Embryonic to pupal period

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


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

Effects of Mutation or Deletion

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

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

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

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

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

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

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

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

Twins regulates Armadillo levels in response to Wg/Wnt signal

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

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

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

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

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

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

Multiple protein phosphatases are required for mitosis in Drosophila

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

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

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

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

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

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

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

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

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

Protein phosphatase 2A (PP2A) plays a major role in dephosphorylating the targets of the major mitotic kinase Cdk1 at mitotic exit, yet how it is regulated in mitotic progression is poorly understood. This study shows that mutations in either the catalytic or regulatory twins/B55 subunit of PP2A act as enhancers of gwlScant, 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 gwlScant; many more embryos survive. Furthermore, heterozygous PP2A mutations make females heterozygous for the strong mutation polo11 partially sterile, even in the absence of gwlScant. 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 gwlScant 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 gwlScant. 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 gwlScant 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 gwlScant 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 gwlScant 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 gwlScant 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 gwlScant 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 gwlScant/+ 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 gwlScant (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 gwlScant 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).


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Varadkar, P., Abbasi, F., Takeda, K., Dyson, J. J. and McCright, B. (2017). PP2A-B56gamma is required for an efficient spindle assembly checkpoint. Cell Cycle 16(12): 1210-1219. PubMed ID: 28562161

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

date revised: 12 January 2018

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