InteractiveFly: GeneBrief

widerborst: Biological Overview | References

BIOLOGICAL OVERVIEW

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 (Sarbassov, 2005b). 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 (Jackson, 2006). 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 (Sarbassov, 2005a). 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 (Pinal, 2006; Martin-Belmonte, 2007). 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) (Hannus, 2002), 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 (Teixeira, 2003; Welte, 2005; Vereshchagina, 2006)], 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 (Ugi, 2004; Strack, 2004). PP2A-B' activity has been implicated in this process (Van Kanegan, 2005). Furthermore, mammalian PP2A can dephosphorylate Akt in vitro (Li, 2003). The phosphorylation state of Thr308 might also be affected by PP2A (Ugi, 2004). 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 (Li, 2002) and the Hedgehog (Hh) pathway (Nybakken, 2005), 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 (Pinal, 2006). 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 (Stocker, 2002). One possible explanation for the current 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); (Viquez, 2006)] 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 (Janssens, 2005). 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 (Viquez, 2006). 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' (Bielinski, 2007; 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 (Gao, 2005), 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 (Holm, 2003; Prusty, 2002; Akimoto, 2005), 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 (Vereshchagina, 2006). 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 (Cavaliere, 2005) is strongly suppressed by wdb. Interestingly, Jünger (2003) has 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).

PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation

The seven-transmembrane protein Smoothened (Smo) and Zn-finger transcription factor Ci/Gli are crucial components in Hedgehog signal transduction that mediates a variety of processes in animal development. In Drosophila, multiple kinases have been identified to regulate Hh signaling by phosphorylating Smo and Ci; however, the phosphatase(s) involved remain obscured. Using an in vivo RNAi screen, PP4 and PP2A were identified as phosphatases that influence Hh signaling by regulating Smo and Ci, respectively. RNAi knockdown of PP4, but not of PP2A, elevates Smo phosphorylation and accumulation, leading to increased Hh signaling activity. Deletion of a PP4-interaction domain (amino acids 626-678) in Smo promotes Smo phosphorylation and signaling activity. It was further found that PP4 regulates the Hh-induced Smo cell-surface accumulation. Mechanistically, it was shown that Hh downregulates Smo-PP4 interaction that is mediated by Cos2. Evidence is provided that PP2A is a Ci phosphatase. Inactivating PP2A regulatory subunit Widerborst (Wdb) by RNAi or by loss-of-function mutation downregulates, whereas overexpressing regulatory subunit upregulates, the level and thus signaling activity of full-length Ci. Furthermore, Wdb counteracts kinases to prevent Ci phosphorylation. Finally, evidence was obtained that Wdb attenuates Ci processing probably by dephosphorylating Ci. Taken together, these results suggest that PP4 and PP2A are two phosphatases that act at different positions of the Hh signaling cascade (Jia, 2009).

The screen used to identify the phosphatases differs from previous screens because an in vivo assay was used to examine Smo expression levels, which is a more direct readout, and because knockdown of specific phosphatase gene(s) involved in Smo dephosphorylation might not affect the pathway activity in a significant way and such gene(s) could have been missed in the previous RNAi screens with cultured cells. This study identified PP4 as a novel Hh signaling component that regulates Smo phosphorylation. The study provides the first evidence for the physiological Smo and Ci phosphatases, and uncovers the underlying mechanism of Smo regulation by phosphatase (Jia, 2009).

This study identified PP4 and PP2A to be negative and positive regulators in the Hh pathway, and it was shown that they exert their roles through Smo and Ci, respectively. Are PP4 and PP2A the only phosphatases in the Hh pathway? Although the data suggest that PP4 is a phosphatase for Smo, the possibility of the involvement of other phosphatase(s) cannot be excluded. Hh induces extensive Smo phosphorylation at numerous Ser/Thr sites, and multiple kinases are involved in these phosphorylation events. It might be possible that multiple phosphatases could be involved. In addition, loss-of-function studies on PP2A regulating Ci are not based on null mutations. This was due to the fact that genetic null mutations of the catalytic and regulatory subunits cause cell lethality. Thus, the results might not be exclusive (Jia, 2009).

Removal of PP4 by RNAi in wing discs induced Smo accumulation in A-compartment cells both near and away from the AP boundary. In addition, PP4 RNAi induced the elevation and anterior expansion of Hh target gene expression. However, the accumulated Smo caused by PP4 RNAi did not ectopically activate Hh target genes in cells away from the AP boundary. In addition, although Smo phosphorylation was potentiated by knocking down PP4 or abolishing Smo-PP4 interaction, the elevated phosphorylation did not suffice to promote Smo cell-surface accumulation. These data suggest that the basal phosphorylation of Smo regulated by PP4 is not sufficient to activate Smo, and that de novo Smo activation still depends on Hh (Jia, 2009).

Previous studies have shown that PKA and CK1 are required for Hh-induced Smo accumulation and signaling activity. Phosphorylation-deficient forms of Smo (with PKA or CK1 sites mutated to Ala) are defective in Hh signaling, whereas SmoSD123, the phosphorylation-mimicking Smo, has potent signaling activity and high level of cell-surface accumulation. Thus, the PKA and CK1 sites are apparently crucial in mediating Smo phosphorylation and activation. Hh treatment may cause increased phosphorylation at these sites. In addition to PKA and CK1 sites, there are many other Ser/Thr residues that are phosphorylated upon Hh stimulation. Although phosphorylation-mimicking mutations at these sites alone did not have discernible effect on Smo, their phosphorylation could modulate the cell-surface accumulation and activity of Smo phosphorylated at the three PKA/CK1 sites, which may at least in part explain why cell-surface accumulation and activity of SmoSD123 is still regulated by Hh. This study found that removing PP4 alone promoted Smo phosphorylation but did not elevate the cell-surface accumulation of Smo. It is possible that high levels of basal Smo phosphorylation in the absence of PP4 do not reach the threshold for promoting Smo cell-surface accumulation. It is also possible that basal Smo phosphorylation mainly occurs at sites other than the crucial PKA/CK1 phosphorylation clusters. In support of this notion, it was found that knockdown PP4 by RNAi promoted SmoSD123 to further accumulate on the cell surface in the absence of Hh (Jia, 2009).

How is Smo phosphorylation regulated? Hh may regulate Smo phosphorylation by regulating the accessibility of its kinase and/or phosphatase. In this study, it was found that Smo interacts with PP4 through amino acids 626-678, a region previously mapped to be a Cos2-interacting domain. It was further found that Smo-PP4 association diminished when Cos2 was knocked down by RNAi. A previous study revealed that Cos2 impedes Hh-induced Smo phosphorylation by interacting with amino acids 626-678 of Smo and Hh-induced phosphorylation of Cos2 at Ser572 dissociates Cos2 from amino acids 626-678 of Smo, thereby alleviating its inhibition on Smo phosphorylation. This study found that Cos2 inhibits Smo phosphorylation by recruiting PP4 and Hh promotes Smo phosphorylation by preventing Cos2-PP4 complex from binding to amino acids 626-678 of Smo. SmoDelta626-678, when not interacting with PP4, could still interact with Cos2 via a Cos2-interaction domain near the Smo C terminus. The Cos2-binding Smo C terminus might not recruit PP4. Taken together, these findings suggest that Hh may promote Smo phosphorylation at least in part by reducing the accessibility of a phosphatase (Jia, 2009).

Phosphorylation of Ci/Gli controls the balance of its activator and repressor activity. This study has demonstrate a role of PP2A in dephosphorylating Ci and attenuating Ci processing. However, it is not known whether Hh regulates PP2A to dephosphorylate Ci. Previous studies have shown that Hh interferes with the Cos2-Ci-kinase protein complex. It is possibly that Hh also regulates Ci phosphatase, or the accessibility of the phosphatase. Future studies should determine whether PP2A interacts with Cos2-Ci and whether such interaction is regulated by Hh (Jia, 2009).

Many aspects of Smo and Ci/Gli regulation are conserved across species. For example, both Drosophila and mammalian Smo proteins undergo a conformational switch in response to Hh stimulation. Ci/Gli proteolysis is mediated by the same set of kinases and E3 ubiquitin ligases. In addition, it has been shown that PP2A is involved in vertebrate Hh signaling, probably by regulating Gli nuclear localization and activity. Therefore, it would be interesting to determine whether PP4 and PP2A play similar roles in regulating phosphorylation of vertebrate Smo and Gli (Jia, 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, Tws, 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).

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 α, β and ε subunits (62%-66% identity) than to the β or γ 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' α/ε 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' α 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' α 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).

Search PubMed for articles about Drosophila Widerborst

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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. PubMed Citation: 12091318

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date revised: 20 April 2012

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