of PP1 or PP2A. In particular, when LTP in area CA1 is induced by strong stimuli it can be blocked by inhibitors of PKA. However, this effect of PKA inhibitors is removed by preincubation of slices with PP1/PP2A inhibitors. This has led to the suggestion that under certain circumstances, PKA may "gate" LTP by suppressing a phosphatase cascade (Winder, 1998 and references).

To investigate the role of phosphatases in synaptic plasticity using genetic approaches, transgenic mice were generated that overexpress a truncated form of calcineurin under the control of the CaMKIIalpha promoter. Mice expressing this transgene show increased calcium-dependent phosphatase activity in the hippocampus. Physiological studies of these mice and parallel pharmacological experiments in wild-type mice reveal a novel, intermediate phase of LTP (I-LTP) in the CA1 region of the hippocampus. This intermediate phase differs from E-LTP by requiring multiple trains for induction and by being dependent on PKA. It differs from L-LTP by not requiring new protein synthesis. These data suggest that calcineurin acts as an inhibitory constraint on I-LTP, one which is relieved by PKA. This inhibitory constraint acts as a gate to regulate the synaptic induction of L-LTP (Winder, 1998 ).

Since phosphatases impose an inhibitory constraint on LTP, these results suggest that PKA is required to suppress phosphatase activity sufficiently to elicit LTP fully. Activation of NMDA receptors increases cAMP levels and PKA activity in CA1 through a calmodulin-dependent process. Therefore, while calcium directly regulates the balance of kinase and phosphatase activity, the generation of cAMP by NMDA-receptor-dependent activation of calcium-sensitive adenylyl cyclases can favor kinases further by inducing a PKA-dependent inactivation of the activation of PP1 by calcineurin, through phosphorylation of I-1. It should be noted, however, that although physiological studies suggest the presence of I-1 or an I-1-like protein in CA1, the histological localization of I-1 in CA1 is somewhat controversial. Because I-1 is a member of a family of proteins that modulate phosphatase function, it is possible that another protein from this family mediates the effects reported here (Winder, 1998 and references).

Hippocampal-dependent memory in mice that express a truncated form of calcineurin was assessed. Mutant mice have normal short-term memory but exhibit a profound and specific defect in long-term memory on both the spatial version of the Barnes maze and on a task requiring the visual recognition of a novel object. To determine whether mutant mice have the capacity for long-term memory, the training protocol was intensified on the spatial version of the Barnes maze by increasing the number of daily training trials. The memory defect is fully reversed, indicating that these mice are capable of forming long-term memory. This rescue experiment suggests that mice overexpressing calcineurin have impaired long-term memory possibly due to a specific defect in the transition between short-term and long-term memory. The memory defect observed was not the result of a developmental abnormality due to the genetic manipulation. In mice in which the expression of the calcineurin transgene is regulated by the tetracycline-controlled transactivator (tTA) system, the spatial memory defect is reversed when the expression of the transgene is repressed by doxycycline. Thus calcineurin has a role in the transition from short- to long-term memory, which correlates with a novel intermediate phase of LTP (Mansuy, 1998).

Regulation of protein phosphatase 2A (PP2A) activity and NMDA receptor (NMDAR) phosphorylation state contribute to the modulation of synaptic plasticity, yet these two mechanisms have not been functionally linked. The NMDAR subunit NR3A is equipped with a unique carboxyl domain that is different from other NMDAR subunits. It is hypothesized that the NR3A C-terminal intracellular domain might serve as synaptic anchor for the phosphatase in the developing CNS. A cDNA library was screened by the yeast two-hybrid method using the NR3A carboxyl domain as the bait. The catalytic subunit of the serine-threonine PP2A was found to be associated with the NR3A carboxyl domain. Immunoprecipitation studies indicate that the NR3A subunit forms a stable complex with PP2A in the rat brain in vivo. Association of PP2A with NMDARs lead to an increase in the phosphatase activity of PP2A and the dephosphorylation of serine 897 of the NMDAR subunit NR1. Stimulation of NMDARs leads to the dissociation of PP2A from the complex and the reduction of PP2A activity. A peptide corresponding to the PP2A-NR3A binding domain functions as a negative regulator of PP2A activity. These data suggest that NMDARs are allosteric modulators of PP2A, which in turn controls their phosphorylation state. The data delineate a mechanistic model of the dynamic regulation of a PP2A-NMDAR signaling complex, mediated by the interaction of NR3A and PP2A, and suggest a novel NMDAR-mediated signaling mechanism. It is possible to speculate that protein kinase A, PP2A, and NMDARs are part of a signaling mechanism linking the history of NMDAR stimulation to the phosphorylation of state of serine 897 of NR1. Along these lines, the finding that NR3A knock-out mice show an increased density of spines in cortical neurons might indicate that such a signaling pathway could be involved in the regulation of the growth of dendritic spines. In fact, NMDAR activity has been implicated in the modulation of dendritic arbors and spines, whereas PP2A has been shown to influence the modification of synaptic structures (Chan, 2001).

BOD1 is required for cognitive function in humans and Drosophila

This study reports a stop-mutation in the BOD1 (Biorientation Defective 1) gene, which co-segregates with intellectual disability in a large consanguineous family. The BOD1 protein is required for proper chromosome segregation, regulating phosphorylation of PLK1 (see Drosophila Polo) substrates by modulating Protein Phosphatase 2A (PP2A; see Drosophila Twins) activity during mitosis. Fibroblast cell lines derived from homozygous BOD1 mutation carriers show aberrant localisation of the cell cycle kinase PLK1 and its phosphatase PP2A at mitotic kinetochores. The relatively normal cell cycle progression observed in cultured cells is in line with the absence of gross structural brain abnormalities in the affected individuals. Moreover, normal adult brain tissues BOD1 expression is maintained at considerable levels, in contrast to PLK1 expression, and evidence is provided for synaptic localization of Bod1 in murine neurons. These observations suggest that BOD1 plays a cell cycle-independent role in the nervous system. To address this possibility, two Drosophila models were established, where neuron-specific knockdown of BOD1 caused pronounced learning deficits and significant abnormalities in synapse morphology. Together these results reveal novel postmitotic functions of BOD1 as well as pathogenic mechanisms that strongly support a causative role of BOD1 deficiency in the aetiology of intellectual disability. Moreover, by demonstrating its requirement for cognitive function in humans and Drosophila evidence is provided for a conserved role of BOD1 in the development and maintenance of cognitive features (Esmaeeli-Nieh, 2016).

PP2A and Apoptosis

The execution phase is an evolutionarily conserved stage of apoptosis that occurs with remarkable temporal and morphological uniformity in most if not all cell types, regardless of the condition used to induce death. Characteristic features of apoptosis such as membrane blebbing, DNA fragmentation, chromatin condensation, and cell shrinkage occur during the execution phase; therefore, there is considerable interest in defining biochemical changes and signaling events early in the execution phase. Since onset of the execution phase is asynchronous across a population with only a small fraction of cells in this stage at any given time, characterizing underlying biochemical changes is difficult. An additional complication is recent evidence suggesting that the execution phase occurs after cells commit to die; thus, agents that modulate events in the execution phase may alter the morphological progression of apoptosis but will not affect the time-course of death. In the present study, a single cell approach was used to study and temporally order biochemical and cytoskeletal events that occur specifically in the execution phase. Microtubules de-acetylate and disassemble as terminally differentiated PC12 cells enter the execution phase following removal of nerve growth factor. Using phosphorylation sensitive antibodies to microtubule-stabilizing protein known as tau, it is shown that tau becomes dephosphorylated near the onset of the execution phase. Low concentrations of okadaic acid inhibit dephosphorylation suggesting a PP2A-li10ke phosphatase is responsible. Transfecting tau into CHO cells to act as a 'reporter' protein shows a similar dephosphorylation of tau by a PP2A-like phosphatase during the execution phase following induction of apoptosis with UV irradiation. Therefore, activation of PP2A phosphatase occurs at the onset of the execution phase in two very different cell types following different initiators of apoptosis; this is consistent with activation of PP2A phosphatase being a common feature of the execution phase of apoptosis. Experiments using either taxol to inhibit microtubule disassembly or okadaic acid to inhibit (tau) dephosphorylation suggest that microtubule disassembly is necessary for (tau) dephosphorylation to occur. Thus, it is propose that an early step in the execution phase (soon after a cell commits to die) is microtubule disassembly, which frees or activates PP2A to dephosphorylate tau as well as other substrates (Mills, 1998).

PP2A and malignancy

Inhibition of protein phosphatase 2A (PP2A) activity has been identified as a prerequisite for the transformation of human cells. However, the molecular mechanisms by which PP2A activity is inhibited in human cancers are currently unclear. In this study, a cellular inhibitor of PP2A with oncogenic activity is described. The protein, designated Cancerous Inhibitor of PP2A (CIP2A), interacts directly with the oncogenic transcription factor c-Myc, inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage-independent cell growth and in vivo tumor formation. The oncogenic activity of CIP2A is demonstrated by transformation of human cells by overexpression of CIP2A. Importantly, CIP2A is overexpressed in two common human malignancies, head and neck squamous cell carcinoma (HNSCC) and colon cancer. Thus, these data show that CIP2A is a human oncoprotein that inhibits PP2A and stabilizes c-Myc in human malignancies (Junttila, 2007).

REFERENCES

Search PubMed for articles about Drosophila twins

Afonso, O., Matos, I., Pereira, A. J., Aguiar, P., Lampson, M. A. and Maiato, H. (2014). Feedback control of chromosome separation by a midzone Aurora B gradient. Science 345: 332-336. PubMed ID: 24925910

Altiok, S., Xu, M. and Spiegelman, B. M. (1997). PPARgamma induces cell cycle withdrawal: inhibition of E2F/DP DNA-binding activity via down-regulation of PP2A. Genes Dev. 11(15): 1987-1998. PubMed Citation: 9271121

Archambault, V. and Glover, D. M. (2009). Polo-like kinases: conservation and divergence in their functions and regulation. Nat. Rev. Mol. Cell Biol. 10: 265-275. PubMed Citation: 19305416

Archambault, V., Zhao, X., White-Cooper, H., Carpenter, A. T. and Glover, D. M. (2007). Mutations in Drosophila Greatwall/Scant reveal its roles in mitosis and meiosis and interdependence with Polo kinase. PLoS Genet 3: e200. PubMed Citation: 17997611

Bajpai, R., et al. (2004). Drosophila Twins regulates Armadillo levels in response to Wg/Wnt signal. Development 131: 1007-1016. 14973271

Berry, M. and Gehring, W. (2000). Phosphorylation status of the SCR homeodomain determines its functional activity: essential role for protein phosphatase 2A,B'. EMBO J. 19: 2946-2957. PubMed Citation: 10856239

Bielinski, V. A. and Mumby, M. C. (2007). Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase. Exp Cell Res 313: 3117-3126. PubMed ID: 17570358

Brownlee, C. W., Klebba, J. E., Buster, D. W. and Rogers, G. C. (2011). The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification. J. Cell Biol. 195(2): 231-43. PubMed Citation: 21987638

Casso, D. J., et al. (2008). A screen for modifiers of Hedgehog signaling in Drosophila melanogaster identifies swm and mts. Genetics 178: 1399-1413. PubMed Citation: 18245841

Castilho, P. V., et al. (2009). The M phase kinase Greatwall (Gwl) promotes inactivation of PP2A/B55delta, a phosphatase directed against CDK phosphosites. Mol. Biol. Cell 20: 4777-4789. PubMed Citation: 19793917

Chabu, C. and Doe, C. Q. (2009). Twins/PP2A regulates aPKC to control neuroblast cell polarity and self-renewal. Dev. Biol. 330(2): 399-405. PubMed Citation: 19374896

Chambon, J. P., Touati, S. A., Berneau, S., Cladiere, D., Hebras, C., Groeme, R., McDougall, A. and Wassmann, K. (2013). The PP2A inhibitor I2PP2A is essential for sister chromatid segregation in oocyte meiosis II. Curr Biol 23: 485-490. PubMed ID: 23434280

Chan, L. Y. and Amon, A. (2009). The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4. Genes Dev. 23(14): 1639-49. PubMed Citation: 19605686

Chan, S. F. and Sucher, N. J. (2001). An NMDA receptor signaling complex with Protein phosphatase 2A. J. Neurosci. 21(20): 7985-7992. 11588171

Chen, F., et al. (2007). Multiple protein phosphatases are required for mitosis in Drosophila. Curr. Biol. 17: 293-303. Medline abstract: 17306545

Creyghton, M. P., et al. (2006). PR130 is a modulator of the Wnt-signaling cascade that counters repression of the antagonist Naked cuticle. Proc. Natl. Acad. Sci. 103: 5397-5402. 16567647

Cygnar, K. D., Gao, X., Pan, D. and Neufeld, T. P. (2005). The phosphatase subunit tap42 functions independently of target of rapamycin to regulate cell division and survival in Drosophila. Genetics 170: 733-740. PubMed ID: 15802506

Cziko, A. M., McCann, C. T., Howlett, I. C., Barbee, S. A., Duncan, R. P., Luedemann, R., Zarnescu, D., Zinsmaier, K. E., Parker, R. R. and Ramaswami, M. (2009). Genetic modifiers of dFMR1 encode RNA granule components in Drosophila. Genetics 182: 1051-1060. PubMed ID: 19487564

Deak, P., Donaldson, M. and Glover, D. M. (2003). Mutations in makos, a Drosophila gene encoding the Cdc27 subunit of the anaphase promoting complex, enhance centrosomal defects in polo and are suppressed by mutations in twins/aar, which encodes a regulatory subunit of PP2A. J. Cell Sci. 116(20): 4147-4158. 12953067

Dobbelaere, J., et al. (2008). A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila. PLoS Biol 6: e224. PubMed Citation: 18798690

Esmaeeli-Nieh, S., et al. (2016). BOD1 is required for cognitive function in humans and Drosophila. PLoS Genet 12: e1006022. PubMed ID: 27166630

Fellner, T., et al. (2003). A novel and essential mechanism determining specificity and activity of protein phosphatase 2A (PP2A) in vivo. Genes Dev. 17: 2138-2150. 12952889

Firulli, B. A., et al. (2003). PKA, PKC, and the Protein phosphatase 2A influence HAND factor function: A mechanism for tissue-specific transcriptional regulation. Molec. Cell 12: 1225-1237. 14636580

Foley, E. A., Maldonado, M. and Kapoor, T. M. M. (2011). Formation of stable attachments between kinetochores and microtubules depends on the B56-PP2A phosphatase. Nat. Cell Biol. 13: 1265-1271. PubMed Citation: 21874008

Forester, C. M., et al. (2007). Control of mitotic exit by PP2A regulation of Cdc25C and Cdk1. Proc. Natl. Acad. Sci. 104: 19867-19872. PubMed Citation: 18056802

Friant, S., Zanolari, B. and Riezman, H. (2000). Increased protein kinase or decreased PP2A activity bypasses sphingoid base requirement in endocytosis. EMBO J. 19: 2834-2844. PubMed Citation: 10856229

Gharbi-Ayachi, A., et al. (2010). The substrate of Greatwall kinase, Arpp19, controls mitosis by inhibiting protein phosphatase 2A. Science 330: 1673-1677. PubMed Citation: 21164014

Garrido, D., Bourouh, M., Bonneil, E., Thibault, P., Swan, A. and Archambault, V. (2020). Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis. PLoS Genet 16(11): e1009184. PubMed ID: 33137813

Gomes, R., et al. (1993). Abnormal anaphase resolution (aar): a locus required for progression through mitosis in Drosophila. J. Cell Sci. 104: 583-593. PubMed Citation: 8505381

Gotz, J., et al. (1998). Delayed embryonic lethality in mice lacking protein phosphatase 2A catalytic subunit calpha. Proc. Natl. Acad. Sci. 95(21): 12370-5. PubMed Citation: 9770493

Gotz, J., et al. (2000). Distinct role of protein phosphatase 2A subunit Calpha in the regulation of E-cadherin and beta-catenin during development. Mech. Dev. 93: 83-93. PubMed Citation: 10781942

Griswold-Prenner, I., et al., (1998). Physical and functional interactions between type I transforming growth factor beta receptors and Balpha, a WD-40 repeat subunit of phosphatase 2A. Mol. Cell. Biol. 18(11): 6595-604. PubMed Citation: 9774674

Groves, M. R., et al. (1999). The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs. Cell 96: 99-110. 9989501

Guo, M., Bier, E., Jan, L.Y. and Jan, Y.N. (1995). tramtrack acts downstream of numb to specify distinct daughter cell fates during asymmetric cell divisions in the Drosophila PNS. Neuron 14(5): 913-25. PubMed Citation: 7748559

Hannus, M., Feiguin, F., Heisenberg, C.-P. and Eaton, S. (2002). Planar cell polarization requires Widerborst, a B' regulatory subunit of protein phosphatase 2A. Development 129: 3493-3503. 12091318

Hatano, Y., et al. (1993). Expression of PP2A B regulatory subunit beta isotype in rat testis. FEBS Lett. 324: 71-5

Heriche, J. K., et al. (1997). Regulation of protein phosphatase 2A by direct interaction with casein kinase 2alpha. Science 276 (5314): 952-955

Hsu, W., Zeng, L. and Costantini, F. (1999). Identification of a domain of Axin that binds to the serine/threonine protein phosphatase 2A and a self-binding domain. J. Biol. Chem. 274(6): 3439-45

Huang, H., Feng, J., Famulski, J., Rattner, J. B., Liu, S. T., Kao, G. D., Muschel, R., Chan, G. K. and Yen, T. J. (2007). Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments. J Cell Biol 177: 413-424. PubMed ID: 17485487

Huang, X. C., Richards, E. M. and Sumners, C. (1996). Mitogen-activated protein kinases in rat brain neuronal cultures are activated by angiotensin II type 1 receptors and inhibited by angiotensin II type 2 receptors. J. Biol. Chem. 271: 15635-41

Ito, A., et al. (2000). A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation. EMBO J. 19(4): 562-571. PubMed ID: 10675325

Jia, G., Liu, Y., Yan, W. and Jia, J. (2009). PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation. Development 136: 307-316. PubMed Citation: 19088085

Jiang, Y. and Broach, J. R. (1999). Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. EMBO J. 18(10): 2782-2792. PubMed ID: 10329624

Jiu, Y., Hasygar, K., Tang, L., Liu, Y., Holmberg, C. I., Burglin, T. R., Hietakangas, V. and Jantti, J. (2013). par-1, atypical pkc and PP2A/B55 sur-6 are implicated in the regulation of exocyst-mediated membrane trafficking in Caenorhabditis elegans. G3 (Bethesda) 4(1): 173-83. PubMed ID: 24192838

Junttila, M. R., et al. (2007). CIP2A inhibits PP2A in human malignancies. Cell 130(1): 51-62. PubMed citation; Online text

Kao, G., Tuck, S., Baillie, D. and Sundaram, M. V. (2004). C. elegans SUR-6/PR55 cooperates with LET-92/protein phosphatase 2A and promotes Raf activity independently of inhibitory Akt phosphorylation sites. Development 131: 755-765

Kara, A., et al. (1998). MPF amplification in Xenopus oocyte extracts depends on a two-step activation of Cdc25 phosphatase. Exp. Cell Res. 244(2): 491-500

Karaiskou, A., Jessus, C., Brassac, T., and Ozon, R. (1999). Phosphatase 2A and polo kinase, two antagonistic regulators of cdc25 activation and MPF auto-amplification. J. Cell Sci. 112: 3747-3756

Kawabe, T., Muslin, A. J. and Korsmeyer, S. J. (1997). HOX 11 interacts with protein phosphatases PP2A and PP1 and disrupts a G2/M cell-cycle checkpoint. Nature 385: 454-458

Kim, M.Y., Bucciarelli, E., Morton, D. G., Williams, B. C., Blake-Hodek, K., Pellacani, C., Von Stetina, J. R., Hu, X., Somma, M. P., Drummond-Barbosa, D. and Goldberg, M. L. (2012). Bypassing the Greatwall-Endosulfine pathway: plasticity of a pivotal cell-cycle regulatory module in Drosophila melanogaster and Caenorhabditis elegans. Genetics 191(4): 1181-97. PubMed Citation: 22649080

Kitagawa, D., et al. (2011). PP2A phosphatase acts upon SAS-5 to ensure centriole formation in C. elegans embryos. Dev. Cell. 20: 550-562. PubMed Citation: 21497765

Kong, M., Fox, C. J., Mu, J., Solt, L., Xu, A., Cinalli, R. M., Birnbaum, M. J., Lindsten, T. and Thompson, C. B. (2004). The PP2A-associated protein alpha4 is an essential inhibitor of apoptosis. Science 306: 695-698. PubMed ID: 15499020

Kotadia, S., et al. (2008). PP2A-dependent disruption of centrosome replication and cytoskeleton organization in Drosophila by SV40 small tumor antigen. Oncogene 27: 6334-6346. PubMed Citation: 18663356

Krishnan, V., et al. (1997). Mediation of Sonic hedgehog-induced expression of COUP-TFII by a protein phosphatase. Science 278(5345): 1947-1950

Lee, J., Kitajima, T. S., Tanno, Y., Yoshida, K., Morita, T., Miyano, T., Miyake, M. and Watanabe, Y. (2008). Unified mode of centromeric protection by shugoshin in mammalian oocytes and somatic cells. Nat Cell Biol 10: 42-52. PubMed ID: 18084284

Lee, T. H., et al. (1991). INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell 64: 415-23

Lee, T. H., Turck, C. and Kirschner, M. W. (1994). Inhibition of cdc2 activation by INH/PP2A. Mol. Biol. Cell 5: 323-338 (1994)

Li, C. and Friedman, J. M. (1999). Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction. Proc. Natl. Acad. Sci. 96: 9677-9682

Li, H., et al. (1997). Protein phosphatase 2A inhibits nuclear telomerase activity in human breast cancer cells. J. Biol. Chem. 272(27): 16729-16732

Li, X., et al. (2001). Protein phosphatase 2A and its B56 regulatory subunit inhibit Wnt signaling in Xenopus. EMBO J. 20: 4122-4131. 11483515

Lin, F. C. and Arndt, K. T. (1995). The role of Saccharomyces cerevisiae type 2A phosphatase in the actin cytoskeleton and in entry into mitosis. EMBO J. 14: 2745-2759

Lin, X. H., et al. (1998). Protein phosphatase 2A is required for the initiation of chromosomal DNA replication. Proc. Natl. Acad. Sci. 95(25): 14693-8

Liu, H., Rankin, S. and Yu, H. (2013). Phosphorylation-enabled binding of SGO1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis. Nat Cell Biol 15: 40-49. PubMed ID: 23242214

Llano, E., Gomez, R., Gutierrez-Caballero, C., Herran, Y., Sanchez-Martin, M., Vazquez-Quinones, L., Hernandez, T., de Alava, E., Cuadrado, A., Barbero, J. L., Suja, J. A. and Pendas, A. M. (2008). Shugoshin-2 is essential for the completion of meiosis but not for mitotic cell division in mice. Genes Dev 22: 2400-2413. PubMed ID: 18765791

Mansuy, I. M., et al. (1998). Restricted and regulated overexpression reveals calcineurin as a key component in the transition from short-term to long-term memory. Cell 92: 39-49

Margolis, S. S., et al. (2006). Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis. Cell 127(4): 759-73. Medline abstract: 17110335

Maton, G., et al. (2005). Differential regulation of Cdc2 and Aurora-A in Xenopus oocytes: a crucial role of phosphatase 2A. J. Cell Sci. 118: 2485-2494. 15923661

Mayer-Jaekel, R. E., et al. (1992). Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila. Mol. Biol. Cell 3: 287-98. PubMed Citation: 1320961

Mayer-Jaekel, R. E., Ohkura, H., et al. (1993). The 55kd regulatory subunit of Drosophila protein phosphatase 2A is required for anaphase. Cell 72: 621-633

McConnell, J. L., Watkins, G. R., Soss, S. E., Franz, H. S., McCorvey, L. R., Spiller, B. W., Chazin, W. J. and Wadzinski, B. E. (2010). Alpha4 is a ubiquitin-binding protein that regulates protein serine/threonine phosphatase 2A ubiquitination. Biochemistry 49: 1713-1718. PubMed ID: 20092282

McCright, B., et al. (1996). The B16 family of protein phosphatase2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm. J. Biol. Chem. 271: 22081-9.

McGuinness, B. E., Hirota, T., Kudo, N. R., Peters, J. M. and Nasmyth, K. (2005). Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells. PLoS Biol 3: e86. PubMed ID: 15737064

Mehsen, H., Boudreau, V., Garrido, D., Bourouh, M., Larouche, M., Maddox, P. S., Swan, A. and Archambault, V. (2018). PP2A-B55 promotes nuclear envelope reformation after mitosis in Drosophila. J Cell Biol 217(12): 4106-4123. PubMed ID: 30309980

Menzel, D., et al. (1995). Protein phosphatase 2A, a potential regulator of actin dynamics and actin-based organelle motility in the green alga Acetabularia. Eur. J. Cell Biol. 67: 179-187

Mills, J., Lee, V. and Pittman, R. (1998). Activation of a PP2A-like phosphatase and dephosphorylation of tau protein characterize onset of the execution phase of apoptosis. J. Cell Sci.111(5): 625-636

Minshull, J., Straight, A., Rudner, A. D., Dernburg, A. F., Belmont, A. and Murray, A. W. (1996). Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast. Curr. Biol. 6: 1609-1620. 8994825

Mayer-Jaekel, R. E., Ohkura, H., Ferrigno, P., Andjelkovic, N., Shiomi, K., Uemura, T., Glover, D. M. and Hemmings, B. A. (1994). Drosophila mutants in the 55 kDa regulatory subunit of protein phosphatase 2A show strongly reduced ability to dephosphorylate substrates of p34cdc2. J. Cell Sci. 107: 2609-2616. 7844174

Moazzen, H., Rosenfeld, R. and Percival-Smith, A. (2009). Non-requirement of a regulatory subunit of Protein Phosphatase 2A, PP2A-B', for activation of Sex comb reduced activity in Drosophila melanogaster. Mech. Dev. 126(8-9): 605-10. PubMed Citation: 19563886

Mochida, S., Maslen, S. L., Skehel, M. and Hunt, T. (2010). Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis. Science 330: 1670-1673. PubMed Citation: 21164013

Moshkin, Y. M., Doyen, C. M., Kan, T. W., Chalkley, G. E., Sap, K., Bezstarosti, K., Demmers, J. A., Ozgur, Z., van Ijcken, W. F. and Verrijzer, C. P. (2013). Histone Chaperone NAP1 Mediates Sister Chromatid Resolution by Counteracting Protein Phosphatase 2A. PLoS Genet 9: e1003719. PubMed ID: 24086141

Nakamura, M, et al. (1994). Musashi, a neural RNA-binding protein required for Drosophila adult external sensory organ development. Neuron 13: 67-81. PubMed Citation: 8043282

Nybakken, K., Vokes, S. A., Lin, T. Y., McMahon, A. P. and Perrimon, N. (2005). A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway. Nat. Genet. 37(12): 1323-32. 16311596

Ogura, K., et al. (2010). Protein phosphatase 2A cooperates with the autophagy-related kinase UNC-51 to regulate axon guidance in Caenorhabditis elegans. Development 137(10): 1657-67. PubMed Citation: 20392746

Okamoto, K., et al. (2002). Cyclin G recruits PP2A to dephosphorylate Mdm2. Molec. Cell 9: 761-771. 11983168

Okumura, E., Morita, A., Wakai, M., Mochida, S., Hara, M. and Kishimoto, T. (2014). Cyclin B-Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase-independent mechanism. J Cell Biol 204: 881-889. PubMed ID: 24616226

Orth, M., Mayer, B., Rehm, K., Rothweiler, U., Heidmann, D., Holak, T. A. and Stemmann, O. (2011). Shugoshin is a Mad1/Cdc20-like interactor of Mad2. EMBO J 30: 2868-2880. PubMed ID: 21666598

Ory, S., et al. (2003). Protein phosphatase 2A positively regulates ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites. Curr. Biol. 13: 1356-1364. 12932319

O'Shaughnessy, R. F., Welti, J. C., Sully, K. and Byrne, C. (2009). Akt-dependent Pp2a activity is required for epidermal barrier formation during late embryonic development. Development 136(20): 3423-31. PubMed Citation: 19762425

Padmanabhan, S., et al. (2009). A PP2A regulatory subunit regulates C. elegans insulin/IGF-1 signaling by modulating AKT-1 phosphorylation. Cell 136(5): 939-51. PubMed Citation: 19249087

Peterson, R. T., et al. (1999). Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycin associated protein. Proc. Natl. Acad. Sci. 96(8): 4438-42. PubMed Citation: 10200280

Petritsch, C., et al. (2000). TGF-beta inhibits p70 S6 kinase via protein phosphatase 2A to induce G1 arrest. Genes Dev. 14: 3093-3101. PubMed Citation: 11124802

Qi, S. T., Wang, Z. B., Ouyang, Y. C., Zhang, Q. H., Hu, M. W., Huang, X., Ge, Z., Guo, L., Wang, Y. P., Hou, Y., Schatten, H. and Sun, Q. Y. (2013). Overexpression of SETbeta, a protein localizing to centromeres, causes precocious separation of chromatids during the first meiosis of mouse oocytes. J Cell Sci 126: 1595-1603. PubMed ID: 23444375

Rangone, H., Wegel, E., Gatt, M. K., Yeung, E., Flowers, A. et al (2011) Suppression of Scant identifies Endos as a substrate of Greatwall Kinase and a negative regulator of Protein Phosphatase 2A in mitosis. PLoS Gen. 7(8): e1002225. PubMed Citation: 21852956

Reilein, A. R., et al. (1998). Regulation of organelle movement in melanophores by protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2A (PP2A). J. Cell Biol. 142(3): 803-13. PubMed Citation: 9700167

Rhyu, M.S., et al. (1994). Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 76: 477-491. PubMed Citation: 8313469

Rattani, A., Wolna, M., Ploquin, M., Helmhart, W., Morrone, S., Mayer, B., Godwin, J., Xu, W., Stemmann, O., Pendas, A. and Nasmyth, K. (2013). Sgol2 provides a regulatory platform that coordinates essential cell cycle processes during meiosis I in oocytes. Elife 2. PubMed ID: 24192037

Sasahara, Y., et al. (1996). Okadaic acid suppresses neural differentiation-dependent expression of the neurofilament-L gene in P19 embryonal carcinoma cells by post-transcriptional modification. J. Biol. Chem. 271: 25960-7. PubMed Citation: 8824230

Seeling, J. M., et al. (1999). Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A. Science 283(5410): 2089-91. PubMed Citation: 10092233

Shiomi, K., et al. (1994). Alternative cell choice induced by low-level expression of a regulator of protein phosphatase 2A in the Drosophila peripheral nervous system. Development 120: 1591-99. PubMed Citation: 8050365

Sieburth, D. S., et al. (1999). A PP2A regulatory subunit positively regulates Ras-mediated signaling during Caenorhabditis elegans vulval induction. Genes Dev. 13: 2562-2569. PubMed Citation: 10521400

Smith, G. D., et al. (1998). Characterization of protein phosphatases in mouse oocytes. Dev. Biol. 204(2): 537-49. PubMed Citation: 9882488

Song, M. H., et al. (2011). Protein phosphatase 2A-SUR-6/B55 regulates centriole duplication in C. elegans by controlling the levels of centriole assembly factors. Dev. Cell. 20: 563-571. PubMed Citation: 21497766

Sontag, E., et al. (1995). A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle. J. Cell Biol. 128: 1131-1144. PubMed Citation: 7896877

Sontag, E., Sontag, J. M. and Garcia, A. (1997). Protein phosphatase 2A is a critical regulator of protein kinase C zeta signaling targeted by SV40 small t to promote cell growth and NF-kappaB activation. EMBO J. 16(18): 5662-5671. PubMed Citation: Su, Y., Ospina, J. K., Zhang, J., Michelson, A. P., Schoen, A. M. and Zhu, A. J. (2011). Sequential phosphorylation of smoothened transduces graded hedgehog signaling. Sci Signal 4: ra43. PubMed ID: 21730325

Tanno, Y., et al. (2010). Phosphorylation of mammalian Sgo2 by Aurora B recruits PP2A and MCAK to centromeres. Genes Dev. 24(19): 2169-79. PubMed Citation: 20889715

Tehrani, M. A., Mumby, M. C. and Kamibayashi, C. (1996). Identification of a novel protein phosphatase 2A regulatory subunit highly expressed in muscle. J. Biol. Chem. 271(9): 5164-70

Tolstykh, T., et al. (2000). Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits. EMBO J. 19: 5682-5691

Tournebize, R, et al. (1997). Distinct roles of PP1 and PP2A-like phosphatases in control of microtubule dynamics during mitosis. EMBO J. 16(18): 5537-5549

Tsukahara, T., Tanno, Y. and Watanabe, Y. (2010). Phosphorylation of the CPC by Cdk1 promotes chromosome bi-orientation. Nature 467: 719-723. PubMed ID: 20739936

Turowski, P, et al. (1995). Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression. J. Cell Biol. 129: 397-410

Uemura, T., Shepherd, S., Ackerman, L., Jan, L.Y. and Jan, Y.N. (1989). numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos. Cell 58: 349-360. PubMed Citation: 2752427

Uemura, T., et al. (1993). Mutations of twins encoding a regulator of protein phosphatase 2A leads to pattern duplication in Drosophila imaginal discs. Genes Dev. 7: 429-440. PubMed Citation: 8383623

Von Stetina, J. R., et al. (2008). alpha-Endosulfine is a conserved protein required for oocyte meiotic maturation in Drosophila. Development 135: 3697-3706. PubMed Citation: 18927152

Vereshchagina, N. and Wilson, C. (2006). Cytoplasmic activated protein kinase Akt regulates lipid-droplet accumulation in Drosophila nurse cells. Development 133(23): 4731-5. PubMed Citation: 17079271

Vereshchagina, N., Ramel, M. C., Bitoun, E. and Wilson, C. (2008). The protein phosphatase PP2A-B' subunit Widerborst is a negative regulator of cytoplasmic activated Akt and lipid metabolism in Drosophila. J. Cell Sci. 121(Pt 20): 3383-92. PubMed Citation: 18827008

Viquez, N. M., et al. (2009). PP2A and GSK-3β act antagonistically to regulate active zone development. J. Neurosci. 29(37): 11484-94. PubMed Citation: 19759297

Vorlaufer, E. and Peters, J. M. (1998). Regulation of the cyclin B degradation system by an inhibitor of mitotic proteolysis. Mol. Biol. Cell 9(7): 1817-1831

Wang, C., et al. (2009). Protein phosphatase 2A regulates self-renewal of Drosophila neural stem cells. Development 136: 2287-2296. PubMed Citation: 19502489

Wang, N., Leung, H. T., Mazalouskas, M. D., Watkins, G. R., Gomez, R. J. and Wadzinski, B. E. (2012). Essential roles of the Tap42-regulated protein phosphatase 2A (PP2A) family in wing imaginal disc development of Drosophila melanogaster. PLoS One 7: e38569. PubMed ID: 22701670

Wang, P., Pinson, X. and Archambault, V. (2011). PP2A-twins is antagonized by greatwall and collaborates with polo for cell cycle progression and centrosome attachment to nuclei in drosophila embryos. PLoS Genet. 7(8): e1002227. PubMed Citation: 21852958

Wang, S. S., et al. (1998). Alterations of the PPP2R1B gene in human lung and colon cancer. Science 282(5387): 284-7. PubMed Citation: 9765152

Wang, Y. and Burke, D. J. (1997). Cdc55p, the B-type regulatory subunit of protein phosphatase 2A, has multiple functions in mitosis and is required for the kinetochore/spindle checkpoint in Saccharomyces cerevisiae. Mol. Cell. Biol. 17: 620-626. 9001215

Wassarman, D. A., et al. (1996). Protein phosphatase 2A positively and negatively regulates Ras1-mediated photoreceptor development in Drosophila. Genes Dev. 10: 272-278. PubMed Citation: 8595878

Wera, S., et al. (1995). Deregulation of translational control of the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A leads to multinucleated cells. J. Biol. Chem. 270: 21374-21381

White-Grindley, E., Li, L., Mohammad Khan, R., Ren, F., Saraf, A., Florens, L. and Si, K. (2014). Contribution of Orb2A stability in regulated amyloid-like oligomerization of Drosophila Orb2. PLoS Biol 12: e1001786. PubMed ID: 24523662

Williams, B. C., Filter, J. J., Blake-Hodek, K. A., Wadzinski, B. E., Fuda, N. J., Shalloway, D. and Goldberg, M. L. (2014). Greatwall-phosphorylated Endosulfine is both an inhibitor and a substrate of PP2A-B55 heterotrimers. Elife 3: e01695. PubMed ID: 24618897

Winder, D. G., et al. (1998). Genetic and pharmacological evidence for a novel, intermediate phase of long-term potentiation suppressed by calcineurin. Cell 92: 25-37

Wu, J., et al. (2000). Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. EMBO J. 19: 5672-5681

Wu, Q., et al. (2007). A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest. Curr. Biol. 17: 213-224. Medline abstract: 17276914

Xu, Z., Cetin, B., Anger, M., Cho, U. S., Helmhart, W., Nasmyth, K. and Xu, W. (2009). Structure and function of the PP2A-shugoshin interaction. Mol Cell 35: 426-441. PubMed ID: 19716788

Xue, C., et al. (1998). Developmental expression and localization of the catalytic subunit of protein phosphatase 2A in rat lung. Dev. Dyn. 211(1): 1-10

Yamagishi, Y., Honda, T., Tanno, Y. and Watanabe, Y. (2010). Two histone marks establish the inner centromere and chromosome bi-orientation. Science 330: 239-243. PubMed ID: 20929775

Yamashita, T., Inui, S., Maeda, K., Hua, D. R., Takagi, K., Fukunaga, K. and Sakaguchi, N. (2006). Regulation of CaMKII by alpha4/PP2Ac contributes to learning and memory. Brain Res 1082: 1-10. PubMed ID: 16516168

Yang, J., Wu, J., Tan, C. and Klein, P. S. (2003). PP2A:B56epsilon is required for Wnt/ß-catenin signaling during embryonic development. Development 130: 5569-5578. 14522869

Yeong, F. M., et al. (2003). Identification of a subunit of a novel Kleisin-ß/SMC complex as a potential substrate of protein phosphatase 2A. Curr. Biol. 13: 2058-2064. 14653995

Yu, H. G. and Koshland, D. (2007). The Aurora kinase Ipl1 maintains the centromeric localization of PP2A to protect cohesin during meiosis. J. Cell Biol. 176(7): 911-8. PubMed citation: 17371833

Yuan, D., Zhou, S., Liu, S., Li, K., Zhao, H., Long, S., Liu, H., Xie, Y., Su, Y., Yu, F. and Li, S. (2020). The AMPK-PP2A axis in insect fat body is activated by 20-hydroxyecdysone to antagonize insulin/IGF signaling and restrict growth rate. Proc Natl Acad Sci U S A 117(17): 9292-9301. PubMed ID: 32277029

Zhong, W. et al. (1996). Asymmetric localization of a mammalian Numb homolog during mouse cortical Neurogenesis. Neuron 17: 43-53

Zhou, X. Z., et al. (2000). Pin1-dependent prolyl isomerization regulates dephosphorylation of Cdc25C and tau proteins. Mol. Cell 6(4): 873-83. 11090625