Greatwall maintains mitosis through regulation of PP2A
2009; Springer Nature; Volume: 28; Issue: 18 Linguagem: Inglês
10.1038/emboj.2009.228
ISSN1460-2075
AutoresSuzanne Vigneron, Estelle Brioudes, Andrew Burgess, Jean‐Claude Labbé, Thierry Lorca, Anna Castro,
Tópico(s)Autophagy in Disease and Therapy
ResumoArticle13 August 2009free access Greatwall maintains mitosis through regulation of PP2A Suzanne Vigneron Suzanne Vigneron Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Montpellier, France Search for more papers by this author Estelle Brioudes Estelle Brioudes Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Montpellier, France Search for more papers by this author Andrew Burgess Andrew Burgess Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Montpellier, France Search for more papers by this author Jean-Claude Labbé Jean-Claude Labbé Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Montpellier, France Search for more papers by this author Thierry Lorca Corresponding Author Thierry Lorca Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Montpellier, France Search for more papers by this author Anna Castro Corresponding Author Anna Castro Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Montpellier, France Search for more papers by this author Suzanne Vigneron Suzanne Vigneron Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Montpellier, France Search for more papers by this author Estelle Brioudes Estelle Brioudes Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Montpellier, France Search for more papers by this author Andrew Burgess Andrew Burgess Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Montpellier, France Search for more papers by this author Jean-Claude Labbé Jean-Claude Labbé Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Montpellier, France Search for more papers by this author Thierry Lorca Corresponding Author Thierry Lorca Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Montpellier, France Search for more papers by this author Anna Castro Corresponding Author Anna Castro Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Montpellier, France Search for more papers by this author Author Information Suzanne Vigneron1, Estelle Brioudes1, Andrew Burgess1, Jean-Claude Labbé1, Thierry Lorca 1,‡ and Anna Castro 1,‡ 1Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, Montpellier, France ‡These authors contributed equally to this work *Corresponding authors. Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, Labellisée Ligue Nationale Contre le Cancer, Universités Montpellier 2 et 1, 1919 Route de Mende, 34293 Montpellier cedex 5, France. Tel.: +33 4 6761 3330; Fax: +33 4 6752 1559; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2009)28:2786-2793https://doi.org/10.1038/emboj.2009.228 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Greatwall (GW) is a new kinase that has an important function in the activation and the maintenance of cyclin B–Cdc2 activity. Although the mechanism by which it induces this effect is unknown, it has been suggested that GW could maintain cyclin B–Cdc2 activity by regulating its activation loop. Using Xenopus egg extracts, we show that GW depletion promotes mitotic exit, even in the presence of a high cyclin B–Cdc2 activity by inducing dephosphorylation of mitotic substrates. These results indicate that GW does not maintain the mitotic state by regulating the cyclin B–Cdc2 activation loop but by regulating a phosphatase. This phosphatase is PP2A; we show that (1) PP2A binds GW, (2) the inhibition or the specific depletion of this phosphatase from mitotic extracts rescues the phenotype induced by GW inactivation and (3) the PP2A-dependent dephosphorylation of cyclin B–Cdc2 substrates is increased in GW-depleted Xenopus egg extracts. These results suggest that mitotic entry and maintenance is not only mediated by the activation of cyclin B–Cdc2 but also by the regulation of PP2A by GW. Introduction The entry into mitosis is driven by the activation of the cell-cycle kinase cyclin B–Cdc2 or MPF. MPF activity oscillates through the cell cycle, peaking at mitosis and dropping during interphase. The primary event controlling MPF activation is the binding of Cdc2 to cyclin B. The expression of cyclin B is restricted to late S and G2 phases and thus, the formation of the complex can only take place during this phase of the cell cycle (Pines and Hunter, 1989; Nurse, 1990). After cyclin B–Cdc2 association, which only yields a partially active complex, the CAK kinase phosphorylates Cdc2 at thr 161. This phosphorylation induces a change in the T loop of Cdc2, making the catalytic cleft fully accessible to ATP (Russo et al, 1996; Draetta, 1997; Fesquet et al, 1997). Finally, Cdc2 is regulated by phosphorylation at thr 14 and tyr 15, which involves a balance of the inhibitory kinases Myt1/Wee1 and the activatory phosphatase Cdc25. Myt1/Wee1 phosphorylate Cdc2 at residues thr 14 and tyr 15 during G2, whereas Cdc25 reverses these inhibitory phosphorylations at mitotic entry (Morgan, 1997). This model proposes that after thr 161 phosphorylation, cyclin B–Cdc2 complexes are held in an inactive state by phosphorylation at Thr 14 and Tyr 15 by Myt1 and Wee1. At the end of the G2 phase, the MPF feedback loop is activated by the abrupt dephosphorylation of these residues by Cdc25. This dephosphorylation promotes an initial activation of cyclin B–Cdc2, which in turns activates Cdc25 and inactivates Wee1 and Myt1 by phosphorylation, resulting in full activation of the cyclin B–Cdc2 complex (Perdiguero and Nebreda, 2004; Perry and Kornbluth, 2007). Apart from CAK, Cdc25, Myt1 and Wee1, a new MPF regulator, GW, has been described. The depletion of GW from metaphase II-arrested Xenopus egg extracts (CSF extracts) induces mitotic exit, whereas the same depletion prevents mitotic entry in cycling extracts. GW kinase has an important function in both, the activation and the maintenance of cyclin B–Cdc2 activity, however, the mechanism by which it regulates this complex is completely unknown (Yu et al, 2006; Zhao et al, 2008). Results Greatwall maintains the mitotic state independently of MPF activity by inhibiting dephosphorylation The removal of GW in mitosis induces the inactivation of MPF concomitantly with phosphorylation of Cdc2 at tyr 15, indicating that it could regulate the MPF feedback loop. To characterize the mechanism by which GW regulates cyclin B–Cdc2 kinase activity, we used CSF extracts depleted of GW alone or co-depleted of Wee1 or Myt1 and GW, and analysed the state of DNA condensation, the phosphorylation of Erp1/Emi2, Cdc27, Cdc25, and tyr 15 of Cdc2 and the activity of cyclin B–Cdc2. Our antibodies efficiently depleted the corresponding proteins from the extracts (Supplementary Figure S1). Moreover, as previously described, GW depletion induced the MPF inactivation, as reflected by dephosphorylation of Erp1/Emi2, Cdc27 and Cdc25; rephosphorylation on tyr 15 of Cdc2 and a decondensation of the DNA (Figure 1A, left panels). This corresponds to a specific effect of GW removal, as the observed phenotype is clearly rescued by the addition of a recombinant wild-type form of GW, but not by a kinase-dead version (Supplementary data, Figure S2A). As tyr 5 of Cdc2 was phosphorylated in the absence of GW, we expected that the co-depletion of Wee1 or Myt1 and GW would reverse this phenotype. However, Wee1/GW co-depletion neither reversed the phosphorylation of tyr 15 nor prevented the MPF inactivation (Figure 1A, right panels), although, a slight delay of this inactivation was observed (compare Figure 1A, H1K, times 0 of CT+ΔGW and ΔWee1+ΔGW). Moreover, all the analysed MPF substrates were dephosphorylated and DNA decondensed under these conditions. From these results, we conclude that Wee1 is not the main target of GW. We next investigated whether GW could maintain MPF activity by inhibiting Myt1 kinase. To test this hypothesis, we co-depleted Myt1 kinase and GW from CSF extracts. Similar results were observed (Figure 1B), therefore, Myt1 is also not the main target of GW. Next, we asked whether this kinase could regulate phosphorylation of tyr 15 of Cdc2 by inhibiting both kinases, Wee1 and Myt1, or by activating Cdc25 phosphatase. To analyse this hypothesis, we co-depleted Wee1 and Myt1 in CSF extracts before GW depletion. When Wee1 and Myt1 co-depletion was followed by the depletion with control antibodies, CSF extracts remained in mitosis, but the triple depletion of Myt1, Wee1 and GW still induced mitotic exit (Figure 2A). However, interestingly, due to the double removal of Myt1 and Wee1, we no longer observed any inhibitory phosphorylation of Cdc2 on tyr 15 and the cyclin B–Cdc2 kinase activity remained high (Figure 2A, left panels). We obtained the same results when Myt1, Wee1, Cdc25 and GW were depleted from the CSF extracts (data not shown). Thus, surprisingly, extracts still exited mitosis in the presence of a high cyclin B–Cdc2 activity. We conclude that GW preserves the mitotic state by a new unknown mechanism that is independent of cyclin B–Cdc2 activity. Moreover, this new mechanism seems to be very rapid, as Cdc27 and Erp1/Emi2 are dephosphorylated immediately after GW depletion. To measure the kinetics of this dephosphorylation, we developed a time-course analysis in which anti-GW antibodies bound to Dynabeads were added to the extract after control or after Myt1–Wee1 co-depletions. Samples were removed at the indicated time points after addition of anti-GW antibodies. The depletion of GW induced a rephosphorylation of Cdc2 on tyr 15 and a decrease of cyclin B–Cdc2 activity (Figure 2B), due to Cdc25 and Wee1 dephosphorylation 5 min after antibody addition (Figure 2C). As expected, prior co-depletion of Myt1 and Wee1 prevented the rephosphorylation of Cdc2 on tyr 15 as well as the decrease of MPF activity. However, dephosphorylation of the different MPF-dependent substrates (Erp1/Emi2, Cdc27 and MAPK) was observed as early as 5 min after antibody addition in both conditions. Figure 1.Co-depletion of GW with Wee1 or Myt1 does not prevent mitotic exit. (A) CSF extracts were co-depleted with control (CT) or anti-Wee1 (ΔWee1) and anti-GW (ΔGW) antibodies. Phosphorylation of the indicated proteins was analysed by western blot. Cyclin B–Cdc2 activity was measured by H1 histone phosphorylation assay (H1K). Finally, chromatin condensation was visualized by light microscopy. Asterisks denote non-specific bands of anti-pTyr 15 antibody. (B) Similar to (A) except that the depletion of Myt1 (ΔMyt1) was studied instead that of Wee1 before GW immunoprecipitation. Bar, 5 μm. Download figure Download PowerPoint Figure 2.GW depletion induces mitotic exit in CSF extracts in the presence of high cyclin B–Cdc2 activity. (A) A triple depletion with Myt1–Wee1–GW antibodies or Myt1–Wee1–Control antibodies were carried out in CSF extracts and the phosphorylation of the indicated proteins, as well as the cyclin B–Cdc2 activity and chromatin condensation were analysed. (B) CSF extracts were immunoprecipitated twice with control antibodies or with anti-Myt1 and anti-Wee1 antibodies. Subsequently, anti-GW antibody-bound Dynabeads were added to the supernatants and samples were removed at the indicated times. * Time-point 0 min in GW immunodepletions of Figure 1 corresponds to time-point 15 min of this figure. (C) Supernatants of GW, Myt1–Wee1 or Myt1–Wee1–GW immunoprecipitates were used to analyse the phosphorylation of the indicated proteins using western blot. Bar 5 μm. Download figure Download PowerPoint These results show that GW maintains phosphorylation of, at least, four different MPF-dependent substrates, that is, Cdc25, Cdc27, MAPK and Erp1/Emi2. To investigate if this protection against phosphorylation is a general response, we analysed the phosphorylation state of four different proteins (Rsk2, Wee1, Cdc25 and Cdc20) whose phosphorylation depends directly or indirectly on cyclin B–Cdc2 activity during mitosis. Most of the analysed proteins (Rsk2, Wee1 and Cdc25 shown in Figure 2C and Cdc27, MAPK and Erp1/Emi2 in Figure 2B) were dephosphorylated after GW depletion. However, this is not the result of a non-specific dephosphorylation, as cyclin B2, Cdc20 (Figure 2C) and Cdc2 (see phospho tyr 15, Figure 2B) conserved their phosphorylation states under these conditions. Finally, we analysed the general cyclin B–Cdc2-dependent phosphorylation state in these extracts by using an antibody directed against the phosphorylated serine of the Cdk consensus motif. As shown in Figure 2C (right panel), a strong signal, corresponding to phosphorylated MPF substrates, was present in CSF as well as Myt1–Wee1 co-depleted extracts, however, this signal decreased markedly in both Myt1–Wee1–GW co-depleted extracts and in interphase extracts. Thus, GW keeps the mitotic state by maintaining phosphorylation of the majority of MPF substrates, although some of them are not subjected to this regulation. The PP1/PP2A inhibitors, microcystin and okadaic acid, rescue the phenotype induced by Greatwall inactivation in CSF extracts The general and rapid dephosphorylation induced by GW removal in CSF extracts suggests that this kinase could act as a phosphatase inhibitor. It has recently been shown that the phosphatase, calcineurin, is required to release CSF extracts from meiotic M phase (Mochida and Hunt, 2007; Nishiyama et al, 2007). In addition, our results show that the overexpression of GW in CSF extracts delays mitotic exit induced by calcium (Supplementary Figure S2B). Thus, one putative target of GW could be calcineurin. We tested the role of calcineurin in this pathway by using the specific inhibitor cyclosporin. However, the inhibition of this phosphatase did not affect the exit of mitosis induced by GW removal, although, as described, it clearly delayed the dephosphorylation of Cdc27 and cyclin B degradation after Ca2+ addition (Supplementary Figure S3B). Thus, GW does not regulate mitosis through calcineurin inhibition. We next questioned whether GW could inhibit PP1 and/or PP2A, the major phosphatases present in Xenopus egg extracts. To test this hypothesis, we used the potent PP1/PP2A phosphatase inhibitor, microcystin, and we investigated whether it could rescue mitotic exit in GW-depleted CSF extracts. To this end, we first depleted GW from CSF extracts and we subsequently added microcystin. Samples were taken just after GW depletion and 0, 30 and 60 min after microcystin addition. The results are shown in Figure 3A. We observed the first dephosphorylation of the different analysed proteins just after GW depletion, followed by a rephosphorylation of these proteins at 30 min after microcystin addition. Moreover, after GW depletion, we observed a phosphorylation on tyr 15 of Cdc2 that was concomitant with Cdc25 and Wee1 dephosphorylation and with a clear decrease in cyclin B–Cdc2 activity. However, 30 min later, tyr 15 was dephosphorylated again, MPF substrates were re-phosphorylated and cyclin B–Cdc2 complex was reactivated. Thus, microcystin rescues the phenotype induced by GW inactivation. Figure 3.Phosphatase inhibitors, microcystin and okadaic acid (OA), rescue the phenotype induced by GW inactivation in CSF extracts. (A) GW-depleted CSF extracts were supplemented with microcystin (1 μM) and the phosphorylation of the indicated proteins as well as the cyclin B–Cdc2 activity were analysed. (B) CSF extracts were devoid of GW and supplemented with increasing doses of OA (from 0.1 to 0.8 μM). (C) GW-depleted CSF extracts were supplemented with 0.5 or 0.75 μM OA. (D) GW-depleted CSF extracts were supplemented with 0.75 μM OA and subsequently supplemented or not with purified PP2A (Upstate). Download figure Download PowerPoint Microcystin is a potent inhibitor of both PP1 and PP2A (MacKintosh et al, 1990; Rivas et al, 2000). To elucidate which of these two phosphatases could be involved in mitotic exit, we used the phosphatase inhibitor, okadaic acid (OA), specificity of which for PP1 and PP2A, at different doses, has been described (Felix et al, 1990). We tested the dose–response of Cdc25 dephosphorylation on OA in GW-depleted CSF extracts, to analyse at what dose this inhibitor was capable to reverse Cdc25 dephosphorylation. As shown in Figure 3B, we observed a complete rephosphorylation of Cdc25, 30 min after GW depletion due to a 600-nM dose of OA. We next tried to determine, more accurately, the minimal dose that was capable of rescuing the GW phenotype. A total of 500 nM OA was sufficient to reverse the dephosphorylation of Cdc27, Cdc25, Wee1 and MAPK, although phosphorylation of the latter was only observed at 60 min probably due to the fact that it is induced indirectly by MPF-dependent phosphorylation of c-Mos (Figure 3C). At this dose, we also observed a dephosphorylation of Cdc2 at tyr 15 and an increase in cyclin B–Cdc2 activity at 20 min after the addition of the drug. As 500 nM of OA has been reported to inhibit 70% of PP2A activity and only 20% of PP1, it is likely that PP2A, rather than PP1, could be involved in the reversion of mitotic exit induced by GW depletion. In agreement with this hypothesis, the addition of the PP1 inhibitor, Inhibitor 2, was not able to reverse GW phenotype in CSF extracts (Supplementary Figure S4). Moreover, we observed a reversion of the effect of OA in GW-depleted CSF extracts when an active form of PP2A phosphatase was added after this phosphatase inhibitor (Figure 3D). GW binds PP2A in human cells and in CSF extracts The results presented above suggest that GW maintains the mitotic state by regulating PP2A activity, suggesting that GW could bind PP2A. To investigate whether GW could associate with PP2A, we co-transfected YFP-tagged GW, and non-tagged PP2A/A and /C subunits in HEK293 cells and we subsequently immunoprecipitated cell lysate with either an anti-YFP or a control antibody. As shown in Figure 4A, both PP2A/A and C subunits were present in the immunoprecipitate when anti-YFP antibody, but not a control antibody, was used. We next co-transfected HEK293 cells with a non-tagged PP2A/A subunit and with either HA-tagged or non-tagged PP2A/C subunit, and the cell lysates were then immunoprecipitated with anti-HA antibodies. The results show that endogenous GW was present in the immunoprecipitate when HA-tagged PP2A/C, but not non-tagged PP2A/C, was used in co-transfection (Figure 4B). Finally, we analysed whether GW and PP2A/A could associate when co-transfected in the absence of PP2A/C overexpression. Under these conditions, we did not observed any association of these two proteins, indicating that PP2A/C subunit is required to mediate GW binding with PP2A (Figure 4C). Thus, GW, PP2A/A and PP2A/C interact in HEK293 cells and this interaction is dependent on the PP2A/C subunit. Figure 4.PP2A binds GW in human cells and CSF extracts. (A) HEK293 cells were co-transfected with YFP-GW, PP2A/A subunit and PP2A/C subunit. Cells were then lysed and immunoprecipitated with anti-GFP antibodies or with control antibodies. The presence of GW, PP2A/A and PP2A/C in 40 ng of total protein of the input and the supernatant, as well as the IP corresponding to 500 μg of total protein were analysed by SDS–PAGE and western blot. (B) HEK293 cells were co-transfected with PP2A/A and either PP2A/C or HA-PP2A/C and immunoprecipitated with anti-HA antibodies. The presence of GW, PP2A/A and PP2A/C was then analysed in the inputs, the supernatants and the Ips. (C) HEK293 cells were co-transfected with YFP-GW and PP2A/A subunit, lysed and immunoprecipitated with anti-GFP or control antibodies as described in Material and methods section. The presence of GW, PP2A/A and PP2A/C was then analysed in the inputs and supernatants by SDS–PAGE and western blot. (D) A total of 50 μl CSF extracts were immunoprecipitated with anti-PP2A/A monoclonal antibodies (6F9) or control antibodies, and the immunoprecipitates as well as a 1.5-μl CSF sample were used to analyse the presence of GW by immunoblotting. The smeared bands present in control IP between 94 and 67 kDa correspond to immunoglobulins in which the heavy and light chains have not been correctly dissociated after boiling. The same amount of CSF extracts were used to immunoprecipitate GW with anti-GW or control antibodies, and the immunoprecipitates as well as a 1.5-μl CSF sample were treated as described above to analyse the presence of PP2A/C. Download figure Download PowerPoint We next analysed whether endogenous GW and PP2A could bind in CSF extracts. To this end, we immunoprecipitated PP2A from CSF extracts by using a monoclonal antibody against the PP2A/A subunit (Kremmer et al, 1997). The results show that GW is clearly present in this immunoprecipitate (Figure 4D, left panel). We next carried out the reverse immunoprecipitation by using anti-GW antibodies. As depicted in Figure 4D (right panel), PP2A was present in the GW IP and completely absent when control antibodies were used, however, unlike the high amount of GW observed in PP2A IP, only a small quantity of PP2A was detected in the GW IP, suggesting that GW does not bind to all PP2A complexes, but probably to one particular sub-complex of this phosphatase. GW maintains the mitotic state by promoting PP2A inhibition The results above show that GW binds PP2A and that the inhibition of this phosphatase rescues the phenotype induced by GW inactivation. To further investigate whether PP2A is the target of GW, we removed this phosphatase from CSF extracts before GW depletion by using a monoclonal antibody directed against PP2A/A subunit (Kremmer et al, 1997). Samples of the PP2A-depleted extracts were taken at the indicated times and used to analyse the phosphorylation of Cdc27, cyclin B2 and tyr 15 of Cdc2 and to measure cyclin B–Cdc2 kinase activity. As shown in Figure 5A (upper panel), 61% of PP2A/A and 83% of PP2A/C were depleted from these extracts. Moreover, this removal clearly prevented the dephosphorylation of Cdc27, as well as phosphorylation of tyr 15 of Cdc2 and cyclin B–Cdc2 inactivation although a small decrease in cyclin B–Cdc2 activity was observed 1 h after GW depletion, probably due to the PP2A left in these extracts (lower panel). Similar results were obtained when PP2A was removed from the extracts by using microcystin–agarose beads (Supplementary Figure S5A). Thus, PP2A depletion rescues the phenotype induced by GW inactivation in CSF extracts. Figure 5.GW maintains the mitotic state by promoting PP2A inhibition. (A) CSF extract was incubated with anti-PP2A/A monoclonal antibodies bound to protein G–Sepharose beads. Three runs of immunodepletion were carried out to remove PP2A. The last supernatant was then depleted of GW by a subsequent immunoprecipitation and used to analyse the phophorylation of Cdc27, Cdc2 and cyclin B2 and to measure cyclin B–Cdc2 activity. The levels of PP2A/A and C were also examined in the three supernatants recovered after PP2A/A immunoprecipitation. (B) Radiolabelled p-mal-cMos was incubated with a PP2A complex obtained from CSF (PP2A CSF) or GW-depleted CSF extracts (PP2A Δ GW). After 1-h incubation, the supernatants were submitted to SDS–PAGE, stained with Coomassie Blue and the phosphorylation of p-mal-cMos revealed by autoradiography. One-tenth of the PP2A immunoprecipitates from CSF (IP PP2A CSF) and GW-depleted CSF extracts (IP PP2A ΔGW) were used to measure the amount of PP2A/C immunoprecipitated in each condition by western blotting. Coomassie Blue staining showing the levels of phosphorylated p-mal-cMos, as well as a scan of this gel using Typhoon Scanner, from the input (10 μl p-mal-cMosp33) and the supernatant of the dephosphorylation reactions with PP2A from CSF (PP2A CSF) and GW-depleted CSF extracts (PP2A ΔGW) are shown. (C) A procedure similar to that followed in (B) except that supernatants were taken at 0, 20, 40, 60 and 80 min of incubation. The gels were scanned using a Typhoon Scanner and quantified by using ImageQuant TL software. Statistical analysis of the results, obtained from three different independent experiments, was performed using unpaired Student's t test. The amounts of phosphorylated p-mal-cMos present at each time were expressed as mean±s.e.m. Statistical difference in the last time point is indicated by an asterisk (*) P<0.0212. Download figure Download PowerPoint Finally, we analysed whether GW modulates the PP2A-dependent dephosphorylation of cyclin B–Cdc2 substrates during mitosis. With this aim, a p-mal-tagged form of the cyclin B–Cdc2 substrate, c-Mos (Castro et al, 2001b), was purified and used as a substrate for PP2A. p-mal-tagged cMos protein was first phosphorylated in the presence of ATPγ33 by a cyclin B–Cdc2 complex immunoprecipitated from CSF extracts. A sample of radiolabelled p-mal-cMosp33 was then incubated with a PP2A complex obtained by immunoprecipitation from either CT or GW-depleted CSF extracts. After 1-h incubation, the level of p-mal-cMos phosphorylation was analysed. The results show that despite the fact that similar amounts of PP2A/C were present in immunoprecipitates from control and GW-depleted CSF extracts (Figure 5B, upper panel) and that the same quantity of p-mal-cMosP33 was incubated with both PP2A IPs (Figure 5B, Coomassie blue staining), a higher decrease of the phosphorylation levels of p-mal-cMosp33 was observed when PP2A was obtained from GW-depleted extracts. The quantification of the autoradiography indicates a threefold decrease in the radiolabelled p-mal-cMosP33 signal when PP2A from GW-depleted extracts was used compared with CT (Supplementary Figure S5B). We next repeated this assay activity as a time course by triplicate and we measured c-Mos phosphorylation at 0, 20, 40, 60 and 80 min. The results of these experiments are shown as the mean value plus error bars in Figure 5C. Confirming the results shown above, dephosphorylation of c-Mos was higher when PP2A was obtained from GW-depleted CSF extracts. This difference was observed after 40 min and was statistically significant at 80 min (*P<0.0212). Thus, these results clearly show that dephosphorylation of cyclin B–Cdc2 substrates by PP2A is regulated by GW in mitotic egg extracts. Discussion It is established that, at mitosis entry, cyclin B–Cdc2 is irreversibly activated and that this irreversibility is directly induced by this complex through a feedback loop. Our results clearly show that the irreversibility of cyclin B–Cdc2 activation is not exclusively induced by the MPF feedback loop. We characterize a pathway controlled by the recently identified GW kinase that acts in parallel to MPF feedback loop and is essential for the irreversibility of cyclin B–Cdc2 activation and for the maintaining of the mitotic state. The GW kinase was first identified at Goldberg's laboratory where it was shown that depletion of this protein from CSF extracts induces mitotic exit concomitantly with rephosphorylation of tyr 15 of Cdc2 and cyclin B–Cdc2 inactivation (Yu et al, 2004, 2006; Zhao et al, 2008). From their results, the authors suggested that GW could maintain the mitotic state by controlling cyclin B–Cdc2 feedback loop. Surprisingly, we found that GW inactivation induces mitotic exit by promoting a rapid dephosphorylation of different mitotic substrates independently of cyclin B–Cdc2 activity. Moreover, although we cannot exclude a direct control of GW on the Myt1–Wee1–Cdc25 pathway, we show that even in the absence of this pathway, GW is still required to maintain the mitotic state. In this light, it is likely that cyclin B–Cdc2 inactivation after GW depletion is not the cause of mitotic exit, but the consequence of Cdc25, Wee1 and Myt1 dephosphorylation. Moreover, we show that this phenotype is reversed by the addition of the phosphatase inhibitors, microcystin and OA, and that this reversion is not observed if additional active PP2A phosphatase is further supplemented. We also present data showing that GW binds PP2A in vivo through its PP2A–C subunit although this association is likely restricted to a specific sub-complex of this phosphatase. Finally, we show that the depletion of PP2A completely rescues the phenotype induced by GW inactivation in CSF extracts and that GW depletion results in an increase of the capacity of PP2A to dephosphorylate cyclin B–Cdc2 substrates. Thus, all these results clearly indicate that GW maintains the mitotic state by regulating PP2A. Until now, mitotic entry and exit was equated to cyclin B–Cdc2 activation and inactivation, respectively, and once this kinase was a
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