InteractiveFly: GeneBrief

greatwall: Biological Overview | References

BIOLOGICAL OVERVIEW

Polo is a conserved kinase that coordinates many events of mitosis and meiosis, but how it is regulated remains unclear. Drosophila females having only one wild-type allele of the polo kinase gene and the dominant Scant mutation produce embryos in which one of the centrosomes detaches from the nuclear envelope in late prophase. Scant creates a hyperactive form of Greatwall (Gwl) with altered specificity in vitro. Greatwall is another protein kinase recently implicated in mitotic entry in Drosophila and Xenopus. Excess Gwl activity in embryos causes developmental failure that can be rescued by increasing maternal Polo dosage, indicating that coordination between the two mitotic kinases is crucial for mitotic progression. Revertant alleles of Scant that restore fertility to polo-Scant heterozygous females are recessive alleles or deficiencies of gwl; they show chromatin condensation defects and anaphase bridges in larval neuroblasts. One recessive mutant allele specifically disrupts a Gwl isoform strongly expressed during vitellogenesis. Females hemizygous for this allele are sterile, and their oocytes fail to arrest in metaphase I of meiosis; both homologues and sister chromatids separate on elongated meiotic spindles with little or no segregation. This allelic series of gwl mutants highlights the multiple roles of Gwl in both mitotic and meiotic progression. These results indicate that Gwl activity antagonizes Polo and thus identify an important regulatory interaction of the cell cycle (Archambault, 2007).

Studies of Greatwall in Xenopus reveal another side to Greatwall function that suggest that the picture in Drosophila is not the whole story. In Xenopus, Greatwall is required for the positive feedback loop that removes inhibitory tyrosine phosphate from the central mitotic regulatory kinase Cdc2. Immunodepletion of Greatwall kinase prevents Xenopus egg extracts from entering or maintaining M phase due to the accumulation of inhibitory phosphorylations on Thr14 and Tyr15 of Cdc2. M phase-promoting factor (MPF) in turn activates Greatwall, implying that Greatwall participates in an MPF autoregulatory loop. Activated Greatwall both accelerates the mitotic G2/M transition in cycling egg extracts and induces meiotic maturation in G2-arrested Xenopus oocytes in the absence of progesterone. Activated Greatwall can induce phosphorylations of Cdc25 in the absence of the activity of Cdc2, Plx1 (Xenopus Polo-like kinase) or mitogen-activated protein kinase, or in the presence of an activator of protein kinase A that normally blocks mitotic entry. The effects of active Greatwall mimic in many respects those associated with addition of the phosphatase inhibitor okadaic acid (OA); moreover, OA allows cycling extracts to enter M phase in the absence of Greatwall. Taken together, these findings support a model in which Greatwall negatively regulates a crucial phosphatase that inhibits Cdc25 activation and M phase induction (Zhao, 2008; full text of article).

Mutations in Drosophila Greatwall reveal its roles in mitosis and meiosis and interdependence with Polo kinase

Reversible protein phosphorylation and periodic protein destruction play major roles in regulating the eukaryotic cell division cycle. The major protein kinase that directs cell division is cyclin-dependent kinase 1 (Cdk1), the active component of Maturation Promoting Factor. The cyclical inactivation of Cdk1 prior to mitotic exit is brought about in part through destruction of its cyclin partner. Two other protein kinase families, the Polo and Aurora families, are known to have critical functions in progression into and through M phase (mitosis and cytokinesis) and functionally interact with each other and also with Cdk1 to mediate their functions (Archambault, 2007).

Polo, originally discovered in Drosophila, exemplifies an evolutionarily conserved mitotic protein kinase. Polo, as well as its close orthologs, has been shown to function in multiple events essential for cell division. Polo was initially found to be essential for centrosome maturation and separation. It promotes recruitment of the γ-tubulin ring complex and phosphorylates Asp to facilitate nucleation of an increased number of dynamic microtubules on mitotic entry. At the G2/M transition, Polo (Polo-like kinase 1 in vertebrates) phosphorylates and activates the Cdc25 phosphatase responsible for removing inhibitory phosphates on Cdk1; this promotes mitotic entry. It also functions at the kinetochore-microtubule interface to monitor tension; the 3F3/2 phospho-epitope seen on kinetochores in the absence of tension is a consequence of Plk1/Plx1 kinase activity in vertebrates. Removal of cohesins from chromosomal arms in mitosis and meiosis also requires phosphorylation of cohesin subunits by Polo kinases. In Drosophila meiosis II, Polo phosphorylates and inactivates the centromeric cohesion protector protein Mei-S332. In addition, Polo is required for cytokinesis. The growing list of Polo kinase substrates is evidence of its role in multiple mitotic events (Archambault, 2007).

It is clear that protein kinases such as Cdk1 and Polo are only part of a large network of protein kinases that regulate cell cycle progression, many of which are as yet poorly characterized. A genome-wide survey found that up to one-third of the protein kinome of Drosophila has some cell cycle role (Bettencourt-Dias, 2004). Depletion of the Gwl kinase from S2 cells by RNA interference (RNAi) led to a mitotic delay characterized by formation of long spindles and scattered chromosomes (Bettencourt-Dias, 2004). Yu (2004) also found a mitotic role for Gwl kinase by characterizing missense hypomorphic mutants. Reduced gwl function results in mitotic defects in larval neuroblasts and tissue culture cells, including delay between late G2 and anaphase onset and chromosome condensation defects. Gwl has close homologs across eukaryotes and more distant homologs in budding and fission yeasts. Indeed, Yu (2006) reported a function for Xenopus Gwl in mitotic entry, as part of the Cdc2/Cdk1 activation loop in oocyte extracts. In that system, Xenopus Gwl is directly activated by cyclin B-Cdc2 and is in turn needed to promote full activation of cyclin B-Cdc2, although the direct target(s) mediating this action is (are) still unknown and indeed no substrates of Gwl are yet known. The primary sequence of Gwl shows that the regions most homologous to other kinases are split by a long intervening sequence of unknown function (Yu, 2004). Despite this recent progress, nothing is known about how activity of this crucial kinase is coordinated with the multiple events of cell cycle progression. Moreover, it is not known how Gwl contributes to the different types of mitotic and meiotic cell cycles of a metazoan (Archambault, 2007).

Elucidation of protein function may be aided through the generation of multiple mutant alleles that can reveal separate functions of individual proteins in multiple cellular processes. Drosophila offers the possibility of such studies and, moreover, allows the study of protein function in different types of cell cycle during its development. This principle was applied to study the gene defined by Scant (Scott of the Antarctic), a gain-of-function, dominant enhancer of maternal-effect embryonic defects of polo mutants. Syncytial embryos derived from females heterozygous for both Scant and polo develop mitotic defects in which a centrosome disassociates from one pole (White-Cooper, 1996) that the Scant mutation is an allele of gwl that introduces a K97M amino-acid substitution into the Gwl protein; this results in a hyperactive kinase with altered specificity in vitro. These results indicate an antagonistic relationship between Gwl and Polo and suggest that their activity has to be coordinated for proper embryonic mitotic function. An allelic series of new gwl mutations reveals multiple functions for the Gwl kinase in both mitosis and female meiosis. These display somatic developmental defects accompanied by chromosome condensation and segregation defects in larval neuroblasts. gwl⁺ encodes two isoforms, only one of which is expressed during vitellogenesis. An allele that specifically prevents the expression of this isoform reveals requirements for Gwl in meiosis and in the maternal contribution to the egg (Archambault, 2007).

The Gwl kinase seems to have multiple roles in progression through mitosis and meiosis. The phenotypes shown by gwl mutants differ at different stages of development reflecting both the nature of different alleles and the variety of ways in which the cell cycle is regulated in Drosophila. Indeed it is these different modes of cell cycle regulation as development proceeds that allow Gwl's multiple functions in cell division to be tackled (Archambault, 2007).

The starting point of this study was the strong genetic interaction between the Scant mutation and polo mutations; heterozygous females lay embryos that die, presumably as a consequence of mitotic failure whose first observed defect is that a single centrosome moves away from the nucleus before nuclear envelope breakdown in all cases examined. This centrosome misbehavior is probably the primary defect; developmental failure probably results from secondary defects of abnormal spindles. It will be interesting to reexamine the phenotype of other maternal-effect mutants showing free centrosomes to see if they disassociate from the nuclear envelope in the same manner. It will also be interesting to find out whether the detaching centrosome always contains either the older or the younger centriole, since they may harbor different amounts of biochemical factors in their pericentriolar material. If so, the history of the centrosome determines its vulnerability to detachment when the Gwl/Polo balance is compromised. The Scant-polo genetic interaction is moderately specific since a screen for mutants reverting the maternal-effect embryonic lethality generated only one third-site interactor among two independent polo⁺ duplication events plus two revertants of the Scant allele itself. Scant encodes a Gwl kinase with a K97M substitution that results in hyperactivity in vitro (albeit with altered specificity on the artificial substrates tested); a wild-type transgene with just this amino acid mutated interacts dominantly with polo mutants, so this amino acid substitution is Scant. Moreover, mothers overexpressing wild-type Gwl kinase in the presence of reduced Polo kinase function produce embryos with the same kinds of defects as Scant. Therefore, the increased activity of Gwl-K97M and not its altered substrate specificity is responsible for the functional interaction between Scant and polo. It follows that a balance between Gwl and Polo activities in embryos is crucial, but because there does not seem to be any such interaction in cell cycles at later stages (in proliferating larval, pupal, or adult tissues), since polo¹¹ +/+ Scant itself has normal viability, the balance appears particularly important for these early embryonic cell cycles. The syncytial nuclear division cycles are unusual in that they comprise rapidly alternating cycles of S phase and M phase without intervening gap (either G1 or G2) phases. A G2 phase is only introduced following cellularization when String (the Cdc25 dual-specificity phosphatase that activates Cdk1) is degraded; its expression then comes under transcriptional regulation in a spatio-temporally defined pattern. The critical balance of Polo and Gwl kinase activities in the syncytium may reflect the absence of a G2 state; mitotic proteins are held on continual standby, awaiting their use in the next cycle, rather than being degraded and resynthesized each cell cycle as is the case for cycles with a G2. Alternatively, centrosome detachment may be frequent in other tissues of polo-Scant flies where it may be better tolerated. However, this is unlikely because only normal centrosome positioning is observed in polo-Scant testes (Archambault, 2007).

This antagonism between Polo and Gwl was not predicted from studies of these enzymes in Xenopus cell-free systems, which have been used to model the entry into mitosis from G2 through the activation of the Cdc25 phosphatase. There the evidence indicates that both Gwl and Plk1 kinases participate in the autoregulatory loop that activates the Cdk1/cyclin B MPF kinase complex (Yu, 2004). This apparent cooperation of the two kinases in this process suggests that the Xenopus cell-free system may be assessing a different aspect of cell cycle progression than in vivo studies on the syncytial cycles of Drosophila embryos. The apparent differences in results may also reflect the different aspects of mitosis under study; activation of Cdk1 in one system and the integrity of the mitotic apparatus in the other. There are few clues about the directionality of the antagonism observed for Polo and Gwl function in fly embryos or whether it involves direct interactions between the two protein kinases. Yu (2006) observed that xPlk1 is capable of phosphorylating xGwl, but that study did not observe changes in xGwl activity or a synergistic effect in combination with cyclin B-Cdc2-mediated phosphorylation. However, any inhibitory effect of Polo kinase on Gwl need not be mediated through regulation of kinase activity but could also occur by regulating Gwl's localization or stability. In this case, reduced dosage of Polo in the fly embryo might provide only a subthreshold activity, insufficient for the efficient downregulation of the hyperactive Gwl kinase encoded by gwl^Scant. That Gwl needs to be downregulated is suggested by its subcellular localization in mitosis. Gwl is enriched in the nucleus in interphase, but it is excluded from the nucleus during prophase, before nuclear envelope breakdown. This could occur through active nuclear export or through degradation. In favor of the latter, it was observed that Gwl is ubiquitinated (Archambault, 2007).

Gwl could also inhibit the function of Polo. In vitro experiments suggest that Gwl does not readily phosphorylate Polo, but it is also possible that phosphorylation of an intermediate substrate by Gwl mediates the hypothetical inhibitory effect. Since Scant causes a decrease in female fertility that is stronger in stronger polo mutant alleles, and the meiotic divisions occur in the embryonic cytoplasm, it is possible that Scant lowers Polo's activity during meiosis. Xiang (2007) has recently observed a functional interaction between polo and matrimony (mtrm); heterozygous mtrm/+ females have an elevated frequency of chromosome nondisjunction in meiosis, and this is suppressed by lowering the polo dosage. Therefore, if Scant acts to lower Polo's activity in female meiosis, then Scant might suppress the increased level of nondisjunction in mtrm heterozygotes. Indeed it does, though Scant needs to be homozygous for the suppression to be detectable, so the possibility that this suppression reflects homozygosity of some closely linked third player rather than Scant itself cannot be eliminated. Furthermore, even in Scant/Scant, the suppression of mtrm/+ is much weaker than halving the dosage of polo⁺ directly. This is consistent with the effects of Scant on fertility; homozygous Scant in a homozygous polo⁺ background is much more fertile than polo¹¹ Scant/+ +, so regardless of how Scant acts to reduce the functional level of polo⁺, one copy of Scant does not reduce the activity of one copy of polo⁺ completely. Nevertheless, the Scant-mtrm interaction result strongly suggests that the polo-Scant (and probably polo-gwl) interaction occurs during female meiosis as well as embryonic mitosis, and the unexpected Scant-mtrm interaction lowering female fertility implies a role for mtrm in embryogenesis (Archambault, 2007).

That Gwl downregulates Polo's function in the embryo is also suggested by the cellular phenotype, which is in line with the known functions of Polo at the centrosome. Moreover, the Scant phenotype is increased by the severity of the polo mutant. Occasional displacement of centrosomes early in mitosis is seen in syncytial embryos derived from heterozygous polo mutant females themselves, and Polo promotes centrosome separation, maturation, and integrity. In Drosophila, Polo phosphorylates Asp and together they promote the recruitment of γ-tubulin to the centrosome. In mammalian cells, Plk1 phosphorylates Nlp, triggering its dissociation from the centrosome and recruitment of several factors. Plk1 also phosphorylates Kizuna, which is required to preserve centrosome cohesion. The detached centrosomes observed in the polo-Scant-derived embryos do not show a reduction in γ-tubulin staining, and astral microtubules nucleate normally. Similar centrosome detachment was observed in Scant/+ and Scant/Scant-derived embryos, albeit with much lower frequencies. Therefore, it seems likely that the partial loss of Polo activity and the gain of Gwl activity both weaken centrosome function in a similar fashion; this is consistent with the mutually antagonistic interaction between polo and Scant mutations. Furthermore, it is noted that centrosome detachment occurs before nuclear envelope breakdown, a time when both Polo and Gwl are enriched around the nuclear envelope in syncytial embryos. This suggests that coordination between the centrosome, microtubules, and the nuclear envelope before nuclear envelope breakdown is sensitive to the balance between Polo and Gwl. Gwl (or cyclin B-Cdk1, which it activates in frog extracts) may share substrates with Polo and regulate them antagonistically in early mitosis (Archambault, 2007).

Centrosome loss can also occur in response to DNA damage, allowing damaged nuclei to fall into the interior of the syncytial embryo and be discarded from the developing fly. This response depends on Mnk/Chk2. The centrosome detachment observed in polo-Scant-derived embryos does not depend on Chk2 and thus seems to arise from a more direct effect on the centrosome-nuclear envelope association (Archambault, 2007).

Hypomorphic gwl mutants do not appear to impact directly upon centrosome behavior in the more conventional cell cycles of the larval central nervous system. Previously, Yu (2004) reported a long delay in late G2 to anaphase in gwl mutant neuroblasts in addition to chromatin condensation defects and has suggested that these defects, particularly undercondensation of chromatin, could all be attributed to the function of Gwl in activating cyclin B-Cdk1, although no direct substrate of Gwl is known. However, the prevalence of condensation defects and anaphase bridges that was observed in gwl mutant neuroblasts, together with the nuclear localization of Gwl in interphase, suggests that Gwl may act directly at the chromosome level. The anaphase bridges observed could be a consequence of tangled chromatids or dicentric chromosomes resulting from telomere fusion or other aberrant DNA damage repair events. When Gwl is depleted from cultured cells, they delay at the spindle assembly checkpoint with high levels of cyclin B and checkpoint proteins at kinetochores. The chromosomes of these cells are scattered upon an elongated spindle as though they have defects in kinetochore function. Since metaphase cells with highly condensed chromosomes accumulate in the larval CNS of gwl mutants, prolonged checkpoint arrest probably also occurs here. However, the polyploid cells seen in the null mutant indicate that cells can slip past the checkpoint without segregating their chromosomes; since this also happens in wild-type neuroblasts in the presence of colchicine, polyploidy is probably not a direct consequence of Gwl failure (Archambault, 2007).

A major role for Gwl kinase in regulating aspects of chromosome behavior is also suggested by the meiotic phenotype seen in gwl^Sr18/Df females. gwl^Sr18 disrupts the only form of Gwl expressed during vitellogenesis without disrupting the second mitotic isoform of Gwl. Therefore gwl^Sr18 provides a unique opportunity to study how loss of Gwl kinase affects vitellogenesis and meiosis. Although gwl^Sr18/Df ovaries develop normally, yolk distribution is abnormal in stages 13-14, females are sterile, and the severe meiotic defects include scattered chromosomes with separated chromatids and elongated spindles (Archambault, 2007).

Scattered chromosomes could result from a number of problems. One possibility is that Gwl is required for proper meiotic recombination; if so, the multiple DNA masses observed could correspond to chromosome fragments resulting from failure to complete chromatid exchange and to repair double-strand breaks. This would also lead to failure to arrest at metaphase I because bivalents would not be held together by chiasmata. This is unlikely for several reasons: (1) if the masses were fragmented chromosomes, they should vary widely in size; they do not; (2) Gwl accumulates in the oocyte nucleus and the nuclei of the nurse cells directly connected to the oocyte at (but not before) stage 8, which is much later than the time of meiotic recombination. However, it is noted that if Gwl is involved in meiotic recombination, the tiny amounts of it present in pachytene (germarial) nuclei could be below the detection limit of the antibody. (3) Two of the five nuclei that accumulate Gwl never entered pachytene; (4) FISH data prove that chromatid cohesion fails in gwl^Sr18 oocytes, and this is sufficient to account for the scattering of DNA masses observed. The number of DNA masses was often higher than six, the maximum expected number for disassociated bivalent chromosomes, disregarding the tiny fourth chromosomes. Therefore, Gwl is required for sister chromatid cohesion in meiosis I. In the absence of Gwl-long, the premature loss of (or failure to establish) arm cohesion would lead to the release of chiasmata if indeed any are formed (Archambault, 2007).

However, neither of these defects alone is expected to lead to complete female sterility. For example, mutants in c(3)G prevent all meiotic recombination but are still partially fertile (Hall, 1972). Mutants in ord do not keep sister chromatid cohesion yet show only a partial loss of female fertility. While the dissolution of sister chromatid cohesion in ord leads to progression through metaphase I into meiosis II, no normal meiosis II figures were seen in gwl^Sr18 oocytes, though it is possible that the elongated bipolar spindles represent attempts to do meiosis II after a failed anaphase I. Thus, the absence of Gwl-long in meiosis does not lead to a simple lack of meiotic recombination nor does it lead only to a premature dissolution of cohesion. The lack of Gwl could lead to a combination of both defects or to yet some other kind of defect that leads to full female sterility. Even if the occasional meiosis succeeds, it is very likely that these embryos would fail to develop because maternal Gwl-long is expected to be required for early embryonic mitoses; indeed, these embryos fail to reach cellular blastoderm (Archambault, 2007).

Most female meioses in gwl^Sr18 have scattered chromosomes on a single elongated spindle; it is thought that the minority (8%) that have multiple bundles of spindle are just the extreme of this scattering, since microtubules are nucleated by the chromatin in the acentriolar oocyte. Failure of karyosome formation might cause this scattering; however, oocytes of earlier stages do at least often form a single karyosome. Mutants that affect the spindle directly such as those affecting the microtubule-associated protein Msps show more spindle defects than chromosome scattering. A mutant disrupting the female germline-specific Cdk1-adaptor Cks30A disrupts the integrity of meiotic spindles in addition to showing chromosome alignment defects, but in this case the chromosome scattering observed is much more modest than that in the gwl^Sr18 mutant (Archambault, 2007).

How does Gwl regulate sister chromatid cohesion? The results suggest that Gwl antagonizes Polo, which is known to negatively regulate sister chromatid cohesion. It is therefore possible that the absence of Gwl during meiosis results in excessive and/or premature Polo activity, leading to premature loss of sister chromatid cohesion. In budding yeast, Polo (Cdc5) promotes the cleavage of the cohesin Scc1 by direct phosphorylation. In meiosis, sister chromatid cohesion is protected at centromeres until anaphase II by Mei-S332 in Drosophila (Shugoshin). Indeed, mei-S332 mutants show premature sister separation in meiosis I. In budding yeast, Shugoshin prevents cleavage of the cohesin Rec8, which replaces Scc1 in meiosis, and Cdc5 is required in meiosis for cleavage of Rec8. In Drosophila, Polo also negatively regulates Mei-S332 activity and localization. Thus, the lack of Gwl in meiosis could lead to premature activity of Polo, which could negatively regulate Mei-S332 and lead to precocious sister separation in meiosis I. This study examined Mei-S332′s localization in gwl^Sr18 hemizygous oocytes and found that Mei-S332 was largely properly localized to centromeres. However, Mei-S332 can be inactivated even when it remains localized at centromeres. Therefore, the possiblity cannot ruled out the possibility that Mei-S332 is being negatively regulated in the absence of Gwl. Alternatively, Gwl could promote sister chromatid cohesion by directly phosphorylating effectors of cohesion. Gwl-long is better than Gwl-short at performing a maternal function and it is suspected that Gwl-long will be a better kinase for a yet unknown maternal substrate (Archambault, 2007).

In conclusion, it appears that Gwl, in common with the other major mitotic protein kinases, has multiple roles in mitotic and meiotic progression. These have been revealed through a series of gwl alleles that exhibit different characteristics and reveal aspects of Gwl kinase function in the different types of cell cycle during Drosophila development. A gain-of-function allele of gwl reveals a requirement for coordinate activity of the Gwl and Polo kinases in the rapidly oscillating M and S phase cycles of early embryos. Partial and total loss of Gwl function leads to frequent chromosome condensation defects and anaphase bridge formation in the conventional division cycles of cells in the larval CNS. Finally, loss of Gwl function in the female germline leads to severe meiotic abnormalities including loss of sister chromatid cohesion. It will be of interest to identify potential binding partners of the Gwl protein kinase both in interphase, when it is present predominantly in the nucleus, and in mitosis, when it moves out to the cytoplasm. This may in turn facilitate the identification of its substrates; this is crucial for understanding exactly how it regulates these various aspects of cell division (Archambault, 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).

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).

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).

Regulation of Greatwall kinase by protein stabilization and nuclear localization

Greatwall (Gwl) functions as an essential mitotic kinase by antagonizing protein phosphatase 2A. This study identified Hsp90, Cdc37 and members of the importin alpha and beta families as the major binding partners of Gwl. Both Hsp90/Cdc37 chaperone and importin complexes associated with the N-terminal kinase domain of Gwl, whereas an intact glycine-rich loop at the N-terminus of Gwl was essential for binding of Hsp90/Cdc37 but not importins. Hsp90 inhibition led to destabilization of Gwl, a mechanism that may partially contribute to the emerging role of Hsp90 in cell cycle progression and the anti-proliferative potential of Hsp90 inhibition. Moreover, in agreement with its importin association, Gwl exhibited nuclear localization in interphase Xenopus S3 cells, and dynamic nucleocytoplasmic distribution during mitosis. KR456/457 was identified as the locus of importin binding and the functional NLS of Gwl. Mutation of this site resulted in exclusion of Gwl from the nucleus. Finally, this study showed that the Gwl nuclear localization is indispensable for the biochemical function of Gwl in promoting mitotic entry (Yamamoto, 2014). Entry into mitosis requires activation of maturation-promoting factor (MPF), a complex of cyclin B and Cdk1. During mitotic entry, MPF activation is triggered by Cdc25-mediated dephosphorylation of Cdk1. In addition to mechanisms that directly control the activity of Cdc25 and MPF, dynamic and regulated nucleocytoplasmic localization of these factors was also demonstrated to be important. In particular, both cyclin B and Cdc25 need to localize to the nucleus to trigger mitotic entry. It has been shown that phosphorylation of cyclin B by Polo-like kinase 1 (Plk1) promotes its nuclear localization. Furthermore, checkpoint kinases, Chk1 and Chk2, phosphorylate Cdc25, and thereby creating a binding site for 14-3-3 proteins which sequestrate Cdc25 in cytoplasm and inhibit M-phase entry. In addition to MPF, another enzyme required for both mitotic entry and maintenance is the Greatwall kinase (Gwl). Recent evidence revealed that Gwl inhibits the activity of protein phosphatase 2A/B55δ, the principal phosphatase acting on Cdk-phosphorylated substrates (Yamamoto, 2014).

The crucial nature of this inhibition was demonstrated by the failure of M-phase maintenance in egg extracts with full activity of MPF and other mitotic kinases but deficient of Gwl function. On the other hand, the presence of Gwl greatly reduced the amount of MPF required for mitotic entry (Yamamoto, 2014).

The mechanism of PP2A/B55δ inhibition by Gwl has been nicely solved with the recent identification of Endosulfine and its related family member, cAMP-regulated phosphoprotein 19 kDa, as substrates of Gwl. These substrates specifically bind to and inhibit PP2A/B55δ when they are phosphorylated by Gwl. While compelling evidence obtained in a variety of experimental systems revealed an essential role of Gwl in mitotic regulation, very little has been learned about how Gwl itself is regulated. It has been shown that Gwl is phosphorylated in M-phase, in quantitative correlation with its kinase activity (Yamamoto, 2014).

Mitotic phosphorylation of Gwl by MPF within its presumptive activation loop is required for Gwl activation, suggesting that MPF acts upstream of Gwl during mitotic entry. It has also been shown that Gwl can be phosphorylated by Plk1, whereas the specific function and regulation of this modification remains to be further clarified (Yamamoto, 2014).

In the search for new regulators of Gwl, a proteomic analysis revealed 2 groups of proteins as the major binding partners of Gwl which were co-purified with Gwl from G2 and M phase Xenopus oocytes. Specifically, Gwl bound both the chaperone complex Hsp90/Cdc37 and importin α/β through its N-terminus. Hsp90 was required to stabilize Gwl in both interphase and M-phase extracts. Furthermore, this study identified the functional NLS in Gwl that mediated its binding to importins. Mutation of amino acid residues KR456/457 within the NLS led to exclusively cytoplasmic localization of the protein in Xenopus S3 cells and interfered its function in promoting mitotic entry in egg extracts (Yamamoto, 2014).

Several heat shock proteins function as molecular chaperones. In particular, Hsp90 has been related to regulators of the cell cycle, including Cdk1, p53, Cdk4, and Plk1. It has been shown that the evolutionarily conserved protein association between Plk1 and Hsp90 was required for the stability of Plk1, and thereby involved in centrosomal functions and metaphase-to-anaphase transition (Yamamoto, 2014).

This study discovered Hsp90 as a major associated partner for Gwl. Further analysis showed that the N-terminal segment of Gwl mediated its association with Hsp90. Interestingly, a glycine-rich loop in the N-terminal Gwl was required for Hsp90 association, and a G41S mutant form of Gwl was deficient in Hsp90 association. Proteomic identification of Gwl-associated proteins also revealed its association with Cdc37, the co-chaperone of Hsp90 involved in recognition of some substrates. Initially identified in budding yeast as a cell division cycle regulator, Cdc37 has been shown to interact with Cdk4, Cdk5, and several other cell cycle factors. Consistently, this study found that G41S Gwl lost the association with Cdc37, confirming the involvement of the glycine-rich loop. Importantly, inhibition of Hsp90 reduced the protein stability of Gwl and suppressed M-phase entry, suggesting that complexing to the Hsp90/Cdc37 chaperone is essential for Gwl function. The study therefore revealed Gwl as a key client protein through which Hsp90/Cdc37 chaperone regulates cell division (Yamamoto, 2014).

This new discovery contributes to better understanding of cell cycle regulation, and yields potential implication to cancer therapy. In fact, Hsp90 is frequently overexpressed in cancer cells, whereas its inhibition exhibits anti-proliferative potentials and is being explored in multiple clinical trials for treatment of various types of cancer. The proper function of cellular enzymes often involves sophisticated regulation of subcellular localization, in addition to enzymatic activity and protein stability. For instance, cyclin B/Cdk1 is imported into the nucleus before nuclear membrane breaks down to promote mitotic entry. Similarly, dynamic subcellular localization of Plk1 and Aurora A has been shown to regulate their functions during mitotic progression. One mechanism to regulate the nuclear localization of a protein is through association with the importin family members. Importin is typically composed of 2 subunits, importin α and importin β. For nuclear transport, importin β either directly binds and transports cargo proteins, or is adapted to the cargo proteins through importin α. This study reports that Gwl associates with both importin α and Importin β, suggesting that the nuclear localization of Gwl is mediated by importin. In cultured Xenopus cells, this study showed that Gwl localized to the nucleus in interphase, and was diffused into the cytoplasm in mitosis. This detailed analysis identified a functional NLS around KR456/457. Mutation of the NLS abolished the association of Gwl with importin α and Importin β, and led to Gwl nuclear exclusion and cytoplasmic sequestration (Yamamoto, 2014).

Independent studies in Drosophila and mammalian cells also characterized the nuclear localization of Gwl. The Drosophila homolog of Gwl contains 2 essential NLS motifs within the central region, mutation of which disrupted its nuclear location. However, neither of these NLS motifs in Drosophila is conserved in vertebrates. The NLS of human Gwl was found around KR444/445, the corresponding residues of KR456/457 in Xenopus Gwl. Thus, the NLS of Xenopus Gwl, as identified in this study, represents a well-conserved mechanism in vertebrate animals (Yamamoto, 2014).

Studies in Drosophila and human cells suggested a pre-mitotic transport of Gwl from nucleus to cytoplasm. It was also observed in Xenopus cells that nuclear Gwl started to diffuse into cytoplasm before centrosomes reached opposite positions. While it remained unclear how this nucleocytoplasmic translocation of Gwl may contribute to the regulation of mitotic entry, previous studies indicated that the translocation was dependent on Gwl phosphorylation by CDK1, and possibly also Plk1 (Yamamoto, 2014).

It was also suggested that the kinase activity of Gwl was required for its nuclear export as a kinase-dead form of Gwl harboring a mutation at the ATP-binding site was deficient in nuclear export. However, a different form of kinase-dead Gwl used in this study exhibited a similar pattern of nucleocytoplasmic translocation as the WT. Notably, this mutant form of Gwl was efficiently phosphorylated in mitosis, as judged by the retarded gel migration. Furthermore, it was observed that Gwl was reconcentrated into the daughter nuclei in telophase, seemingly before mitotic chromosomes are fully decondensed. By comparison, the NLS-deficient form of Gwl remained diffused in the cytoplasm during mitotic exit (Yamamoto, 2014).

An important lesson learned from previous studies on Cdk1/cyclin B, Plk1, Aurora kinases, and other mitotic regulators was that their subcellular localization often constitutes an essential mechanism to ensure the proper function of these factors. Regulation of Gwl nuclear localization is functionally important as the NLS-deficient Gwl was unable to fully rescue the mitotic defects caused by Gwl-depletion in both Drosophila and mammalian cells (Yamamoto, 2014).

To shed new light on the role of Gwl nuclear localization, whether the NLS is required for the biochemical function of Gwl was investigated. It has been shown that Gwl promotes mitotic entry through phosphorylation of Ensa/Arpp-19, which subsequently inhibit PP2A/B55δ. Interestingly, the results showed that the NLS-deficient Gwl failed to promote mitotic entry in extracts supplemented with sperm nuclei, despite that this mutant possesses intact and functional kinase domains and promoted mitotic entry in extracts devoid of nucleus formation. Notably, a number of previous studies also characterized the nuclear import of cyclin B1 and Cdc25C in Xenopus egg extracts (Yamamoto, 2014).

These studies argue that even though Xenopus egg extracts can biochemically undergo cell cycle progression in the absence of nuclei, nucleocytoplasmic regulation of the cell cycle is recapitulated in this system when sperm nuclei are reconstituted. As another example, although Xenopus egg extracts support DNA replication without nuclei, it has been shown that the NLS of cyclin E is required for DNA replication in Xenopus egg extracts with the supplementation of sperm nuclei. Moreover, the nuclear import of cyclin B1 is required for mitotic entry in Xenopus oocytes, despite that enucleated Xenopus oocytes still exhibit mitotic activation of Cdk1 (Yamamoto, 2014).

While future analyses are required to reveal how the nuclear localization of Gwl contributes to its biochemical function, a number of possibilities are proposed. First, Gwl may phosphorylate its substrates in the nucleus to promote mitotic entry. Alternatively, Gwl may be initially activated in the nucleus prior to its nuclear export during the early stage of mitotic entry. The latter possibility may be attributed to the nuclear localization of its activating signal, such as MPF activity, or a lower level of inhibitory activity in the nucleus, such as phosphatases that target Gwl. Finally, Gwl negatively regulates the DNA damage checkpoint in Xenopus egg extracts. It is thus possible that the nuclear localization of Gwl contributes to mitotic entry by suppressing an inhibitory signal, such as the cell cycle checkpoint governing DNA damage or incomplete DNA replication (Yamamoto, 2014).

Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila

Mutations in the Drosophila gene greatwall cause improper chromosome condensation and delay cell cycle progression in larval neuroblasts. Chromosomes are highly undercondensed, particularly in the euchromatin, but nevertheless contain phosphorylated histone H3, condensin, and topoisomerase II. Cells take much longer to transit the period of chromosome condensation from late G2 through nuclear envelope breakdown. Mutant cells are also subsequently delayed at metaphase, due to spindle checkpoint activity. These mutant phenotypes are not caused by spindle aberrations, by global defects in chromosome replication, or by activation of a caffeine-sensitive checkpoint. The Greatwall proteins in insects and vertebrates are located in the nucleus and belong to the AGC family of serine/threonine protein kinases; the kinase domain of Greatwall is interrupted by a long stretch of unrelated amino acids (Yu, 2004; full text of article).

Two bistable switches govern M phase entry

The abrupt and irreversible transition from interphase to M phase is essential to separate DNA replication from chromosome segregation. This transition requires the switch-like phosphorylation of hundreds of proteins by the cyclin-dependent kinase 1 (Cdk1):cyclin B (CycB) complex (see Drosophila Cdk1). Previous studies have ascribed these switch-like phosphorylations to the auto-activation of Cdk1:CycB through the removal of inhibitory phosphorylations on Cdk1-Tyr15. The positive feedback in Cdk1 activation creates a bistable switch that makes mitotic commitment irreversible. Cdk1 auto-activation was found to be dispensable for irreversible, switch-like mitotic entry due to a second mechanism, whereby Cdk1:CycB inhibits its counteracting phosphatase (PP2A:B55). This study shows, in Xenopus egg extracts, that the PP2A:B55-inhibiting Greatwall (Gwl)-endosulfine (ENSA) pathway (see Drosophila Greatwall) is both necessary and sufficient for switch-like phosphorylations of mitotic substrates. Using purified components of the Gwl-ENSA pathway in a reconstituted system, a sharp Cdk1 threshold was found for phosphorylation of a luminescent mitotic substrate. The Cdk1 threshold to induce mitotic phosphorylation is distinctly higher than the Cdk1 threshold required to maintain these phosphorylations-evidence for bistability. A combination of mathematical modeling and biochemical reconstitution show that the bistable behavior of the Gwl-ENSA pathway emerges from its mutual antagonism with PP2A:B55. These results demonstrate that two interlinked bistable mechanisms provide a robust solution for irreversible and switch-like mitotic entry (Mochida, 2016).

Search PubMed for articles about Drosophila Greatwall

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 ID: 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(11): e200. PubMed ID: 17997611

Bettencourt-Dias, M., et al. (2004). Genome-wide survey of protein kinases required for cell cycle progression. Nature 432: 980-987. PubMed ID: 15616552

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 ID: 19793917

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

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 ID: 18056802

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

Hall, J. C. (1972). Chromosome segregation influenced by two alleles of the meiotic mutant c(3)G in Drosophila melanogaster. Genetics 71: 367-400. PubMed ID: 4624918

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 ID: 22649080

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 ID: 18663356

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 ID: 21164013

Mochida, S., Rata, S., Hino, H., Nagai, T. and Novak, B. (2016). Two bistable switches govern M phase entry. Curr Biol 26(24): 3361-3367. PubMed ID: 27889260

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

Oshimori, N., Ohsugi, M. and Yamamoto, T. (2006). The Plk1 target Kizuna stabilizes mitotic centrosomes to ensure spindle bipolarity. Nat. Cell Biol. 8: 1095-1101. PubMed ID: 16980960

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 ID: 21852956

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 ID: 18927152

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

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 ID: 21852958

White-Cooper, H., Carmena, M., Gonzalez, C. and Glover, D. M. (1996). Mutations in new cell cycle genes that fail to complement a multiply mutant third chromosome of Drosophila. Genetics 144: 1097-1111. PubMed ID: 8913753

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

Xiang, Y, et al. (2007) The inhibition of polo kinase by matrimony facilitates G2 arrest in the meiotic cell cycle. PLoS Biol. (12): e323. PubMed ID: 18052611

Yamamoto, T. M., Wang, L., Fisher, L. A., Eckerdt, F. D. and Peng, A. (2014). Regulation of Greatwall kinase by protein stabilization and nuclear localization. Cell Cycle 13(22): 3565-3575. PubMed ID: 25483093

Yu, J., et al. (2004). Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila. J. Cell Biol. 164(4): 487-92. PubMed ID: 14970188

Yu, J., Zhao, Y., Li, Z., Galas, S. and Goldberg. M. L. (2006). Greatwall kinase participates in the Cdc2 autoregulatory loop in Xenopus egg extracts. Mol. Cell 22(1): 83-91. PubMed ID: 16600872

Zhao, Y., Haccard, O., Wang, R., Yu, J., Kuang, J., Jessus, C. and Goldberg, M. L. (2008). Roles of greatwall kinase in the regulation of cdc25 phosphatase. Mol. Biol. Cell 19(4): 1317-27. PubMed ID: 18199678

date revised: 15 April 2020

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