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PP1 initiates the dephosphorylation of MASTL, triggering mitotic exit and bistability in human cells
Abstract
Entry into mitosis is driven by the phosphorylation of thousands of substrates, under the master control of Cdk1. During entry into mitosis, Cdk1, in collaboration with MASTL kinase, represses the activity of the major mitotic protein phosphatases, PP1 and PP2A, thereby ensuring mitotic substrates remain phosphorylated. For cells to complete and exit mitosis, these phosphorylation events must be removed, and hence, phosphatase activity must be reactivated. This reactivation of phosphatase activity presumably requires the inhibition of MASTL, however, it is not currently understood how or what deactivates MASTL. In this study, we identified that PP1 is associated with and capable of partially dephosphorylating and deactivating MASTL during mitotic exit. Using mathematical modelling we were able to confirm that deactivation of MASTL is essential for mitotic exit. Furthermore, small decreases in Cdk1 activity during metaphase are sufficient to initiate the reactivation of PP1, which in turn partially deactivates MASTL to release inhibition of PP2A and hence create a feedback loop. This feedback loop drives complete deactivation of MASTL, ensuring a robust switch-like activation of phosphatase activity during mitotic exit.
Supplementary resource (1)
... PP1 and PP2A are critically important for mitotic and meiotic M-phase progression and exit. In mitosis, PP1 and PP2A have unique temporal and spatial regulation, and work in concert to oppose CDK1 activity, preventing M-phase entry, and promoting metaphase-to-anaphase transition [6][7][8][9]. In mammalian oocytes, inhibition of PP1/PP2A with reagents, such as Okadaic Acid and Calyculin A causes major meiotic abnormalities, and oocyte-specific conditional loss of PP2A causes substantial defects in chromosome segregation and M-phase progression [10][11][12][13][14][15][16][17][18][19]. ...
... Conversely, no change in PP1c(T320) phosphorylation was observed following PP1 activation (Figure 2g; p = 0.9478). These results are consistent with the observation of Rogers et al. of fast and substantial loss of H3T3 phosphorylation in mitotic cells treated with 40 µM of the first-generation PP1 disrupting peptide (PDP3), but only a slow and modest loss of PP1c(T320) phosphorylation [7]. Their findings and ours suggest that distinct PP1 substrates have different sensitivities to dephosphorylation following PP1 activation [7,46]. ...
... These results are consistent with the observation of Rogers et al. of fast and substantial loss of H3T3 phosphorylation in mitotic cells treated with 40 µM of the first-generation PP1 disrupting peptide (PDP3), but only a slow and modest loss of PP1c(T320) phosphorylation [7]. Their findings and ours suggest that distinct PP1 substrates have different sensitivities to dephosphorylation following PP1 activation [7,46]. ...
Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-phase transitions. Protein Phosphatase 1 (PP1) is one of these phosphatases, with oscillations in PP1 activity driving mitotic M-phase. Evidence from a variety of experimental systems also points to roles in meiosis. Here, we report that PP1 is important for M-phase transitions through mouse oocyte meiosis. We employed a unique small-molecule approach to inhibit or activate PP1 at distinct phases of mouse oocyte meiosis. These studies show that temporal control of PP1 activity is essential for the G2/M transition, metaphase I/anaphase I transition, and the formation of a normal metaphase II oocyte. Our data also reveal that inappropriate activation of PP1 is more deleterious at the G2/M transition than at prometaphase I-to-metaphase I, and that an active pool of PP1 during prometaphase is vital for metaphase I/anaphase I transition and metaphase II chromosome alignment. Taken together, these results establish that loss of oscillations in PP1 activity causes a range of severe meiotic defects, pointing to essential roles for PP1 in female fertility, and more broadly, M-phase regulation.
... Degradation of Cyclin B upon transition into anaphase initiates the cascade that activates PP2A-B55. Decreasing CDK1-Cyclin B activity is proposed to render PP1 active to dephosphorylate and partially inactivate GWL [553][554][555][556] (Figure 3A). This means that the phosphorylations that are ultimately removed from ENSA and Arpp19 by PP2A-B55 now fail to be efficiently replaced, hence allowing PP2A-B55 to target CDK1-phosphorylated substrates [552]. ...
... This means that the phosphorylations that are ultimately removed from ENSA and Arpp19 by PP2A-B55 now fail to be efficiently replaced, hence allowing PP2A-B55 to target CDK1-phosphorylated substrates [552]. In that respect, PP2A-B55 further dephosphorylates GWL to ensure its complete inactivation and thereby maintain elevated PP2A-B55 activity to promote mitotic exit [553][554][555][556] (Figure 3A). ...
... Notably, both phosphorylations are targeted by PP1 itself. Thus, declining CDK1-Cyclin B activity following SAC silencing is thought to be sufficient to allow PP1 autodephosphorylation and I-1 dephosphorylation, thereby resulting in increased activation of PP1 to levels that drive mitotic exit [553][554][555][556]560]. This sequential activation of PP1 and PP2A-B55 occurring in animal cells resembles a phosphatase relay reported in fission yeast, which also operates as a timer for the orderly dephosphorylation of mitotic substrates at the end of division [559]. ...
Mitosis requires extensive rearrangement of cellular architecture and of subcellular structures so that replicated chromosomes can bind correctly to spindle microtubules and segregate towards opposite poles. This process originates two new daughter nuclei with equal genetic content and relies on highly-dynamic and tightly regulated phosphorylation of numerous cell cycle proteins. A burst in protein phosphorylation orchestrated by several conserved kinases occurs as cells go into and progress through mitosis. The opposing dephosphorylation events are catalyzed by a small set of protein phosphatases, whose importance for the accuracy of mitosis is becoming increasingly appreciated. This review will focus on the established and emerging roles of mitotic phosphatases, describe their structural and biochemical properties, and discuss recent advances in understanding the regulation of phosphatase activity and function.
... Interestingly, we could further observe that proteolysis of Cyclin B also occurs during prometaphase but at a significant lower rate ( Figure 6G,H and Video 14). It was recently proposed that a small reduction in CDK1/Cyclin B activity is sufficient to allow PP1 auto-reactivation, which consequently triggers a feedback loop that ensures robust phosphatase activity (Grallert et al., 2015;Rogers et al., 2016). Thus, the modest decline in Cyclin B that takes place before metaphase might be critical to permit PP1-mediated inactivation of Mps1 and SAC silencing. ...
... However, several lines of evidence indicate that the SAC is switched off under condition of high CDK1/Cyclin B activity (Mirchenko and Uhlmann, 2010;Oliveira et al., 2010;Kamenz and Hauf, 2014;Rattani et al., 2014;Vázquez-Novelle et al., 2014), hence challenging the impact that CDK1/Cyclin B-mediated regulation of PP1 might have on SAC silencing. Interestingly, recent mathematical modelling support that small decreases in CDK1/Cyclin B activity are sufficient to initiate PP1 re-activation of and trigger a positive feedback loop that ensures robust phosphatase activity (Rogers et al., 2016). Therefore, slow Cyclin Figure 6G,H). ...
... Taking our results together with previously reported work (Dohadwala et al., 1994;Yamano et al., 1994;Liu et al., 2010;Mochida and Hunt, 2012;Grallert et al., 2015;Qian et al., 2015;Rogers et al., 2016), we propose a model that controls Mps1 activation in a timely manner and perfectly coordinated with mitotic progression. Elevated CDK1 and Aurora B activities during prometaphase, respectively repress PP1 activity and avert the phosphatase localization at unattached kinetochores. ...
Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.
... This is achieved by a PP1-PP2A relay switch (Grallert et al., 2014), where loss of CDK1 activity is driven by APC cdc20 ubiquitination and destruction of cyclin B. Interestingly, MASTL promotes cyclin B recruitment to the APC/C, which likely helps ensure a robust bistable switch at the metaphase-anaphase transition (Voets and Wolthuis, 2015). Loss of CDK1 activity relieves its inhibitory phosphorylation of T320 on PP1, allowing PP1 to auto-dephosphorylate T320 and subsequently partially dephosphorylate and reduce MASTL activity (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016a). This releases inhibition of PP2A-B55, which in turn begins removing CDK1 phosphorylation events, including those on MASTL (Rogers et al., 2016a) and MPS1 (Diril et al., 2016), thereby creating a positive feedback loop and a bistable mitotic exit switch. ...
... Loss of CDK1 activity relieves its inhibitory phosphorylation of T320 on PP1, allowing PP1 to auto-dephosphorylate T320 and subsequently partially dephosphorylate and reduce MASTL activity (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016a). This releases inhibition of PP2A-B55, which in turn begins removing CDK1 phosphorylation events, including those on MASTL (Rogers et al., 2016a) and MPS1 (Diril et al., 2016), thereby creating a positive feedback loop and a bistable mitotic exit switch. PP2A-B55 and FCP1 then complete the dephosphorylation and deactivation of MASTL during mitotic exit (Hégarat et al., 2014;Monica Della et al., 2015). ...
... Taken together these results highlight the importance of maintaining a tight control over the balance between PP2A-B55 and MASTL to ensure that mitotic phosphorylation and mitotic fidelity is maintained. In support, we showed using mathematical modeling, that MASTL overexpression disrupts PP2A-B55 reactivation timing, delaying mitotic exit (Rogers et al., 2016a;Rogers et al., 2018). ...
MASTL kinase is a master regulator of mitosis, essential for ensuring that mitotic substrate phosphorylation is correctly maintained. It achieves this through the phosphorylation of alpha-endosulfine and subsequent inhibition of the tumor suppressor PP2A-B55 phosphatase. In recent years MASTL has also emerged as a novel oncogenic kinase that is upregulated in a number of cancer types, correlating with chromosome instability and poor patient survival. While the chromosome instability is likely directly linked to MASTL’s control of mitotic phosphorylation, several new studies indicated that MASTL has additional effects outside of mitosis and beyond regulation of PP2A-B55. These include control of normal DNA replication timing, and regulation of AKT/mTOR and Wnt/β-catenin oncogenic kinase signaling. In this review, we will examine the phenotypes and mechanisms for how MASTL, ENSA, and PP2A-B55 deregulation drives tumor progression and metastasis. Finally, we will explore the rationale for the future development of MASTL inhibitors as new cancer therapeutics.
... Based on the "inhibition by unfair competition" model, the key event for activating PP2A-B55 is inactivation of Mastl through dephosphorylation. Removal of Cdk1 sites on Mastl is initiated by PP1, and then once PP2A-B55 is activated, it can also dephosphorylate Mastl (Heim et al., 2015;Ma et al., 2016b;Rogers et al., 2016;Ren et al., 2017). There is some disagreement on which Mastl phosphorylation sites are dephosphorylated by PP1, and it is also unclear if a specific PP1 holoenzyme is responsible because multiple PP1 regulatory subunits have been identified in Mastl purifications (Rogers et al., 2016;Ren et al., 2017). ...
... Removal of Cdk1 sites on Mastl is initiated by PP1, and then once PP2A-B55 is activated, it can also dephosphorylate Mastl (Heim et al., 2015;Ma et al., 2016b;Rogers et al., 2016;Ren et al., 2017). There is some disagreement on which Mastl phosphorylation sites are dephosphorylated by PP1, and it is also unclear if a specific PP1 holoenzyme is responsible because multiple PP1 regulatory subunits have been identified in Mastl purifications (Rogers et al., 2016;Ren et al., 2017). The Fcp1 phosphatase has also been implicated in dephosphorylation of ENSA and Mastl, but given the essential role of Fcp1 in dephosphorylating the RNA polymerase C-terminal domain, these data are difficult to interpret (Visconti et al., 2012;Hégarat et al., 2014;Williams et al., 2014). ...
... At metaphase, when the APC/C-Cdc20 complex is activated and initiates cyclin B1 degradation and thereby Cdk1 inactivation, PP1 autodephosphorylates, leading to its activation (Wu et al., 2009). Modeling suggests that Cdk1 activity has to be reduced by 90% before PP1 gets activated; however, this seems inconsistent with how fast PP2A-B55 is activated and the reported rates of cyclin B1 degradation (Clute and Pines, 1999;Cundell et al., 2013;Rogers et al., 2016). This inconsistency is possibly explained by the fact that the stoichiometry of Thr320 phosphorylation is 60% in prometaphase-arrested cells, which would not be sufficient to fully inhibit PP1 (Olsen et al., 2010). ...
The accurate segregation of genetic material to daughter cells during mitosis depends on the precise coordination and regulation of hundreds of proteins by dynamic phosphorylation. Mitotic kinases are major regulators of protein function, but equally important are protein phosphatases that balance their actions, their coordinated activity being essential for accurate chromosome segregation. Phosphoprotein phosphatases (PPPs) that dephosphorylate phosphoserine and phosphothreonine residues are increasingly understood as essential regulators of mitosis. In contrast to kinases, the lack of a pronounced peptide-binding cleft on the catalytic subunit of PPPs suggests that these enzymes are unlikely to be specific. However, recent exciting insights into how mitotic PPPs recognize specific substrates have revealed that they are as specific as kinases. Furthermore, the activities of PPPs are tightly controlled at many levels to ensure that they are active only at the proper time and place. Here, I will discuss substrate selection and regulation of mitotic PPPs focusing mainly on animal cells and explore how these actions control mitosis, as well as important unanswered questions.
... PP2A-B55, Fcp1, PP2A-B56 or PP1, which are all reported to be involved in Cdc20 dephosphorylation in various contexts (Castilho et al., 2009;Craney et al., 2016;Hein et al., 2017;Kim et al., 2017;Labit et al., 2012;Lee et al., 2017;Mochida et al., 2009;Visconti et al., 2012). However, it is probably not an indirect effect of impaired PP2A-B55 reactivation, because cyclin-B degradation, which sets the timer for it, is only mildly affected by CaN inhibition in some experiments that showed a strong effect on Cdc20 phosphorylation (e.g. Figure 16A) (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016). In addition, it has been reported that meiotic and mitotic exit in Xenopus extracts are generally not affected by depletion of Fcp1, although Cdc20 was not directly probed in this context (Ma et al., 2016). ...
... Our data suggest that PP1 is this phosphatase and we therefore add another substrate to its already wide spectrum (Heim et al., 2015). In the meantime, other reports have confirmed this conclusion for meiosis and mitosis of Xenopus laevis as well as for mitosis of cultured human cells (Ma et al., 2016;Rogers et al., 2016). ...
... The important role of PP1 for Gwl inactivation, which we unveiled in Xenopus laevis, was later shown to be conserved in higher vertebrates. Similar to what we observed in extracts of early Xenopus embryos, it was shown that in cultured human cells PP1 is the critical phosphatase to initiate Gwl inactivation during mitotic exit (Rogers et al., 2016). Notably, dephosphorylation of Ser875 of human Gwl (corresponds to Ser883 of Xenopus Gwl) is not occurring during mitotic exit and PP1 rather seems to dephosphorylate cyclin-B/Cdk1 target sites (McCloy et al., 2015;Rogers et al., 2016). ...
... Obviously, these phosphorylation events need to be tightly modulated by counteracting phosphatases in accordance with the transition of cell cycle stages. In fact, several recent studies suggested that both PP2A and PP1 are involved in the dephosphorylation of Gwl (25)(26)(27)(28)(29)(30). ...
... These events, together, lead to dephosphorylation of mitotic phosphoproteins and reorganization of cellular structures to the interphase state. Given the role of Gwl in regulation of PP2A/B55, it has been suggested that de-activation of Gwl kinase is an essential event of mitotic exit (27,28,30). To delineate the involvement of PP1-dependent dephosphorylation of Gwl Ser-883 in mitotic exit, we investigated the fine kinetics of Ser-883 dephosphorylation in CSF egg extracts that undergo synchronized mitotic exit following the addition of calcium. ...
... In this study, we have shown that PP1, particularly PP1?, bound Gwl, and was responsible for the dephosphorylation of Gwl at its Ser-883 autophosphorylation site. Our results validated and extended the findings of several recent studies concluding that the dephosphorylation and inactivation of Gwl during mitotic exit is mediated by PP1 (27,28,30). In the present study, we were able to generate a Gwl Ser-883 phospho-specific antibody, and use it to demonstrate directly the role of PP1 in Gwl Ser-883 dephosphorylation in Xenopus egg extracts. ...
Greatwall (Gwl) kinase plays an essential role in regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily-conserved serine residue near the carboxyl-terminus (Ser-883 in Xenopus). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phospho-mimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association, and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M-phase. Interestingly, upregulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation.
... At prophase, before nuclear envelope breakdown, activated Gwl will move to the cytoplasm where it will phosphorylate ENSA and inhibit PP2A-B55 (Alvarez-Fernandez et al., Wang et al., 2013;Larouche et al., 2021). However, since as reported below, Gwl is a PP2A-B55 substrate (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016;Ren et al., 2017), this model does not explain how Gwl phosphorylation and activity is maintained in the cytoplasm to induce ENSA phosphorylation in the presence of a fully active phosphatase. Alternatively, ENSA can be partially localized in the nucleus, as previously described for human ENSA (Charrasse et al., 2017), where a pool of this protein would be phosphorylated by Cyclin B/Cdk1-activated Gwl. ...
... Active PP1 induces dephosphorylation of Gwl on Ser875 and decreases phospho-Arpp19-ENSA enabling the activation of a threshold level of PP2A-B55 that will subsequently dephosphorylate Gwl on its Thr194-Thr207 Tloop activatory sites. Gwl will be now completely inactivated and will trigger a negative feedback loop resulting in the overall reactivation of PP2A-B55 and mitotic exit (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016;Ren et al., 2017). Besides PP1 and PP2A-B55, FCP1 also dephosphorylates Gwl on other Cyclin B/Cdk1 phosphosites including Ser90 and S453, however, the exact role of these sites in the control of kinase activity has not been reported (Della Monica et al., 2015). ...
Cells require major physical changes to induce a proper repartition of the DNA. Nuclear envelope breakdown, DNA condensation and spindle formation are promoted at mitotic entry by massive protein phosphorylation and reversed at mitotic exit by the timely and ordered dephosphorylation of mitotic substrates. This phosphorylation results from the balance between the activity of kinases and phosphatases. The role of kinases in the control of mitosis has been largely studied, however, the impact of phosphatases has long been underestimated. Recent data have now established that the regulation of phosphatases is crucial to confer timely and ordered cellular events required for cell division. One major phosphatase involved in this process is the phosphatase holoenzyme PP2A-B55. This review will be focused in the latest structural, biochemical and enzymatic insights provided for PP2A-B55 phosphatase as well as its regulators and mechanisms of action.
... Our next goal was to functionally characterize the candidate substrate proteins from the SILAC screen. To further refine the list of potential substrates, we crossreferenced the phosphorylated proteins with our previously published list of proteins that co-immunoprecipitated (Co-IP) with MASTL from mitotic HeLa cell extracts 55 and MASTL substrates predicted by Hermida et al. 41 . There were 11 proteins that were present in all three datasets, an additional 11 common to our SILAC screen and Hermida's dataset, and a further 15 common to SILAC screen and our previous Co-IP (Fig. 3a, Supplementary Table S4). ...
... For MASTL, substrates were further refined by cross-referencing with previously identified proteins that coimmunoprecipitated with MASTL from mitotic HeLa cell extracts 55 . For the AKT1 + YB1 in vitro kinase assay, two (n = 2) biological replicates were performed, and for MASTL + ENSA and MASTL + hnRNPM in vitro kinase assays, three (n = 3) independent biological replicates were performed. ...
Microtubule-associated serine/threonine kinase-like (MASTL) has emerged as a critical regulator of mitosis and as a potential oncogene in a variety of cancer types. To date, Arpp-19/ENSA are the only known substrates of MASTL. However, with the roles of MASTL expanding and increased interest in development of MASTL inhibitors, it has become critical to determine if there are additional substrates and what the optimal consensus motif for MASTL is. Here we utilized a whole cell lysate in vitro kinase screen combined with stable isotope labelling of amino acids in cell culture (SILAC) to identify potential substrates and the residue preference of MASTL. Using the related AGC kinase family members AKT1/2, the kinase screen identified several known and new substrates highly enriched for the validated consensus motif of AKT. Applying this method to MASTL identified 59 phospho-sites on 67 proteins that increased in the presence of active MASTL. Subsequent in vitro kinase assays suggested that MASTL may phosphorylate hnRNPM, YB1 and TUBA1C under certain in vitro conditions. Taken together, these data suggest that MASTL may phosphorylate several additional substrates, providing insight into the ever-increasing biological functions and roles MASTL plays in driving cancer progression and therapy resistance.
... Our next goal was to functionally characterize the candidate substrate proteins from the in cellulo screen. To further re ne the list of potential substrates, we cross-referenced the phosphorylated proteins with our previously published list of proteins that co-immunoprecipitated (Co-IP) with MASTL from mitotic HeLa cell extracts 53 and MASTL substrates predicted by Hermida et al 41 . There were 11 proteins that were present in all three datasets, an additional 11 common to our in cellulo assay and Hermida's dataset, and a further 15 common to our in cellulo assay and our previous Co-IP (Fig. 3A, Supplementary Table S4). ...
... Further stringency was applied using a Student's t-test, with P < 0.05 taken as signi cant. For MASTL, substrates were further re ned by cross-referencing with previously identi ed proteins that co-immunoprecipitated with MASTL from mitotic HeLa cell extracts 53 . ...
MASTL (microtubule-associated serine/threonine kinase-like) has emerged as a critical regulator of mitosis and as a potential oncogene in a variety of cancer types. To date, Arpp-19/ENSA are the only known substrates of MASTL. However, with the roles of MASTL expanding and increased interest in development of MASTL inhibitors, it has become critical to determine if there are additional substrates and what the optimal consensus motif for MASTL is. Here we utilized a whole cell lysate ( in cellulo ) kinase screen approach combined with stable isotope labelling of amino acids in cell culture to identify potential substrates and a consensus motif of MASTL. Using the related AGC kinase family members AKT1/2, the in cellulo assay identified several known and new substrates highly enriched with the validated consensus motif for AKT. Applying this method to MASTL identified 59 phospho-sites on 26 novel proteins significantly increased in the presence of active MASTL. Subsequent in vitro kinase assays confirmed that MASTL was capable of phosphorylating hnRNPM, YB1, RPS6, TUBA1C, and RPL36A under some conditions. Taken together, these data suggest that MASTL may phosphorylate several additional substrates, providing insight into the ever-increasing biological functions and roles MASTL plays in driving cancer progression and therapy resistance.
... Également, elle initie la réactivation de PP2A-B55d, qui conduira à la déphosphorylation de l'ensemble des substrats mitotiques. En effet, PP1 déphosphoryle Greatwall sur un résidu activateur, la T883, ce qui fait chuter l'activité de la kinaseMa et al., 2016;Ren et al., 2017;Rogers et al., 2016). Une fraction de PP2A-B55d est alors réactivée. ...
... Une fraction de PP2A-B55d est alors réactivée. Comme PP2A-B55d déphosphoryle Greatwall sur des résidus supplémentaires ainsi que son substrat, Arpp19, sur la S67, elle se retrouve libérée de l'action inhibitrice de Greatwall/Arpp19 et est totalement réactivéeMa et al., 2016;Ren et al., 2017;Rogers et al., 2016) . Chez la levure, PP1 est également capable de se lier à PP2A-B55 par l'intermédiaire de la sous-unité régulatrice B55 qui possède un motif RVxF. ...
Ma thèse a visé à comprendre comment la cellule germinale femelle, ou ovocyte, reprend la division méiotique. Dans tout le règne animal, l’ovocyte est bloqué en prophase de première division méiotique et reprend la division au moment de l’ovulation, sous l’effet d’un signal externe, la progestérone chez le xénope. Cette hormone déclenche une voie de signalisation qui aboutit après 3 à 5 heures à l’activation de la kinase Cdk1. Les premières molécules activées de Cdk1 déclenchent une boucle d’auto-amplification permettant son activation totale et l’entrée en division. Cette boucle repose sur des phosphorylations catalysées par Cdk1 et ne fonctionne que si la phosphatase qui contrecarre son activité, PP2A-B55δ, est inhibée. Cette inhibition dépend de la protéine Arpp19, un inhibiteur spécifique de PP2A-B55δ quand elle est phosphorylée sur la S67 par la kinase Greatwall. Arpp19 a un second rôle lors de la reprise de la méiose. Chez tous les vertébrés, l’arrêt en prophase est maintenu par une forte activité de PKA. Chez le xénope, Arpp19 est phosphorylée par PKA sur un résidu distinct de celui ciblé par Greatwall, la S109. En réponse à la progestérone, l’inhibition de PKA provoque la déphosphorylation d’Arpp19, ce qui permet d'enclencher la voie de signalisation conduisant à l'activation de Cdk1. Une phosphatase est donc requise pour déphosphoryler Arpp19 sur S109 et lever le verrou exercé sur l'activation de Cdk1 en prophase. Mon travail a visé à élucider l’identité moléculaire de cette phosphatase inconnue. Par des approches de biochimie et protéomique, j’ai identifié cette phosphatase comme étant PP2A-B55δ. Par des approches fonctionnelles dans des extraits acellulaires et des ovocytes, j’ai établi que PP2A-B55δ est active en prophase. La phosphorylation d’Arpp19 sur la S109 dépend d'une balance entre les activités de PKA et PP2A-B55δ, en faveur de la kinase. En réponse à la progestérone, l’activité de PP2A-B55δ n’est pas affectée et l’inhibition de PKA suffit à la déphosphorylation d’Arpp19. PP2A-B55δ orchestre donc la levée de l’arrêt en prophase et l'entrée en phase M en agissant à deux périodes et sur deux sites distincts d’Arpp19, respectivement ciblés par PKA et Greatwall.
... 61 PP2A-B55 promotes its own release from this inhibition by dephosphorylating Arpp19/ ENSA and by dephosphorylating Gwl, [62][63][64] although PP1 also significantly contributes to Gwl dephosphorylation. [63][64][65] Therefore, there are at least two double-negative feedback loops that regulate PP2A-B55 and could potentially generate bistability: one between PP2A-B55 and Gwl, and one between PP2A-B55 and the stoichiometric inhibitors ENSA and Arpp19 ( Figure 4A). We measured PP2A-B55 activity based on the release of inorganic phosphate from a 32 P-labeled peptide encompassing the serine 50 site of Cdc20 ( Figure S2A). ...
... However, although PP2A-B55 has been shown to dephosphorylate Gwl, it has more recently been shown that the activity of Gwl is mainly regulated by PP1 rather than PP2A-B55. [63][64][65] Furthermore, the hyperphosphorylation of Gwl only weakly correlates with its activity. 63 Therefore, we asked whether the observed bistability in Gwl phosphorylation actually translated into bistability in Gwl activity. ...
The phosphorylation of mitotic proteins is bistable, which contributes to the decisiveness of the transitions into and out of M phase. The bistability in substrate phosphorylation has been attributed to bistability in the activation of the cyclin-dependent kinase Cdk1. However, more recently it has been suggested that bistability also arises from positive feedback in the regulation of the Cdk1-counteracting phosphatase PP2A-B55. Here, we demonstrate biochemically using Xenopus laevis egg extracts that the Cdk1-counteracting phosphatase PP2A-B55 functions as a bistable switch, even when the bistability of Cdk1 activation is suppressed. In addition, Cdk1 regulates PP2A-B55 in a biphasic manner; low concentrations of Cdk1 activate PP2A-B55 and high concentrations inactivate it. As a consequence of this incoherent feedforward regulation, PP2A-B55 activity rises concurrently with Cdk1 activity during interphase and suppresses substrate phosphorylation. PP2A-B55 activity is then sharply downregulated at the onset of mitosis. During mitotic exit, Cdk1 activity initially falls with no obvious change in substrate phosphorylation; dephosphorylation then commences once PP2A-B55 spikes in activity. These findings suggest that changes in Cdk1 activity are permissive for mitotic entry and exit but that the changes in PP2A-B55 activity are the ultimate trigger.
... Many of the cohort of PP1-dependent sites we have identified are not currently assigned as substrates of a particular phosphatase (Figure 7-source data 1), and will be useful to explain the functions of PPP1CA/C in mitosis and mitotic exit. In agreement with the idea that PP1 lies upstream of PP2A-B55 and thus controls its activation (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016), some of these sites, including those on PRC1 and ENSA, are known PP2A-B55 substrates which are indirectly affected by removal of PP1 (Cundell et al., 2016). For the intermediate sites with half-lives <15 min in the control, the motifs show minor exclusion of phospho-threonine with similar, but slightly reduced, surrounding sequence preferences to that seen in the early clusters ( Figure 7B). ...
... PP2A-B55 inhibition is maintained during this period by the BEG pathway. Once both PP1 and the required interactors are dephosphorylated, PP1 can then oppose the CDK1 phosphorylation of MASTL and initiate PP2A-B55 activation (Rogers et al., 2016). Thus, two waves of phosphatase activity co-ordinate the early events of mitotic exit. ...
APC/C-mediated proteolysis of cyclin B and securin promotes anaphase entry, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-counteracting phosphatases PP1 and PP2A-B55, allowing wide-spread dephosphorylation of substrates. Meanwhile, continued APC/C activity promotes proteolysis of other mitotic regulators. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution proteomics, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid proteolysis of cyclin B, securin and geminin at the metaphase-anaphase transition, followed by slow proteolysis of other substrates. Dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent categories with unique sequence motifs. We conclude that dephosphorylation initiated by selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit.
... Many of the cohort of PP1-dependent sites we have identified are not currently assigned as substrates of a particular phosphatase (Figure 7-source data 1), and will be useful to explain the functions of PPP1CA/C in mitosis and mitotic exit. In agreement with the idea that PP1 lies upstream of PP2A-B55 and thus controls its activation (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016), some of these sites, including those on PRC1 and ENSA, are known PP2A-B55 substrates which are indirectly affected by removal of PP1 (Cundell et al., 2016). For the intermediate sites with half-lives <15 min in the control, the motifs show minor exclusion of phospho-threonine with similar, but slightly reduced, surrounding sequence preferences to that seen in the early clusters ( Figure 7B). ...
... PP2A-B55 inhibition is maintained during this period by the BEG pathway. Once both PP1 and the required interactors are dephosphorylated, PP1 can then oppose the CDK1 phosphorylation of MASTL and initiate PP2A-B55 activation (Rogers et al., 2016). Thus, two waves of phosphatase activity co-ordinate the early events of mitotic exit. ...
APC/C-mediated proteolysis of cyclin B and securin promotes anaphase entry, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-counteracting phosphatases PP1 and PP2A-B55, allowing wide-spread dephosphorylation of substrates. Meanwhile, continued APC/C activity promotes proteolysis of other mitotic regulators. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution proteomics, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid proteolysis of cyclin B, securin and geminin at the metaphase-anaphase transition, followed by slow proteolysis of other substrates. Dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent categories with unique sequence motifs. We conclude that dephosphorylation initiated by selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit.
... However, due to the temporal properties of its regulatory mechanism PP2A-B55 only becomes active in anaphase B when cyclin B levels fall below a threshold level (Cundell et al., 2013;Cundell et al., 2016). PP1 has been reported to be involved in the activation of PP2A-B55 through the dephosphorylation of the Gwl/MASTL kinase (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016;Ren et al., 2017), and could thus be considered an upstream regulator of PP2A-B55. PP2A-B55 action on CDC20 is therefore most likely restricted to a later point in anaphase, perhaps at the switch to APC/C regulation by CDH1 and destruction of late anaphase substrates. ...
... By contrast, as the APC/C becomes active downstream of checkpoint silencing, PP1 promotes dephosphorylation of CDC20, and decisively tips the balance towards APC/C CDC20 activation, destabilisation of the mitotic state, and consequently mitotic exit. The APC/C CDC20 active state is then consolidated by PP2A-B55, the activation of which is also initiated by PP1 (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016;Ren et al., 2017), ensuring that CDC20 is completely dephosphorylated in anaphase. ...
Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase promoting complex/cyclosome and its co-activator CDC20 (APC/C CDC20 ) form the main ubiquitin E3 ligase for these two proteins. APC/C CDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylation. Here, we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilises cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1-phosphorylation defective CDC20 6A mutants. These CDC20 6A cells show a normal spindle checkpoint response, and rapidly destroy cyclin B once all chromosomes have aligned and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase by promoting APC/C CDC20 -dependent destruction of cyclin B in human cells.
... However, due to the temporal properties of its regulatory mechanism PP2A-B55 only becomes active in anaphase B when cyclin B1 levels fall below a threshold level (Cundell et al., 2013;Cundell et al., 2016). PP1 has been reported to be involved in the activation of PP2A-B55 through the dephosphorylation of the Gwl/MASTL kinase (Heim et al., 2015;Ma et al., 2016;Ren et al., 2017;Rogers et al., 2016), and could thus be considered an upstream regulator of PP2A-B55. PP2A-B55 action on CDC20 is therefore most likely restricted to a later point in anaphase, perhaps at the switch to APC/C regulation by CDH1 and destruction of late anaphase substrates. ...
... This APC/C CDC20 active state is then consolidated by the action of PP2A-B55, the activation of which is initiated by PP1 (Heim et al., 2015;Ma et al., 2016;Ren et al., 2017;Rogers et al., 2016), ensuring that CDC20 is completely dephosphorylated in anaphase (Figure 9). ...
Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20 form the main ubiquitin E3 ligase for these proteins. APC/C-CDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylations. Here we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilises cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1-phosphorylation defective CDC20-6A mutants. These CDC20-6A cells show a normal spindle checkpoint response, but once all chromosomes have aligned rapidly degrade cyclin B and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase by promoting APC/C-CDC20-dependent destruction of cyclin B in human cells.
... These half-lives were all doubled or extended to beyond the window of the experiment and corresponded to the majority of non-stable phospho-sites observed within the analysis. Unsurprisingly, some of these sites are known PP2A-B55 substrates, as PP1 lies upstream of PP2A-B55 activation (Cundell et al., 2016;Rogers et al., 2016). Nevertheless, many of these sites are not currently assigned as substrates of a particular phosphatase, indicative of the mitotic function of PPP1CA/C suggested in the literature (Wu et al., 2009). ...
... As cyclin B is degraded, PP1 rapidly responds globally to the changes in CDK1 activity becoming PP2A-B55 inhibition is maintained during this period by the BEG pathway. Once dephosphorylated, PP1 can then oppose the CDK1 phosphorylation of MASTL and initiate PP2A-B55 activation (Rogers et al., 2016). This would create two waves of phosphatase activation to co-ordinate the early events of mitotic exit. ...
APC/C-mediated proteolysis of cyclin B and securin promotes entry into anaphase, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-opposing phosphatases PP1 and PP2A-B55 leading to dephosphorylation of substrates crucial for mitotic exit. Meanwhile, continued APC/C activity is required to target various proteins, including Aurora and Polo kinases, for degradation. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution mass spectrometry, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid (~5min half-life) proteolysis of cyclin B, securin and geminin at the metaphase to anaphase transition, followed by slow proteolysis (>60 min half-life) of other mitotic regulators. Protein dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent fast, intermediate and slow categories with unique sequence motifs. We conclude that dephosphorylation initiated by the selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit.
... First, in early anaphase, degradation of CyclinB reduces Cdk1/CyclinB activity levels, which leads to the dephosphorylation of protein phosphatase 1 (PP1) on its Cdk1/CyclinB phosphorylation site at the C-terminus. Dephosphorylation and reactivation of PP1 partially inactivates Greatwall by promoting its dephosphorylation of the S875 residue (in Mastl), which in turn partially releases ENSA and ARPP-19 inhibition of PP2A/B55 [60][61][62]. This is sufficient to promote Greatwall dephosphorylation on T194 and S875 residues by PP2A/B55 phosphatase activity [60][61][62], resulting in full inhibition of Greatwall kinase activity and in its translocation to the nucleus [55]. ...
... Dephosphorylation and reactivation of PP1 partially inactivates Greatwall by promoting its dephosphorylation of the S875 residue (in Mastl), which in turn partially releases ENSA and ARPP-19 inhibition of PP2A/B55 [60][61][62]. This is sufficient to promote Greatwall dephosphorylation on T194 and S875 residues by PP2A/B55 phosphatase activity [60][61][62], resulting in full inhibition of Greatwall kinase activity and in its translocation to the nucleus [55]. Recent studies have also described an additional layer of phosphatase regulation of Greatwall-Endosulfine because the RNA polymerase II C-terminal domain phosphatase (Fcp1) dephosphorylates Greatwall, ENSA, and ARPP-19 [63,64]. ...
During the cell cycle, hundreds of proteins become phosphorylated and dephosphorylated, indicating that protein kinases and protein phosphatases play a central role in its regulation. It has been widely recognized that oscillation in cyclin-dependent kinase (CDK) activity promotes DNA replication, during S-phase, and chromosome segregation, during mitosis. Each CDK substrate phosphorylation status is defined by the balance between CDKs and CDK-counteracting phosphatases. In fission yeast and animal cells, PP2A/B55 is the main protein phosphatase that counteracts CDK activity. PP2A/B55 plays a key role in mitotic entry and mitotic exit, and it is regulated by the Greatwall-Endosulfine (ENSA) molecular switch that inactivates PP2A/B55 at the onset of mitosis, allowing maximal CDK activity at metaphase. The Greatwall-ENSA-PP2A/B55 pathway is highly conserved from yeast to animal cells. In yeasts, Greatwall is negatively regulated by nutrients through TORC1 and S6 kinase, and couples cell growth, regulated by TORC1, to cell cycle progression, driven by CDK activity. In animal cells, Greatwall is phosphorylated and activated by Cdk1 at G2/M, generating a bistable molecular switch that results in full activation of Cdk1/CyclinB. Here we review the current knowledge of the Greatwall-ENSA-PP2A/B55 pathway and discuss its role in cell cycle progression and as an integrator of nutritional cues.
... The balance is shifted towards PP1 autoactivation by dephosphorylation of its C-terminal tail and I-1 [108][109][110]. Free PP1 then dephosphorylates and partially inactivates Greatwall [224][225][226], ceasing the supply of phosphorylated ENSA/Arpp19. Any PP2A-B55 dephosphorylated p-ENSA/p-Arpp19 inhibitor molecules are no longer replenished. ...
... PP2A-B55 completes Greatwall inactivation by full dephosphorylation of the protein. This positive feedback loop, coupled with the rest of the system architecture makes mitotic exit a bistable transition [226]. Once liberated, PP2A-B55 is thought to dephosphorylate hundreds of substrates in conjunction with PP1 and PP2A-B56. ...
Accurate division of cells into two daughters is a process that is vital to propagation of life. Protein phosphorylation and selective degradation have emerged as two important mechanisms safeguarding the delicate choreography of mitosis. Protein phosphatases catalyze dephosphorylation of thousands of sites on proteins, steering the cells through establishment of the mitotic phase and exit from it. A large E3 ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) becomes active during latter stages of mitosis through G1 and marks hundreds of proteins for destruction. Recent studies have revealed the complex interregulation between these two classes of enzymes. In this review, we highlight the direct and indirect mechanisms by which phosphatases and the APC/C mutually influence each other to ensure accurate spatiotemporal and orderly progression through mitosis, with a particular focus on recent insights and conceptual advances.
... This correlated with a significant increase in the presence of anaphase chromosome bridges ( Figure S2C). To understand how increased MASTL expression could be delaying mitosis, we utilised our previously developed mathematical model of mitotic exit [21]. Using this model, we simulated a doubling of MASTL activity to mimic a twofold increase in MASTL expression (Fig. 2e). ...
... Therefore, to better understand how MASTL overexpression could be driving breast cancer, we undertook an unbiased phosphoproteomic screen using stable isotope labelling of amino acids in cell culture (SILAC) (Fig. 3a). Asynchronous MCF10A control and MASTLexpressing cells were grown in light or heavy (Lys-8, Arg-10) amino acids, as previously described [21,25]. We identified 10,024 phosphopeptides across the five replicates, with good correlation between each run ( Figure S3A). ...
MASTL kinase is essential for correct progression through mitosis, with loss of MASTL causing chromosome segregation errors, mitotic collapse and failure of cytokinesis. However, in cancer MASTL is most commonly amplified and overexpressed. This correlates with increased chromosome instability in breast cancer and poor patient survival in breast, ovarian and lung cancer. Global phosphoproteomic analysis of immortalised breast MCF10A cells engineered to overexpressed MASTL revealed disruption to desmosomes, actin cytoskeleton, PI3K/AKT/mTOR and p38 stress kinase signalling pathways. Notably, these pathways were also disrupted in patient samples that overexpress MASTL. In MCF10A cells, these alterations corresponded with a loss of contact inhibition and partial epithelial–mesenchymal transition, which disrupted migration and allowed cells to proliferate uncontrollably in 3D culture. Furthermore, MASTL overexpression increased aberrant mitotic divisions resulting in increased micronuclei formation. Mathematical modelling indicated that this delay was due to continued inhibition of PP2A-B55, which delayed timely mitotic exit. This corresponded with an increase in DNA damage and delayed transit through interphase. There were no significant alterations to replication kinetics upon MASTL overexpression, however, inhibition of p38 kinase rescued the interphase delay, suggesting the delay was a G2 DNA damage checkpoint response. Importantly, knockdown of MASTL, reduced cell proliferation, prevented invasion and metastasis of MDA-MB-231 breast cancer cells both in vitro and in vivo, indicating the potential of future therapies that target MASTL. Taken together, these results suggest that MASTL overexpression contributes to chromosome instability and metastasis, thereby decreasing breast cancer patient survival.
... Cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and stained as described. 39 FIJI-ImageJ and Adobe Photoshop CC were used for image handling. The nuclear fluorescence intensity was calculated using FIJI-ImageJ (v1.51c) as previously described. ...
... The nuclear fluorescence intensity was calculated using FIJI-ImageJ (v1.51c) as previously described. 39 Sonic Hedgehog in small cell lung cancer A Szczepny et al overexpression in a transgenic mouse tumor model. 22,23 These results suggest that upregulation of Ccnb1 can induce chromosomal instability in addition the deregulation of the cell cycle induced by the combined loss of both Tp53 and Rb1. ...
Hedgehog (Hh) signaling regulates cell fate and self-renewal in development and cancer. Canonical Hh signaling is mediated by Hh ligand binding to the receptor Patched (Ptch), which in turn activates Gli-mediated transcription through Smoothened (Smo), the molecular target of the Hh pathway inhibitors used as cancer therapeutics. Small cell lung cancer (SCLC) is a common, aggressive malignancy with universally poor prognosis. Although preclinical studies have shown that Hh inhibitors block the self-renewal capacity of SCLC cells, the lack of activating pathway mutations have cast doubt over the significance of these observations. In particular, the existence of autocrine, ligand-dependent Hh signaling in SCLC has been disputed. In a conditional Tp53;Rb1 mutant mouse model of SCLC, we now demonstrate a requirement for the Hh ligand Sonic Hedgehog (Shh) for the progression of SCLC. Conversely, we show that conditional Shh overexpression activates canonical Hh signaling in SCLC cells, and markedly accelerates tumor progression. When compared to mouse SCLC tumors expressing an activating, ligand-independent Smo mutant, tumors overexpressing Shh exhibited marked chromosomal instability and Smoothened-independent upregulation of Cyclin B1, a putative non-canonical arm of the Hh pathway. In turn, we show that overexpression of Cyclin B1 induces chromosomal instability in mouse embryonic fibroblasts lacking both Tp53 and Rb1. These results provide strong support for an autocrine, ligand-dependent model of Hh signaling in SCLC pathogenesis, and reveal a novel role for non-canonical Hh signaling through the induction of chromosomal instability.Oncogene advance online publication, 5 June 2017; doi:10.1038/onc.2017.173.
... Phosphatase PP1, one of the seven members of the phosphoprotein phosphatase (PPP) family, has been proposed to initiate the mitotic exit regulator PP2A: B55 [68,69]. PPP1R7 (SDS22) and ENSA are critical regulators of PP1 and PP2A: B55, respectively, and both are essential for the proper completion of mitosis. ...
Cancer risk after ionizing radiation (IR) is assumed to be linear with the dose; however, for low doses, definite evidence is lacking. Here, using temporal multi-omic systems analyses after a low (LD; 0.1 Gy) or a high (HD; 1 Gy) dose of X-rays, we show that, although the DNA damage response (DDR) displayed dose proportionality, many other molecular and cellular responses did not. Phosphoproteomics uncovered a novel mode of phospho-signaling via S12-PPP1R7, and large-scale dephosphorylation events that regulate mitotic exit control in undamaged cells and the G2/M checkpoint upon IR in a dose-dependent manner. The phosphoproteomics of irradiated DNA double-strand breaks (DSBs) repair-deficient cells unveiled extended phospho-signaling duration in either a dose-dependent (DDR signaling) or independent (mTOR-ERK-MAPK signaling) manner without affecting signal magnitude. Nascent transcriptomics revealed the transcriptional activation of genes involved in NRF2-regulated antioxidant defense, redox-sensitive ERK-MAPK signaling, glycolysis and mitochondrial function after LD, suggesting a prominent role for reactive oxygen species (ROS) in molecular and cellular responses to LD exposure, whereas DDR genes were prominently activated after HD. However, how and to what extent the observed dose-dependent differences in molecular and cellular responses may impact cancer development remain unclear, as the induction of chromosomal damage was found to be dose-proportional (10–200 mGy).
... Thus, APC/C activation allows sister chromatids to segregate in anaphase ( Figure 2B). After anaphase onset, the decreased Cyclin B levels cannot sustain Gwl activation by CDK1, and Gwl is inactivated by PP1 (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016;Ren et al., 2017). The time needed for these events and for PP2A-B55 to dephosphorylate Endosulfines defines a delay between anaphase and late mitotic events that depend on PP2A-B55. ...
In most animal cell types, the interphase nucleus is largely disassembled during mitotic entry. The nuclear envelope breaks down and chromosomes are compacted into separated masses. Chromatin organization is also mostly lost and kinetochores assemble on centromeres. Mitotic protein kinases play several roles in inducing these transformations by phosphorylating multiple effector proteins. In many of these events, the mechanistic consequences of phosphorylation have been characterized. In comparison, how the nucleus reassembles at the end of mitosis is less well understood in mechanistic terms. In recent years, much progress has been made in deciphering how dephosphorylation of several effector proteins promotes nuclear envelope reassembly, chromosome decondensation, kinetochore disassembly and interphase chromatin organization. The precise roles of protein phosphatases in this process, in particular of the PP1 and PP2A groups, are emerging. Moreover, how these enzymes are temporally and spatially regulated to ensure that nuclear reassembly progresses in a coordinated manner has been partly uncovered. This review provides a global view of nuclear reassembly with a focus on the roles of dephosphorylation events. It also identifies important open questions and proposes hypotheses.
... As reported before, MASTL kinase could enhance the CDK1 mitotic phosphorylation events during mitosis [44]. On the other hand, CDK1 inactivation reduced its ability in phosphorylation inhabitation, which is followed by a decline in MASTL activity [45][46][47]. Thus, mitotic phosphorylation might decline during mitosis in the jejunum tissue of young calves with ruminal acidosis. ...
Diet-induced acidosis imposes a health risk to young calves. In this study, we aimed to investigate the host jejunum transcriptome changes, along with its microbial community variations, using our established model of feed-induced ruminal acidosis in young calves. Eight bull calves were randomly assigned to two diet treatments beginning at birth (a starch-rich diet, Aci; a control diet, Con). Whole-transcriptome RNA sequencing was performed on the jejunum tissues collected at 17 weeks of age. Ribosomal RNA reads were used for studying microbial community structure variations in the jejunum. A total of 853 differentially expressed genes were identified (402 upregulated and 451 downregulated) between the two groups. The cell cycle and the digestion and absorption of protein in jejunal tissue were affected by acidosis. Compared to the control, genera of Campylobacter, Burkholderia, Acidaminococcus, Corynebacterium, and Olsenella significantly increased in abundance in the Aci group, while Lachnoclostridium and Ruminococcus were significantly lower in the Aci group. Expression changes in the AXL gene were associated with the abundance variations of a high number of genera in jejunum. Our study provided a snapshot of the transcriptome changes in the jejunum and its associated meta-transcriptome changes in microbial communities in young calves with feed-induced acidosis.
... PP2A-B55 activity is regulated by the Cdk1-MASTL-ARPP19 pathway in which Cdk1 activates the MASTL kinase that then phosphorylates ARPP19/ENSA proteins, which bind and inactivate PP2A-B55 (Gharbi-Ayachi et al., 2010;Mochida et al., 2010;Vigneron et al., 2009). The activation of PP2A-B55 is initiated by a decrease in Cdk1 activity, which activates PP1 to initiate dephosphorylation and inactivation of the MASTL kinase (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016). ...
Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1–Cdk1 independently inhibits APC/C–Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.
... Furthermore, although there have been no in vitro MASTL data on gastric cancer, knockdown of MASTL expression did reduce proliferation and induce apoptosis of other human cancer cells, such as thyroid [18] and colon cancer cells [13] , which is consistent with our current data. The function of the MASTL enzyme in human cancers could be the upregulation of tumor cell cycle progression [19,20] and metastasis [10] , and resistance to chemotherapy [13] and radiotherapy [11,21] . MASTL is active in the cell cycle progression to induce the transition of G 2 to cell mitosis by inhibiting the activity of tumor suppressor PP2A-B55 phosphatase [20] . ...
Microtubule-associated serine/threonine kinase (MASTL) functions to regulate chromosome condensation and mitotic progression. Therefore, aberrant MASTL expression is commonly implicated in various human cancers. This study analyzed MASTL expression in gastric cancer vs. adjacent normal tissue for elucidating the association with clinicopathological data from patients. This work was then extended to investigate the effects of MASTL knockdown on tumor cells in vitro. The level of MASTL expression in gastric cancer tissue was assessed from the UALCAN, GEPIA, and Oncomine online databases. Lentivirus carrying MASTL or negative control shRNA was infected into gastric cancer cells. RT-qPCR, Western blotting, cell viability, cell counting, flow cytometric apoptosis and cell cycle, and colony formation assays were performed. MASTL was upregulated in gastric cancer tissue compared to the adjacent normal tissue, and the MASTL expression was associated with advanced tumor stage, Helicobacter pylori infection and histological subtypes. On the other hand, knockdown of MASTL expression significantly reduced tumor cell viability and proliferation, and arrested cell cycle at G2/M stage but promoted tumor cells to undergo apoptosis. At protein level, knockdown of MASTL expression enhanced levels of cleaved PARP1, cleaved caspase-3, Bax and p-ERK1/2 expression, but downregulated expression levels of BCL-2 and p-NF-κB-p65 protein in AGS and MGC-803 cells. MASTL overexpression in gastric cancer tissue may be associated with gastric cancer development and progression, whereas knockdown of MASTL expression reduces tumor cell proliferation and induces apoptosis. Further study will evaluate MASTL as a potential target of gastric cancer therapeutic strategy.
... PP2A-B55 activity is regulated by the Cdk1-MASTL-ARPP19 pathway in which Cdk1 activates the MASTL kinase that then phosphorylates ARPP19/ENSA proteins which bind and inactivate PP2A-B55 (Gharbi-Ayachi et al., 2010;Mochida et al., 2010;Vigneron et al., 2009). The activation of PP2A-B55 is initiated by a decrease of Cdk1 activity which activates PP1 to initiate dephosphorylation and inactivation of the MASTL kinase (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016). ...
Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets Cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by Cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 gets activated prior to Cyclin B1 degradation. Here we show that the MCC component BubR1 harbours both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively our work reveals how Cdc20 can be dephosphorylated in the presence of Cyclin B1-Cdk1 activity without causing premature anaphase onset.
... First, phosphorylation might inhibit PP1 due to direct competition with other phosphorylated substrates for binding to the active site. [8,16,17] Second, phosphorylation of the PxTPP sequence could induce interactions of the intrinsically disordered C-tail with PP1 on an allosteric site to induce inhibitory conformational changes. Third, phosphorylation of the PxTPP sequence could affect interactions of PP1 with other proteins. ...
Phosphoprotein phosphatase‐1 (PP1) is a key player in the regulation of phospho‐serine (pSer) and phospho‐threonine (pThr) dephosphorylation and is involved in a large fraction of cellular signaling pathways. Aberrant activity of PP1 has been linked to many diseases, including cancer and heart failure. Besides a well‐established activity control by regulatory proteins, an inhibitory function for phosphorylation (p) of a Thr residue in the C‐terminal intrinsically disordered tail of PP1 has been demonstrated. The associated phenotype of cell‐cycle arrest was repeatedly proposed to be due to autoinhibition of PP1 through either conformational changes or substrate competition. Here, we use PP1 variants created by mutations and protein semisynthesis to differentiate between these hypotheses. Our data support the hypothesis that pThr exerts its inhibitory function by mediating protein complex formation rather than by a direct mechanism of structural changes or substrate competition.
... Following the decreased activity of CDK1, inhibition of PP1 is relieved, triggering FCP1 activation, which in turn dephosphorylates MASTL. Similarly, the increasing activity of PP1 enhances the activation of PP2A/B55 [7,[12][13][14][15]. This creates a bistable switch to exit mitosis [16,17]. ...
Progression through mitosis is balanced by the timely regulation of phosphorylation and dephosphorylation events ensuring the correct segregation of chromosomes before cytokinesis. This balance is regulated by the opposing actions of CDK1 and PP2A, as well as the Greatwall kinase/MASTL. MASTL is commonly overexpressed in cancer, which makes it a potential therapeutic anticancer target. Loss of Mastl induces multiple chromosomal errors that lead to the accumulation of micronuclei and multilobulated cells in mitosis. Our analyses revealed that loss of Mastl leads to chromosome breaks and abnormalities impairing correct segregation. Phospho-proteomic data for Mastl knockout cells revealed alterations in proteins implicated in multiple processes during mitosis including double-strand DNA damage repair. In silico prediction of the kinases with affected activity unveiled NEK2 to be regulated in the absence of Mastl. We uncovered that, RAD51AP1, involved in regulation of homologous recombination, is phosphorylated by NEK2 and CDK1 but also efficiently dephosphorylated by PP2A/B55. Our results suggest that MastlKO disturbs the equilibrium of the mitotic phosphoproteome that leads to the disruption of DNA damage repair and triggers an accumulation of chromosome breaks even in noncancerous cells.
... In addition to its role in mitotic entry, MASTL plays an important role in mitotic exit, where its deactivation has been seen to be an essential requirement for the cells to exit from mitosis (13,14). This inactivation of MASTL results in the reactivation of PP2A, which is also essential for the mitotic exit (15)(16)(17). ...
Microtubule associated serine threonine like kinase (MASTL), also known as Greatwall kinase (Gwl), has an important role in the regulation of mitosis. By inhibiting protein phosphatase 2A, it plays a crucial role in activating one of the most important mitotic kinases known as cyclin dependent kinase1 (CDK1). MASTL has been seen to be up regulated in various types of cancers and is also involved in tumor recurrence. It is activated by CDK1 through phosphorylations in the activation/T-loop but the complete mechanism of its activation is still unclear. Here we report that AKT phosphorylates MASTL at T299 residue which plays a critical role in its activation. Our results suggest that AKT increases CDK1 mediated phosphorylation and hence activity of MASTL which in turn promotes mitotic progression through PP2A inhibition. We also show that the oncogenic potential of AKT is augmented by MASTL activation as the AKT mediated proliferation in colorectal cell lines can be attenuated by inhibiting and/or silencing MASTL. In brief, we report that AKT has an important role in the progression of mitosis in mammalian cells and it does so through the phosphorylation and activation of MASTL.
... did include I-1 in a mathematical model of striatal medium spiny neurons andRogers et al. (2016) incorporated I-1 in modeling the control of PP1 in mitosis where phosphatases play a fundamental role(Wu et al., 2009;Holder et al., 2019;Moura and Conde, 2019). ...
... The best example for this is PP2A with its B55 regulatory subunit (PP2A:B55), which is tightly regulated by Greatwall (Gwl) kinase [13] via its substrates ENSA and ARPP19 that become potent PP2A:B55 inhibitors upon phosphorylation [14,15]. Gwl itself is activated by Cdk1-dependent phosphorylation [16], which is reversed by PP1 [17][18][19] and PP2A:B55 [6,20], and the latter creates a mutual antagonism. Reconstitution of the Gwl-ENSA-PP2A:B55 pathway in vitro confirmed these interactions and revealed that PP2A:B55 has a bistable activity with respect to Cdk1 activity [6] ( Figure 1B). ...
... The best example for this is PP2A with its B55 regulatory subunit (PP2A:B55), which is tightly regulated by Greatwall (Gwl) kinase [13] via its substrates ENSA and ARPP19 that become potent PP2A:B55 inhibitors upon phosphorylation [14,15]. Gwl itself is activated by Cdk1-dependent phosphorylation [16], which is reversed by PP1 [17][18][19] and PP2A:B55 [6,20], and the latter creates a mutual antagonism. Reconstitution of the Gwl-ENSA-PP2A:B55 pathway in vitro confirmed these interactions and revealed that PP2A:B55 has a bistable activity with respect to Cdk1 activity [6] ( Figure 1B). ...
Distinct protein phosphorylation levels in interphase and M phase require tight regulation of Cdk1 activity [1, 2]. A bistable switch, based on positive feedback in the Cdk1 activation loop, has been proposed to generate different thresholds for transitions between these cell-cycle states [3, 4, 5]. Recently, the activity of the major Cdk1-counteracting phosphatase, PP2A:B55, has also been found to be bistable due to Greatwall kinase-dependent regulation [6]. However, the interplay of the regulation of Cdk1 and PP2A:B55 in vivo remains unexplored. Here, we combine quantitative cell biology assays with mathematical modeling to explore the interplay of mitotic kinase activation and phosphatase inactivation in human cells. By measuring mitotic entry and exit thresholds using ATP-analog-sensitive Cdk1 mutants, we find evidence that the mitotic switch displays hysteresis and bistability, responding differentially to Cdk1 inhibition in the mitotic and interphase states. Cdk1 activation by Wee1/Cdc25 feedback loops and PP2A:B55 inactivation by Greatwall independently contributes to this hysteretic switch system. However, elimination of both Cdk1 and PP2A:B55 inactivation fully abrogates bistability, suggesting that hysteresis is an emergent property of mutual inhibition between the Cdk1 and PP2A:B55 feedback loops. Our model of the two interlinked feedback systems predicts an intermediate but hidden steady state between interphase and M phase. This could be verified experimentally by Cdk1 inhibition during mitotic entry, supporting the predictive value of our model. Furthermore, we demonstrate that dual inhibition of Wee1 and Gwl kinases causes loss of cell-cycle memory and synthetic lethality, which could be further exploited therapeutically.
... Upon anaphase entry, active PP1 promotes dephosphorylation of S875 and partial inactivation of Gwl, which in turn results in a partial activation of PP2A-B55. Active PP2A-B55 will then dephosphorylate Gwl on both T194 and S875 residues (human numbering) and fully inactivate this kinase (see poster) (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016). Fcp1 additionally targets dephosphorylation of Gwl on S90 and S453 (Della Monica et al., 2015). ...
Mitosis is controlled by a subtle balance between kinase and phosphatase activities that involve the master mitotic kinase cyclin- B-Cdk1 and its antagonizing protein phosphatase 2A-B55 (PP2AB55). Importantly, the Greatwall (Gwl; known as Mastl in mammals, Rim15 in budding yeast and Ppk18 in fission yeast) kinase pathway regulates PP2A-B55 activity by phosphorylating two proteins, cAMPregulated phosphoprotein 19 (Arpp19) and α-endosulfine (ENSA). This phosphorylation turns these proteins into potent inhibitors of PP2A-B55, thereby promoting a correct timing and progression of mitosis. In this Cell Science at a Glance article and the accompanying poster, we discuss how Gwl is regulated in space and time, and how the Gwl-Arpp19-ENSA-PP2A-B55 pathway plays an essential role in the control of M and S phases from yeast to human. We also summarize how Gwl modulates oncogenic properties of cells and how nutrient deprivation influences Gwl activity.
... To this end, the antimitotic function of PP1 has been well established as an essential mechanism that promotes mitotic exit (42,49). As an example, recent studies discovered a role of PP1 in the dephosphorylation of MASTL (also known as Greatwall kinase) during mitotic exit (36,(52)(53)(54). On the other hand, portions of PP1 bind specific mitotic structures and substrates, and play an active role in promoting several key steps of mitotic progression. ...
Mitotic progression is regulated largely by reversible phosphorylation events that are mediated by mitotic kinases and phosphatases. Protein phosphatase 1 (PP1) has been shown to play a crucial role in regulation of mitotic entry, progression, and exit. We previously observed, in Xenopus egg extracts, that phosphatase 1 nuclear targeting subunit (PPP1R10/PNUTS) acts as a mitotic regulator by negatively modulating PP1. This study investigates the role of PNUTS in mitotic progression in mammalian cells, and demonstrates that PNUTS expression is elevated in mitosis and depletion partially blocks mitotic entry. Cells which enter mitosis after PNUTS knockdown exhibit frequent chromosome mis-segregation. Aurora A/B kinase complexes and several kinetochore components are identified as PNUTS-associated proteins. PNUTS depletion suppresses the activation of Aurora A/B kinases, and disrupts the spatiotemporal regulation of the chromosomal passenger complex (CPC). PNUTS dynamically localizes to kinetochores, and is required for the activation of the spindle assembly checkpoint. Finally, PNUTS depletion sensitizes the tumor cell response to Aurora inhibition, suggesting that PNUTS is a potential drug target in combination anti-cancer therapy.
Implications:
Delineation of how PNUTS governs the mitotic activation and function of Aurora kinases will improve the understanding of the complex phospho-regulation in mitotic progression, and suggest new options to enhance the therapeutic efficacy of Aurora inhibitors.
... Once mitosis is complete, the cell must exit mitosis, and to do this, the prevailing phosphorylation(s) has to be removed. Removal is suggested to be accomplished by reversing the inhibitory effect of MASTL on phosphatases by PP1 [27]. Our data are well aligned with the understanding of the regulatory role for MASTL in cell cycle regulation in colon cancer cells, given that inhibiting MASTL was sufficient to inhibit cell cycle progression and mitosis. ...
Abstract Background Chemotherapeutic agents that modulate cell cycle checkpoints and/or tumor-specific pathways have shown immense promise in preclinical and clinical studies aimed at anti-cancer therapy. MASTL (Greatwall in Xenopus and Drosophila), a serine/threonine kinase controls the final G2/M checkpoint and prevents premature entry of cells into mitosis. Recent studies suggest that MASTL expression is highly upregulated in cancer and confers resistance against chemotherapy. However, the role and mechanism/s of MASTL mediated regulation of tumorigenesis remains poorly understood. Methods We utilized a large patient cohort and mouse models of colon cancer as well as colon cancer cells to determine the role of Mastl and associated mechanism in colon cancer. Results Here, we show that MASTL expression increases in colon cancer across all cancer stages compared with normal colon tissue (P
... In addition to its role at mitotic entry, MASTL plays an important role in mitotic exit as well, where upon its deactivation has been seen to be an essential requirement for the cells to exit from mitosis [9,10]. Being so critical for mitosis, MASTL has been investigated in various types of carcinomas and has been seen to be up regulated in a . ...
Microtubule associated serine threonine like kinase (MASTL) has been recently identified as an important regulator of mitosis. By inhibiting protein phosphatase 2A, it plays a crucial role in activating one of the most important mitotic kinases known as cyclin dependent kinase1 (CDK1). MASTL has been seen to be up regulated in various types of cancers and is involved in tumor recurrence. It is activated by CDK1 through its auto regulatory loop but the complete mechanism of its activation is still unclear. In this study, we evaluated the regulation of MASTL via AKT during mitosis. Here we report that AKT phosphorylates MASTL at T299 which plays a critical role in its activation. Our results suggest that AKT increases CDK1 mediated phosphorylation and hence activity of MASTL which in turn promotes cell proliferation. We also show that the oncogenic potential of AKT is augmented by MASTL activation as the AKT mediated oncogenesis in colorectal cell lines can be attenuated by inhibiting and/or silencing MASTL. In brief, we report that AKT has an important role in the progression of mitosis in mammalian cells and it does so through the phosphorylation and activation of MASTL.
... Currently, there is nothing known about the identity of any of the phosphatase(s) responsible for dephosphorylation of MAST3. Recent studies of Gwl and MASTL have identified specific roles of both PP1 and PP2A in their regulation (Heim et al., 2015;Ma et al., 2016;Rogers et al., 2016;Wang et al., 2016). Modeling studies also support a potential role for phosphatase action upon Gwl as part of a negative feedback process that could contribute to its regulation (Vinod and Novak, 2015)(see also below). ...
ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition.
... Declining cyclin B levels (marked by X) reduce CDK1 activity, which further removes inhibition of APC/C, creating a negative feedback loop. Reduced CDK1 activity allows PP1 to reactivate, which then dephosphorylates MASTL (Rogers et al., 2016), leading to the reactivation of PP2A-B55 and PP2A-B56 (Grallert et al., 2015). The specifi c dephosphorylation of key phosphosites drives the early events of mitotic exit (Cundell et al., 2016). ...
During mitosis, a cell divides its duplicated genome into two identical daughter cells. This process must occur without errors to prevent proliferative diseases (e.g., cancer). A key mechanism controlling mitosis is the precise timing of more than 32,000 phosphorylation and dephosphorylation events by a network of kinases and counterbalancing phosphatases. The identity, magnitude, and temporal regulation of these events have emerged recently, largely from advances in mass spectrometry. Here, we show phosphoevents currently believed to be key regulators of mitosis. For an animated version of this SnapShot, please see http://www.cell.com/cell/enhanced/odonoghue2.
... CycA-dependent kinases have been suggested as triggers for mitotic entry, as they have considerable kinase activity in interphase [21,22], consistent with our observation that Cdk2:CycA can activate the Gwl-ENSA pathway. On mitotic exit, in contrast, dephosphorylation of Gwl by PP1 [16,23,24] could initiate the reverse transition by supporting the PP2A:B55 auto-activation. Whether the design principle of interlinked toggle switches with a pair of triggers is a generic feature of other decision-making pathways in living cells is a question for future experimental and modeling studies. ...
Unreduced gametes, that are important for species evolution and agricultural development, are generally believed to be formed by meiotic defects. However, we found that male diploid loach (Misgurnus anguillicaudatus) could produce not only haploid sperms, but also unreduced sperms, after cyclin-dependent kinase 1 gene (cdk1, one of the most important kinases in regulating cell mitosis) deletion. Observations on synaptonemal complexes of spermatocyte in prophase of meiosis and spermatogonia suggested that the number of chromosomes in some spermatogonia of cdk1-/- loach doubled, leading to unreduced diploid sperm production. Then, transcriptome analysis revealed aberrant expressions of some cell cycle-related genes (such as ppp1c and gadd45) in spermatogonia of cdk1-/- loach relative to wild-type loach. An in vitro and in vivo experiment further validated that Cdk1 deletion in diploid loach resulted in mitotic defects, leading to unreduced diploid sperm formation. In addition, we found that cdk1-/- zebrafish could also produce unreduced diploid sperms. This study provides important information on revealing the molecular mechanisms behind unreduced gamete formation through mitotic defects, and lays the foundation for a novel strategy for fish polyploidy creation by using cdk1 mutants to produce unreduced sperms, which can then be used to obtain polyploidy, proposed to benefit aquaculture.
The nucleus undergoes dramatic structural and functional changes during cell division. With the entry into mitosis, in human cells the nuclear envelope breaks down, chromosomes rearrange into rod-like structures which are collected and segregated by the spindle apparatus. While these processes in the first half of mitosis have been intensively studied, much less is known about the second half of mitosis, when a functional nucleus reforms in each of the emerging cells. Here we review our current understanding of mitotic exit and nuclear reformation with spotlights on the links to cancer biology.
Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-Phase transitions. Protein Phosphatase 1 (PP1) is one of these phosphatases, with oscillations in PP1 activity driving mitotic M-Phase. Evidence from a variety of experimental systems also points to roles in meiosis. Here we report that PP1 is important for M-Phase transitions through mouse oocyte meiosis. We employed a unique small-molecule approach to inhibit or activate PP1 at distinct phases of mouse oocyte meiosis. These studies show that temporal control of PP1 activity is essential for G2/M transition, metaphase I/anaphase I transition, and the formation of a normal metaphase II oocyte. Our data also reveal that inappropriate activation of PP1 is more deleterious at G2/M transition than at prometaphase I-to-metaphase I, and that an active pool of PP1 during prometaphase is vital for metaphase I/anaphase I transition and metaphase II chromosome alignment. Taken together, these results establish that loss of oscillations in PP1 activity causes a range of severe meiotic defects, pointing to essential roles for PP1 in female fertility, and more broadly, M-Phase regulation.
Summary statement
Altering the normal cyclical activity of the phosphatase PP1 in oocytes causes a range of severe meiotic defects, pointing to essential roles for PP1 in M-Phase entry, progression, and exit.
The phosphorylation of mitotic proteins is bistable, which contributes to the decisiveness of the transitions into and out of M phase. The bistability in substrate phosphorylation has been attributed to bistability in the activation of the cyclin-dependent kinase Cdk1. However, more recently it has been suggested that bistability also arises from positive feedback in the regulation of the Cdk1-counteracting phosphatase, PP2A-B55. Here, we demonstrate biochemically using Xenopus laevis egg extracts that the Cdk1-counteracting phosphatase PP2A-B55 functions as a bistable switch, even when the bistability of Cdk1 activation is suppressed. In addition, Cdk1 regulates PP2A-B55 in a biphasic manner; low concentrations of Cdk1 activate PP2A-B55 and high concentrations inactivate it. As a consequence of this incoherent feedforward regulation, PP2A-B55 activity rises concurrently with Cdk1 activity during interphase and suppresses substrate phosphorylation. PP2A-B55 activity is then sharply downregulated at the onset of mitosis. During mitotic exit Cdk1 activity initially falls with no obvious change in substrate phosphorylation; dephosphorylation then commences once PP2A-B55 spikes in activity. These findings suggest that changes in Cdk1 activity are permissive for mitotic entry and exit, but the changes in PP2A-B55 activity are the ultimate trigger.
APC/C-mediated proteolysis of cyclin B and securin promotes anaphase entry, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-counteracting phosphatases PP1 and PP2A-B55, allowing wide-spread dephosphorylation of substrates. Meanwhile, continued APC/C activity promotes proteolysis of other mitotic regulators. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution proteomics, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid proteolysis of cyclin B, securin and geminin at the metaphase-anaphase transition, followed by slow proteolysis of other substrates. Dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent categories with unique sequence motifs. We conclude that dephosphorylation initiated by selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit.
Here we will review the evidence showing that mitotic exit is initiated by regulated proteolysis and then driven by the PPP‐family of phosphoserine/threonine phosphatases. Rapid APC/CCDC20 and ubiquitin‐dependent proteolysis of cyclin B and securin initiates sister chromatid separation, the first step of mitotic exit. Because proteolysis of Aurora and Polo family kinases dependent on APC/CCDH1 is relatively slow, this creates a new regulatory state, anaphase, different to G2 and M‐phase. We will discuss how the CDK1‐counteracting phosphatases PP1 and PP2A‐B55, together with Aurora and Polo kinases, contribute to the temporal regulation and order of events in the different stages of mitotic exit from anaphase to cytokinesis. For PP2A‐B55 these timing properties are created by the ENSA‐dependent inhibitory pathway and differential recognition of phosphoserine and phosphothreonine. Finally, we will discuss how Aurora B and PP2A‐B56 are needed for the spatial regulation of anaphase spindle formation and how APC/C‐dependent destruction of PLK1 acts as a timer for abscission, the final event of cytokinesis. This article is protected by copyright. All rights reserved.
Dynamic changes in protein phosphorylation govern the transitions between different phases of the cell division cycle. A “tug of war” between highly conserved protein kinases and the family of phosphoprotein phosphatases (PPP) establishes the phosphorylation state of proteins, which controls their function. More than three-quarters of all proteins are phosphorylated at one or more sites in human cells, with the highest occupancy of phosphorylation sites seen in mitosis. Spatial and temporal regulation of opposing kinase and PPP activities is crucial for accurate execution of the mitotic program. The role of mitotic kinases has been the focus of many studies, while the contribution of PPPs was for a long time underappreciated and is just emerging. Misconceptions regarding the specificity and activity of protein phosphatases led to the belief that protein kinases are the primary determinants of mitotic regulation, leaving PPPs out of the limelight. Recent studies have shown that protein phosphatases are specific and selective enzymes, and that their activity is tightly regulated. In this review, we discuss the emerging roles of PPPs in mitosis and their regulation of and by mitotic kinases, as well as mechanisms that determine PPP substrate recognition and specificity.
Cell division is tightly regulated to disentangle copied chromosomes in an orderly manner and prevent loss of genome integrity. During mitosis, transcriptional activity is limited and post-translational modifications (PTMs) are responsible for functional protein regulation. Essential mitotic regulators, including polo-like kinase 1 (PLK1) and cyclin-dependent kinases (CDK), as well as the anaphase-promoting complex/cyclosome (APC/C), are members of the enzymatic machinery responsible for protein modification. Interestingly, communication between PTMs ensures the essential tight and timely control during all consecutive phases of mitosis. Here, we present an overview of current concepts and understanding of crosstalk between PTMs regulating mitotic progression.
The transitions between phases of the cell cycle have evolved to be robust and switch-like, which ensures temporal separation of DNA replication, sister chromatid separation, and cell division. Mathematical models describing the biochemical interaction networks of cell cycle regulators attribute these properties to underlying bistable switches, which inherently generate robust, switch-like, and irreversible transitions between states. We have recently presented new mathematical models for two control systems that regulate crucial transitions in the cell cycle: mitotic entry and exit,¹ Mochida S, Rata S, Hino H, Nagai T, Novák B. Two Bistable Switches Govern M Phase Entry. Curr Biol. 2016;26:3361–3367[Crossref], [PubMed], [Web of Science ®] [Google Scholar] and the mitotic checkpoint.² Mirkovic M, Hutter LH, Novák B, Oliveira RA. Premature sister chromatid separation is poorly detected by the spindle assembly checkpoint as a result of system-level feedback. Cell Rep. 2015;13:469–478[Crossref], [Web of Science ®] [Google Scholar] Each of the two control systems is characterized by two interlinked bistable switches. In the case of mitotic checkpoint control these switches are mutually activating, whereas in the case of the mitotic entry/exit network the switches are mutually inhibiting. In this Perspective we describe the qualitative features of these regulatory motifs and show that having two interlinked bistable mechanisms further enhances robustness and irreversibility. We speculate that these network motifs also underlie other cell cycle transitions and cellular transitions between distinct biochemical states.
During M phase, Endosulfine (Endos) family proteins are phosphorylated by Greatwall kinase (Gwl), and the resultant pEndos inhibits the phosphatase PP2A-B55, which would otherwise prematurely reverse many CDK-driven phosphorylations. We show here that PP2A-B55 is the enzyme responsible for dephosphorylating pEndos during M phase exit. The kinetic parameters for PP2A-B55's action on pEndos are orders of magnitude lower than those for CDK-phosphorylated substrates, suggesting a simple model for PP2A-B55 regulation that we call inhibition by unfair competition. As the name suggests, during M phase PP2A-B55's attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. When Gwl is inactivated during the M phase-to-interphase transition, the dynamic balance changes: pEndos dephosphorylated by PP2A-B55 cannot be replaced, so the phosphatase can refocus its attention on CDK-phosphorylated substrates. This mechanism explains simultaneously how PP2A-B55 and Gwl together regulate pEndos, and how pEndos controls PP2A-B55.
When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A-B55alpha complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A-B55alpha functions downstream of Cdk1 inactivation. PP2A-B55alpha isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-beta1, and RNAi depletion of importin-beta1 delayed mitotic exit synergistically with PP2A-B55alpha. This demonstrates that PP2A-B55alpha and importin-beta1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.
Calyculin-A (CA), okadaic acid (OA), and tautomycin (TAU) are potent inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A)
and are widely used on cells in culture. Despite their well characterized selectivity in vitro, their exact intracellular effects on PP1 and PP2A cannot be directly deduced from their extracellular concentration because
their cell permeation properties are not known. Here we demonstrate that, due to the tight binding of the inhibitors to PP1
and/or PP2A, their cell penetration could be monitored by measuring PP1 and PP2A activities in cell-free extracts. Treatment
of MCF7 cells with 10 nm CA for 2 h simultaneously inhibited PP1 and PP2A activities by more than 50%. A concentration of 1 μm OA was required to obtain a similar time course of PP2A inhibition in MCF7 cells to that observed with 10 nm CA, whereas PP1 activity was unaffected. PP1 was predominantly inhibited in MCF7 cells treated with TAU but even at 10 μm TAU PP1 inhibition was much slower than that observed with 10 nm CA. Furthermore, binding of inhibitors to PP2Ac and/or PP1c in MCF7 cells led to differential posttranslational modifications
of the carboxyl termini of the proteins as demonstrated by Western blotting. OA and CA, in contrast to TAU, induced demethylation
of the carboxyl-terminal Leu309 residue of PP2Ac. On the other hand, CA and TAU, in contrast to OA, elicited a marked decrease in immunoreactivity of the
carboxyl terminus of the α-isoform of PP1c, probably reflecting proteolysis of the protein. These results suggest that in
MCF7 cells OA selectively inhibits PP2A and TAU predominantly affects PP1, a conclusion supported by their differential effects
on cytokeratins in this cell line.
The cyclin E oncogene activates CDK2 to drive cells from G1 to S phase of the cell cycle to commence DNA replication. It coordinates essential cellular functions with the cell cycle including histone biogenesis, splicing, centrosome duplication and origin firing for DNA replication. The two E-cyclins, E1 and E2, are assumed to act interchangeably in these functions. However recent reports have identified unique functions for cyclins E1 and E2 in different tissues, and particularly in breast cancer.
Cyclins E1 and E2 localise to distinct foci in breast cancer cells as well as co-localising within the cell. Both E-cyclins are found in complex with CDK2, at centrosomes and with the splicing machinery in nuclear speckles. However cyclin E2 uniquely co-localises with NPAT, the main activator of cell-cycle regulated histone transcription. Increased cyclin E2, but not cyclin E1, expression is associated with high expression of replication-dependent histones in breast cancers.
The preferential localisation of cyclin E1 or cyclin E2 to distinct foci indicates that each E-cyclin has unique roles. Cyclin E2 uniquely interacts with NPAT in breast cancer cells, and is associated with higher levels of histones in breast cancer. This could explain the unique correlations of high cyclin E2 expression with poor outcome and genomic instability in breast cancer.
The widespread reorganization of cellular architecture in mitosis is achieved through extensive protein phosphorylation, driven by the coordinated activation of a mitotic kinase network and repression of counteracting phosphatases. Phosphatase activity must subsequently be restored to promote mitotic exit. Although Cdc14 phosphatase drives this reversal in budding yeast, protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activities have each been independently linked to mitotic exit control in other eukaryotes. Here we describe a mitotic phosphatase relay in which PP1 reactivation is required for the reactivation of both PP2A-B55 and PP2A-B56 to coordinate mitotic progression and exit in fission yeast. The staged recruitment of PP1 (the Dis2 isoform) to the regulatory subunits of the PP2A-B55 and PP2A-B56 (B55 also known as Pab1; B56 also known as Par1) holoenzymes sequentially activates each phosphatase. The pathway is blocked in early mitosis because the Cdk1-cyclin B kinase (Cdk1 also known as Cdc2) inhibits PP1 activity, but declining cyclin B levels later in mitosis permit PP1 to auto-reactivate. PP1 first reactivates PP2A-B55; this enables PP2A-B55 in turn to promote the reactivation of PP2A-B56 by dephosphorylating a PP1-docking site in PP2A-B56, thereby promoting the recruitment of PP1. PP1 recruitment to human, mitotic PP2A-B56 holoenzymes and the sequences of these conserved PP1-docking motifs suggest that PP1 regulates PP2A-B55 and PP2A-B56 activities in a variety of signalling contexts throughout eukaryotes.
Activation of anaphase-promoting complex/cyclosome (APC/C(Cdc20)) by Cdc20 is delayed by the spindle assembly checkpoint (SAC). When all kinetochores come under tension, the SAC is turned off and APC/C(Cdc20) degrades cyclin B and securin, which activates separase [1]. The latter then cleaves cohesin holding sister chromatids together [2]. Because cohesin cleavage also destroys the tension responsible for turning off the SAC, cells must possess a mechanism to prevent SAC reactivation during anaphase, which could be conferred by a dependence of the SAC on Cdk1 [3-5]. To test this, we analyzed mouse oocytes and embryos expressing nondegradable cyclin B together with a Cdk1-resistant form of separase. After biorientation and SAC inactivation, APC/C(Cdc20) activates separase but the resulting loss of (some) cohesion is accompanied by SAC reactivation and APC/C(Cdc20) inhibition, which aborts the process of further securin degradation. Cyclin B is therefore the only APC/C(Cdc20) substrate whose degradation at the onset of anaphase is necessary to prevent SAC reactivation. The mutual activation of tension sensitive SAC and Cdk1 creates a bistable system that ensures complete activation of separase and total downregulation of Cdk1 when all chromosomes have bioriented.
Entry into mitosis is triggered by activation of Cdk1 and inactivation of its counteracting phosphatase PP2A/B55. Greatwall kinase inactivates PP2A/B55 via its substrates Ensa and ARPP19. Both Greatwall and Ensa/ARPP19 are regulated by phosphorylation, but the dynamic regulation of Greatwall activity and the phosphatases that control Greatwall kinase and its substrates are poorly understood. To address these questions we applied a combination of mathematical modelling and experiments using phospho-specific antibodies to monitor Greatwall, Ensa/ARPP19 and Cdk substrate phosphorylation during mitotic entry and exit. We demonstrate that PP2A/B55 is required for Gwl dephosphorylation at the essential Cdk site Thr194. Ensa/ARPP19 dephosphorylation is mediated by the RNA Polymerase II carboxy terminal domain phosphatase Fcp1. Surprisingly, inhibition or depletion of neither Fcp1 nor PP2A appears to block dephosphorylation of the bulk of mitotic Cdk1 substrates during mitotic exit. Taken together our results suggest a hierarchy of phosphatases coordinating Greatwall, Ensa/ARPP19 and Cdk substrate dephosphorylation during mitotic exit.
Cytokinesis follows separase activation and chromosome segregation. This order is ensured in budding yeast by the mitotic exit network (MEN), where Cdc14p dephosphorylates key conserved Cdk1-substrates exemplified by the anaphase spindle-elongation protein Ase1p. However, in metazoans, MEN and Cdc14 function is not conserved. Instead, the PP2A-B55α/ENSA/Greatwall (BEG) pathway controls the human Ase1p ortholog PRC1. In this pathway, PP2A-B55 inhibition is coupled to Cdk1-cyclin B activity, whereas separase inhibition is maintained by cyclin B concentration. This creates two cyclin B thresholds during mitotic exit. Simulation and experiments using PRC1 as a model substrate show that the first threshold permits separase activation and chromosome segregation, and the second permits PP2A-B55 activation and initiation of cytokinesis. Removal of the ENSA/Greatwall (EG) timer module eliminates this second threshold, as well as associated delay in PRC1 dephosphorylation and initiation of cytokinesis, by uncoupling PP2A-B55 from Cdk1-cyclin B activity. Therefore, temporal order during mitotic exit is promoted by the metazoan BEG pathway.
Progression through the eukaryotic cell cycle is characterized by specific transitions, where cells move irreversibly from stage i-1 of the cycle into stage i. These irreversible cell cycle transitions are regulated by underlying bistable switches, which share some common features. An inhibitory protein stalls progression, and an activatory protein promotes progression. The inhibitor and activator are locked in a double-negative feedback loop, creating a one-way toggle switch that guarantees an irreversible commitment to move forward through the cell cycle, and it opposes regression from stage i to stage i-1. In many cases, the activator is an enzyme that modifies the inhibitor in multiple steps, whereas the hypo-modified inhibitor binds strongly to the activator and resists its enzymatic activity. These interactions are the basis of a reaction motif that provides a simple and generic account of many characteristic properties of cell cycle transitions. To demonstrate this assertion, we apply the motif in detail to the G1/S transition in budding yeast and to the mitotic checkpoint in mammalian cells. Variations of the motif might support irreversible cellular decision-making in other contexts.
Loss of cell division cycle 2 (Cdc2, also known as Cdk1) activity after cyclin B degradation is necessary, but not sufficient, for mitotic exit. Proteins phosphorylated by Cdc2 and downstream mitotic kinases must be dephosphorylated. We report here that protein phosphatase-1 (PP1) is the main catalyst of mitotic phosphoprotein dephosphorylation. Suppression of PP1 during early mitosis is maintained through dual inhibition by Cdc2 phosphorylation and the binding of inhibitor-1. Protein kinase A (PKA) phosphorylates inhibitor-1, mediating binding to PP1. As Cdc2 levels drop after cyclin B degradation, auto-dephosphorylation of PP1 at its Cdc2 phosphorylation site (Thr 320) allows partial PP1 activation. This promotes PP1-regulated dephosphorylation at the activating site of inhibitor-1 (Thr 35) followed by dissociation of the inhibitor-1–PP1 complex and then full PP1 activation to promote mitotic exit. Thus, Cdc2 both phosphorylates multiple mitotic substrates and inhibits their PP1-mediated dephosphorylation.
Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.
The first cell-penetrating peptide
that activates protein phosphatase-1 (PP1) by disrupting a subset of PP1 complexes in living cells has been developed. Activated PP1 rapidly dephosphorylates its substrates, counteracting kinase activity inside cells. Activation of PP1 can thus be a novel approach to study PP1 function and to counteract Ser/Thr kinase activity under pathologically increased kinase signaling.
The atypical AGC kinase Greatwall (Gwl) mediates a pathway that prevents the precocious removal of phosphorylations added
to target proteins by M phase-promoting factor (MPF); Gwl is thus essential for M phase entry and maintenance. Gwl itself
is activated by M phase-specific phosphorylations that are investigated here. Many phosphorylations are nonessential, being
located within a long nonconserved region, any part of which can be deleted without effect. Using mass spectrometry and mutagenesis,
we have identified 3 phosphorylation sites (phosphosites) critical to Gwl activation (pT193, pT206, and pS883 in Xenopus laevis) located in evolutionarily conserved domains that differentiate Gwl from related kinases. We propose a model in which the
initiating event for Gwl activation is phosphorylation by MPF of the proline-directed sites T193 and T206 in the presumptive
activation loop. After this priming step, Gwl can intramolecularly phosphorylate its C-terminal tail at pS883; this site probably
plays a role similar to that of the tail/Z motif of other AGC kinases. These events largely (but not completely) explain the
full activation of Gwl at M phase.
Large numbers of mass spectrometry proteomics studies are being conducted to understand all types of biological processes.
The size and complexity of proteomics data hinders efforts to easily share, integrate, query and compare the studies. The
Model Organism Protein Expression Database (MOPED, htttp://moped.proteinspire.org) is a new and expanding proteomics resource
that enables rapid browsing of protein expression information from publicly available studies on humans and model organisms.
MOPED is designed to simplify the comparison and sharing of proteomics data for the greater research community. MOPED uniquely
provides protein level expression data, meta-analysis capabilities and quantitative data from standardized analysis. Data
can be queried for specific proteins, browsed based on organism, tissue, localization and condition and sorted by false discovery
rate and expression. MOPED empowers users to visualize their own expression data and compare it with existing studies. Further,
MOPED links to various protein and pathway databases, including GeneCards, Entrez, UniProt, KEGG and Reactome. The current
version of MOPED contains over 43 000 proteins with at least one spectral match and more than 11 million high certainty spectra.
Greatwall kinase has been identified as a key element in M phase initiation and maintenance in Drosophila, Xenopus oocytes/eggs, and mammalian cells. In M phase, Greatwall phosphorylates endosulfine and related proteins that bind to and inhibit protein phosphatase 2A/B55, the principal phosphatase for Cdk-phosphorylated substrates. We show that Greatwall binds active PP2A/B55 in G2 phase oocytes but dissociates from it when progesterone-treated oocytes reach M phase. This dissociation does not require Greatwall kinase activity or phosphorylation at T748 in the presumptive T loop of the kinase. A mutant K71M Greatwall, also known as Scant in Drosophila, induces M phase in the absence of progesterone when expressed in oocytes, despite its reduced stability and elevated degradation by the proteasome. M phase induction by Scant Greatwall requires protein synthesis but is not associated with altered binding or release of PP2A/B55 as compared to wild-type Greatwall. However, in vitro studies with Greatwall proteins purified from interphase cells indicate that Scant, but not wild-type Greatwall, has low but detectable activity against endosulfine. These results demonstrate progesterone-dependent regulation of the PP2A/B55-Greatwall interaction during oocyte maturation and suggest that the cognate Scant Greatwall mutation has sufficient constitutive kinase activity to promote M phase in Xenopus oocytes.
Here we investigate the mechanisms regulating Greatwall (Gwl), a serine/threonine kinase essential for promoting the correct
timing of mitosis. We identify Gwl as a unique AGC kinase that, unlike most AGC members, appears to be devoid of a hydrophobic
motif despite the presence of a functional hydrophobic pocket. Our results suggest that Gwl activation could be mediated by
the binding of its hydrophobic pocket to the hydrophobic motif of another AGC kinase. Our molecular modeling and mutagenic
analysis also indicate that Gwl displays a conserved tail/linker site whose phosphorylation mediates kinase activation by
promoting the interaction of this phosphorylated residue with two lysines at the N terminus. This interaction could stabilize
the αC-helix and maintain kinase activity. Finally, the different phosphorylation sites on Gwl are identified, and the role
of each one in the regulation of Gwl kinase activity is determined. Our data suggest that only the phosphorylation of the
tail/linker site, located outside the putative T loop, appears to be essential for Gwl activation. In summary, our results
identify Gwl as a member of the AGC family of kinases that appears to be regulated by unique mechanisms and that differs from
the other members of this family.
Entry into mitosis in eukaryotes requires the activity of cyclin-dependent kinase 1 (Cdk1). Cdk1 is opposed by protein phosphatases
in two ways: They inhibit activation of Cdk1 by dephosphorylating the protein kinases Wee1 and Myt1 and the protein phosphatase
Cdc25 (key regulators of Cdk1), and they also antagonize Cdk1’s own phosphorylation of downstream targets. A particular form
of protein phosphatase 2A (PP2A) containing a B55δ subunit (PP2A- B55δ) is the major protein phosphatase that acts on model
CDK substrates in Xenopus egg extracts and has antimitotic activity. The activity of PP2A-B55δ is high in interphase and low in mitosis, exactly opposite
that of Cdk1. We report that inhibition of PP2A-B55δ results from a small protein, known as α-endosulfine (Ensa), that is
phosphorylated in mitosis by the protein kinase Greatwall (Gwl). This converts Ensa into a potent and specific inhibitor of
PP2A-B55δ. This pathway represents a previously unknown element in the control of mitosis.
CDC14 was originally identified by L. Hartwell in his famous screen for genes that regulate the budding yeast cell cycle. Subsequent work showed that Cdc14 belongs to a family of highly conserved dual-specificity phosphatases that are present in a wide range of organisms from yeast to human. Human CDC14B is even able to fulfill the essential functions of budding yeast Cdc14. In budding yeast, Cdc14 counteracts the activity of cyclin dependent kinase (Cdk1) at the end of mitosis and thus has important roles in the regulation of anaphase, mitotic exit and cytokinesis. On the basis of the functional conservation of other cell-cycle genes it seemed obvious to assume that Cdc14 phosphatases also have roles in late mitosis in mammalian cells and regulate similar targets to those found in yeast. However, analysis of the human Cdc14 proteins (CDC14A, CDC14B and CDC14C) by overexpression or by depletion using small interfering RNA (siRNA) has suggested functions that are quite different from those of ScCdc14. Recent studies in avian and human somatic cell lines in which the gene encoding either Cdc14A or Cdc14B had been deleted, have shown - surprisingly - that neither of the two phosphatases on its own is essential for viability, cell-cycle progression and checkpoint control. In this Commentary, we critically review the available data on the functions of yeast and vertebrate Cdc14 phosphatases, and discuss whether they indeed share common functions as generally assumed.
Entry into mitosis depends on the activity of cyclin-dependent kinases (CDKs). Conversely, exit from mitosis occurs when mitotic cyclins are degraded, thereby extinguishing CDK activity. Exit from mitosis must also require mitotic phosphoproteins to revert to their interphase hypophosphorylated forms, but there is a controversy about which phosphatase(s) is/are responsible for dephosphorylating the CDK substrates. We find that PP2A associated with a B55 delta subunit is relatively specific for a model mitotic CDK substrate in Xenopus egg extracts. The phosphatase activity measured by this substrate is regulated during the cell cycle--high in interphase and suppressed during mitosis. Depletion of PP2A-B55 delta (in interphase) from 'cycling' frog egg extracts accelerated their entry into mitosis and kept them indefinitely in mitosis. When PP2A-B55 delta was depleted from mitotic extracts, however, exit from mitosis was hardly delayed, showing that other phosphatase(s) are also required for mitotic exit. Increasing the concentration of PP2A-B55 delta in extracts by adding recombinant enzyme inhibited the entry into mitosis. This form of PP2A seems to be a key regulator of entry into and exit from mitosis.
The eukaryotic cell cycle comprises an ordered series of events, orchestrated by the activity of cyclin-dependent kinases (Cdks), leading from chromosome replication during S phase to their segregation in mitosis. The unidirectionality of cell-cycle transitions is fundamental for the successful completion of this cycle. It is thought that irrevocable proteolytic degradation of key cell-cycle regulators makes cell-cycle transitions irreversible, thereby enforcing directionality. Here we have experimentally examined the contribution of cyclin proteolysis to the irreversibility of mitotic exit, the transition from high mitotic Cdk activity back to low activity in G1. We show that forced cyclin destruction in mitotic budding yeast cells efficiently drives mitotic exit events. However, these remain reversible after termination of cyclin proteolysis, with recovery of the mitotic state and cyclin levels. Mitotic exit becomes irreversible only after longer periods of cyclin degradation, owing to activation of a double-negative feedback loop involving the Cdk inhibitor Sic1 (refs 4, 5). Quantitative modelling suggests that feedback is required to maintain low Cdk activity and to prevent cyclin resynthesis. Our findings demonstrate that the unidirectionality of mitotic exit is not the consequence of proteolysis but of systems-level feedback required to maintain the cell cycle in a new stable state.
The decision to enter mitosis is mediated by a network of proteins that regulate activation of the cyclin B-Cdk1 complex. Within this network, several positive feedback loops can amplify cyclin B-Cdk1 activation to ensure complete commitment to a mitotic state once the decision to enter mitosis has been made. However, evidence is accumulating that several components of the feedback loops are redundant for cyclin B-Cdk1 activation during normal cell division. Nonetheless, defined feedback loops become essential to promote mitotic entry when normal cell cycle progression is perturbed. Recent data has demonstrated that at least three Plk1-dependent feedback loops exist that enhance cyclin B-Cdk1 activation at different levels. In this review, we discuss the role of various feedback loops that regulate cyclin B-Cdk1 activation under different conditions, the timing of their activation, and the possible identity of the elusive trigger that controls mitotic entry in human cells.
Calyculin-A (CA), okadaic acid (OA), and tautomycin (TAU) are potent inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A) and are widely used on cells in culture. Despite their well characterized selectivity in vitro, their exact intracellular effects on PP1 and PP2A cannot be directly deduced from their extracellular concentration because their cell permeation properties are not known. Here we demonstrate that, due to the tight binding of the inhibitors to PP1 and/or PP2A, their cell penetration could be monitored by measuring PP1 and PP2A activities in cell-free extracts. Treatment of MCF7 cells with 10 nM CA for 2 h simultaneously inhibited PP1 and PP2A activities by more than 50%. A concentration of 1 microM OA was required to obtain a similar time course of PP2A inhibition in MCF7 cells to that observed with 10 nM CA, whereas PP1 activity was unaffected. PP1 was predominantly inhibited in MCF7 cells treated with TAU but even at 10 microM TAU PP1 inhibition was much slower than that observed with 10 nM CA. Furthermore, binding of inhibitors to PP2Ac and/or PP1c in MCF7 cells led to differential posttranslational modifications of the carboxyl termini of the proteins as demonstrated by Western blotting. OA and CA, in contrast to TAU, induced demethylation of the carboxyl-terminal Leu309 residue of PP2Ac. On the other hand, CA and TAU, in contrast to OA, elicited a marked decrease in immunoreactivity of the carboxyl terminus of the alpha-isoform of PP1c, probably reflecting proteolysis of the protein. These results suggest that in MCF7 cells OA selectively inhibits PP2A and TAU predominantly affects PP1, a conclusion supported by their differential effects on cytokeratins in this cell line.
Recently, it has been shown that cyclin B1 was degraded mainly before the onset of anaphase in mammalian cells. When a nondegradable
form of cyclin B1 was introduced into cells, the metaphase-anaphase transition was blocked. This blockage was not due to a
failure in activating anaphase-promoting complex, nor was it due to a failure of degradation of securin. To resolve the question
of whether this blockage by overexpressing the nondegradable form of cyclin B1 is physiologically relevant or not, we developed
a novel method to estimate the relative protein level of the overexpressed cyclin B1 mutant within an individual cell. We
found that a low level of nondegradable cyclin B1 (less than 30% of the endogenous cyclin B1) was sufficient to block the
metaphase-anaphase transition, implying that the blockage of anaphase onset by the nondegradable cyclin B1 was not due to
an artifact of excessive M-phase-promoting factor activity. This result suggests that, in mammalian cells, the majority of
cyclin B1 must be destroyed before the cell can enter anaphase.
Cycles of protein phosphorylation are fundamental in regulating the progression of the eukaryotic cell through its division cycle. Here we test the complement of Drosophila protein kinases (kinome) for cell cycle functions after gene silencing by RNA-mediated interference. We observed cell cycle dysfunction upon downregulation of 80 out of 228 protein kinases, including most kinases that are known to regulate the division cycle. We find new enzymes with cell cycle functions; some of these have family members already known to phosphorylate microtubules, actin or their associated proteins. Additionally, depletion of several signalling kinases leads to specific mitotic aberrations, suggesting novel roles for familiar enzymes. The survey reveals the inter-digitation of systems that monitor cellular physiology, cell size, cellular stress and signalling processes with the basic cell cycle regulatory machinery.
The disassembly of the mitotic spindle and exit from mitosis require the inactivation of Cdk1. Here, we show that expression of nondegradable cyclinB1 causes dose-dependent mitotic arrest phenotypes. By monitoring chromosomes in living cells, we determined that pronounced overexpression of stable cyclinB1 entailed metaphase arrest without detectable sister chromatid separation, while moderate overexpression arrested cells in a pseudometaphase state, in which separated sister chromatids were kept at the cellular equator by a bipolar 'metaphase-like' spindle. Chromosomes that left the pseudometaphase plate became pulled back and individual kinetochores were found to be merotelically attached to both spindle poles in stable cyclinB1 arrested cells. Inactivation of the chromokinesin hKid, by RNAi or antibody microinjection, prevented the formation of stable bipolar spindles and the 'metaphase-like' alignment of chromosomes in cells expressing stable cyclinB1. These experiments show that cyclinB1 is able to maintain a bipolar spindle even after sister chromatids had become separated and suggest an important role of hKid in this process. Cells expressing low levels of nondegradable cyclinB1 progressed further in mitosis and arrested in telophase.
Inhibitor 1 (I-1) is a protein inhibitor of protein phosphatase 1 (PP1), the predominating Ser/Thr phosphatase in the heart. Non-phosphorylated I-1 is inactive, whereas I-1 phosphorylated by protein kinase A (PKA) at Thr35 is a potent PP1 inhibitor. The phosphatases that dephosphorylate I-1Thr35 and thus deactivate I-1 in the heart are not established. Here we overexpressed I-1 in neonatal rat cardiac myocytes with recombinant adenovirus and determined phosphorylation of I-1, and one of the major target proteins of PKA/PP1 in the heart, phospholamban (PLB), by Western blot with phospho-specific antibodies. Incubation with the calcineurin inhibitor cyclosporine A or okadaic acid, used at a concentration preferentially inhibiting phosphatase 2A (PP2A), increased significantly I-1Thr35 (approximately 2- to 6-fold) and PLB Ser16 phosphorylation (approximately 2-fold). The results indicate that calcineurin and PP2A act to maintain a low basal level of phosphorylated (active) I-1 in living cardiac myocytes. Calcineurin may constitute a cross-talk between calcium- and cAMP-dependent pathways.
CDK1 is a nonredundant cyclin-dependent kinase (CDK) with an essential role in mitosis, but its multiple functions still are poorly understood at a molecular level. Here we identify a selective small-molecule inhibitor of CDK1 that reversibly arrests human cells at the G2/M border of the cell cycle and allows for effective cell synchronization in early mitosis. Inhibition of CDK1 during cell division revealed that its activity is necessary and sufficient for maintaining the mitotic state of the cells, preventing replication origin licensing and premature cytokinesis. Although CDK1 inhibition for up to 24 h is well tolerated, longer exposure to the inhibitor induces apoptosis in tumor cells, suggesting that selective CDK1 inhibitors may have utility in cancer therapy.
• apoptosis
• cancer therapy
• cell cycle
• cytokinesis
• replication origin
The PP2A phosphatase is often inactivated in cancer and is considered as a tumour suppressor. A new pathway controlling PP2A activity in mitosis has been recently described. This pathway includes the Greatwall (GWL) kinase and its substrates endosulfines. At mitotic entry, GWL is activated and phosphorylates endosulfines that then bind and inhibit PP2A. We analysed whether GWL overexpression could participate in cancer development. We show that GWL overexpression promotes cell transformation and increases invasive capacities of cells through hyperphosphorylation of the oncogenic kinase AKT. Interestingly, AKT hyperphosphorylation induced by GWL is independent of endosulfines. Rather, GWL induces GSK3 kinase dephosphorylation in its inhibitory sites and subsequent SCF-dependent degradation of the PHLPP phosphatase responsible for AKT dephosphorylation. In line with its oncogenic activity, we find that GWL is often overexpressed in human colorectal tumoral tissues. Thus, GWL is a human oncoprotein that promotes the hyperactivation of AKT via the degradation of its phosphatase, PHLPP, in human malignancies.
We describe an unprecedented reaction between peptide selenoesters and peptide dimers bearing N-terminal selenocystine that proceeds in aqueous buffer to afford native amide bonds without the use of additives. The selenocystine-selenoester ligations are complete in minutes, even at sterically hindered junctions, and can be used in concert with one-pot deselenization chemistry. Various pathways for the transformation are proposed and probed through a combination of experimental and computational studies. Our new reaction manifold is showcased in the total synthesis of two proteins from Mycobacterium tuberculosis; a catalytically active chorismate mutase enzyme and the secreted T-cell antigenic protein ESAT-6.
Entry into mitosis is mediated by the phosphorylation of key cell cycle regulators by cyclin-dependent kinase 1 (Cdk1). In Xenopus embryos, the M-phase-promoting activity of Cdk1 is antagonized by protein phosphatase PP2A-B55. Hence, to ensure robust cell cycle transitions, Cdk1 and PP2A-B55 must be regulated so that their activities are mutually exclusive. The mechanism underlying PP2A-B55 inactivation at mitotic entry is well understood: Cdk1-activated Greatwall (Gwl) kinase phosphorylates Ensa/Arpp19, thereby enabling them to bind to and inhibit PP2A-B55. However, the re-activation of PP2A-B55 during mitotic exit, which is essential for cell cycle progression, is less well understood. Here, we identify protein phosphatase PP1 as an essential component of the PP2A-B55 re-activation pathway in Xenopus embryo extracts. PP1 initiates the re-activation of PP2A-B55 by dephosphorylating Gwl. We provide evidence that PP1 targets the auto-phosphorylation site of Gwl, resulting in efficient Gwl inactivation. This step is necessary to facilitate subsequent complete dephosphorylation of Gwl by PP2A-B55. Thus, by identifying PP1 as the phosphatase initiating Gwl inactivation, our study provides the molecular explanation for how Cdk1 inactivation is coupled to PP2A-B55 re-activation at mitotic exit.
The presence or absence of a phosphorylation on a substrate at any particular point in time is a functional readout of the balance in activity between the regulatory kinase and the counteracting phosphatase. Understanding how stable or short-lived a phos- phorylation site is required for fully appreciating the biological consequences of the phosphorylation. Our current understanding of kinases and their substrates is well established; however, the role phosphatases play is less understood. Therefore, we utilized a phosphatase dependent model of mitotic exit to identify potential substrates that are preferentially dephosphorylated. Using this method, we identified 416,000 phosphosites on 43300 unique proteins, and quantified the temporal phosphorylation changes that occur during early mitotic exit (McCloy et al., 2015 [1]). Furthermore, we annotated the majority of these phosphorylation sites with a high confidence upstream kinase using published, motif and prediction based methods. The results from this study have been deposited into the ProteomeXchange repository with identifier PXD001559. Here we provide additional analysis of this dataset; for each of the major mitotic kinases we identified motifs that correlated strongly with phosphorylation status. These motifs could be used to predict the stability of phosphorylated residues in proteins of interest, and help infer potential functional roles for uncharacterized phosphorylations. In addition, we provide valida- tion at the single cell level that serine residues phosphorylated by Cdk are stable during phosphatase dependent mitotic exit. In summary, this unique dataset contains information on the temporal mitotic stability of thousands of phosphorylation sites regulated by dozens of kinases, and information on the potential preference that phosphatases have at both the protein and individual phosphosite level. The compellation of this data provides an invaluable resource for the wider research community.
Entry into mitosis is driven by the coordinated phosphorylation of thousands of proteins. For the cell to complete mitosis and divide into two identical daughter cells it must regulate dephosphorylation of these proteins in a highly ordered, temporal manner. There is currently a lack of a complete understanding of the phosphorylation changes that occur during the initial stages of mitotic exit in human cells. Therefore, we performed a large unbiased, global analysis to map the very first dephosphorylation events that occur as cells exit mitosis. We identified and quantified the modification of >16,000 phosphosites on >3,300 unique proteins during early mitotic exit, providing up to 8-fold greater resolution than previous studies. The data have been deposited to the ProteomeXchange with identifier PXD001559. Only a small fraction (~10%) of phosphorylation sites were dephosphorylated during early mitotic exit and these occurred on proteins involved in critical early exit events, including organization of the mitotic spindle, the spindle assembly checkpoint, and reformation of the nuclear envelope. Surprisingly this enrichment was observed across all kinase consensus motifs, indicating that it is independent of the upstream phosphorylating kinase. Therefore, dephosphorylation of these sites is likely determined by the specificity of phosphatase/s rather than the activity of kinase/s. Dephosphorylation was significantly affected by the amino acids at and surrounding the phosphorylation site, with several unique evolutionarily conserved amino acids correlating strongly with phosphorylation status. These data provide a potential mechanism for the specificity of phosphatases, and how they co-ordinate the ordered events of mitotic exit. In summary, our results provide a global overview of the phosphorylation changes that occur during the very first stages of mitotic exit, providing novel mechanistic insight into how phosphatase/s specifically regulate this critical transition.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
ABSTRACT Greatwall (Gwl) functions as an essential mitotic kinase by antagonizing protein phosphatase 2A. In this study we identified Hsp90, Cdc37 and members of the importin α and β families as the major binding partners of Gwl. Both Hsp90/Cdc37 chaperone and importin complexes associated with the N-terminal kinase domain of Gwl, whereas an intact glycine-rich loop at the N-terminus of Gwl was essential for binding of Hsp90/Cdc37 but not importins. We found that Hsp90 inhibition led to destabilization of Gwl, a mechanism that may partially contribute to the emerging role of Hsp90 in cell cycle progression and the anti-proliferative potential of Hsp90 inhibition. Moreover, in agreement with its importin association, Gwl exhibited nuclear localization in interphase Xenopus S3 cells, and dynamic nucleocytoplasmic distribution during mitosis. We identified KR456/457 as the locus of importin binding and the functional NLS of Gwl. Mutation of this site resulted in exclusion of Gwl from the nucleus. Finally, we showed that the Gwl nuclear localization is indispensable for the biochemical function of Gwl in promoting mitotic entry.
Entry and progression through mitosis has traditionally been linked directly to the activity of cyclin-dependent kinase 1 (Cdk1). In this study we utilized low doses of the Cdk1-specific inhibitor, RO3306 from early G 2 phase onwards. Addition of low doses of RO3306 in G 2 phase induced minor chromosome congression and segregation defects. In contrast, mild doses of RO3306 during G 2 phase resulted in cells entering an aberrant mitosis, with cells fragmenting centrosomes and failing to fully disassemble the nuclear envelope. Cells often underwent cytokinesis and metaphase simultaneously, despite the presence of an active spindle assembly checkpoint, which prevented degradation of cyclin B1 and securin, resulting in the random partitioning of whole chromosomes. This highly aberrant mitosis produced a significant increase in the proportion of viable polyploid cells present up to 3 days post-treatment. Furthermore, cells treated with medium doses of RO3306 were only able to reach the threshold of Cdk1 substrate phosphorylation required to initiate nuclear envelope breakdown, but failed to reach the levels of phosphorylation required to correctly complete pro-metaphase. Treatment with low doses of Okadaic acid, which primarily inhibits PP2A, rescued the mitotic defects and increased the number of cells that completed a normal mitosis. This supports the current model that PP2A is the primary phosphatase that counterbalances the activity of Cdk1 during mitosis. Taken together these results show that continuous and subtle disruption of Cdk1 activity from G 2 phase onwards has deleterious consequences on mitotic progression by disrupting the balance between Cdk1 and PP2A.
Protein phosphatase-1 (PP1) is a major Ser/Thr phosphatase that is involved in numerous cellular processes. PP1-disrupting peptides (PDPs) are selective chemical tools used to study PP1. They generate catalytically active PP1 inside cells but do not bind to the closely related PP2A. Here, we show that PDPs also do not act directly on PP2B, thus demonstrating the selectivity of PDPs toward PP1. We present PDPs with different properties, enabling reversible versus permanent activation of PP1. We also show that Ca(2+) spiking is an acute effect caused by PDP-induced activation of PP1. The Ca(2+) is released from internal stores. Our data show that PDPs can be used as selective chemical genetics tools to study acute and long-term effects of PP1 activation in intact cells, and PDPs will therefore be valuable tools to study PP1 biology.
Acquired resistance to the anti-estrogen tamoxifen remains a significant challenge in breast cancer management. In this study we used an integrative approach to characterize global protein expression and tyrosine-phosphorylation events in tamoxifen-resistant MCF7 breast cancer cells (TamR) compared with parental controls. Quantitative mass-spectrometry and computational approaches were combined to identify perturbed signalling networks, and candidate regulatory proteins were functionally interrogated by siRNA-mediated knockdown. Network analysis revealed that cellular metabolism was perturbed in TamR cells, together with pathways enriched for proteins associated with growth-factor, cell-cell and cell-matrix-initiated signalling. Consistent with known roles for Ras/MAPK and PI3-kinase signalling in tamoxifen resistance, tyrosine phosphorylated MAPK1, SHC1 and PIK3R2 were elevated in TamR cells. Phosphorylation of the tyrosine kinase Yes and expression of the actin-binding protein MARCKS were elevated 2- and 8-fold in TamR cells respectively, and were selected for further analysis. Knockdown of either protein in TamR cells had no effect on anti-estrogen-sensitivity, but significantly decreased cell motility. MARCKS expression was significantly higher in breast cancer cell lines than normal mammary epithelial cells and in ER-negative versus ER-positive breast cancer cell lines. In primary breast cancers, cytoplasmic MARCKS staining was significantly higher in basal-like and HER2 cancers than in luminal cancers, and was independently predictive of poor survival in multivariate analyses of the whole cohort (p<0.0001) and in ER-positive patients (p=0.0005). These findings provide network-level insights into the molecular alterations associated with the tamoxifen-resistant phenotype and identify MARCKS as a potential biomarker of therapeutic responsiveness that may assist stratification of patients for optimal therapy. This article is protected by copyright. All rights reserved.
Despite the complexity and variety of biological oscillators, their core design invariably includes an essential negative feedback loop. In the Xenopus laevis embryonic cell cycle oscillator, this loop consists of the kinase cyclin B-Cdk1 and the ubiquitin ligase APC/C(Cdc20); active Cdk1 activates APC/C(Cdc20), which then brings about cyclin B degradation and inactivates Cdk1. Here we ask how this negative feedback loop functions quantitatively, with the aim of understanding what mechanisms keep the Cdk1-APC/C(Cdc20) system from settling into a stable steady state with intermediate levels of Cdk1 and APC/C(Cdc20) activity. We found that the system operates as a time-delayed, digital switch, with a time lag of ∼ 15 min between Cdk1 and APC/C(Cdc20) activation and a tremendously high degree of ultrasensitivity (nH≈17). Computational modelling shows how these attributes contribute to the generation of robust, clock-like oscillations. Principles uncovered here may also apply to other activator-repressor oscillators and help in designing robust synthetic clocks.
Fixing Broken DNA
Some physiological processes, such as immunoglobulin class switching and telomere attrition, result in double-stranded DNA breaks. The DNA damage repair protein, 53BP1, prevents nucleolytic processing of these breaks, but the proteins it partners with to do this are unknown (see the Perspective by Lukas and Luka s ). Di Virgilio et al. (p. 711 , published online 10 January), using mass spectroscopy–based methods, and Zimmermann et al. (p. 700 , published online 10 January), using a telomere-based assay, identify Rif1 as a 53BP1 phosphorylation- and DNA damage–dependent interaction partner. Mice with a B cell–specific deletion in Rif1 showed impaired immunoglobulin class switching. Rif1-deficient cells exhibited extensive 5′-3′ resection at DNA ends, with enhanced genetic instability. Thus, Rif1 partners with 53BP1 to promote the proper repair of double-stranded DNA breaks.
After sister chromatid splitting at anaphase onset, exit from mitosis comprises an ordered series of events. Dephosphorylation of numerous mitotic substrates, which were phosphorylated by cyclin-dependent kinase (Cdk), is thought to bring about mitotic exit, but how temporal ordering of mitotic exit events is achieved is poorly understood. Here, we show, using budding yeast, that dephosphorylation of Cdk substrates involved in sequential mitotic exit events occurs with ordered timing. We test different models of how ordering might be achieved by modulating Cdk and Cdk-counteracting phosphatase Cdc14 activities in vivo, as well as by kinetic analysis of Cdk substrate phosphorylation and dephosphorylation in vitro. Our results suggest that the gradual change of the phosphatase to kinase ratio over the course of mitotic exit is read out by Cdk substrates that respond by dephosphorylation at distinct thresholds. This provides an example and a mechanistic explanation for a quantitative model of cell-cycle progression.
Reversible protein phosphorylation is an essential aspect of mitosis and forms the basis of nuclear envelope breakdown, chromosome condensation and spindle assembly. Through global phosphoproteomic analysis, it has become clear that overall protein phosphorylation and phosphosite occupancy is most abundant during mitosis. At mitotic exit, this abundant phosphorylation must be reversed, and this process requires massive and rapid protein dephosphorylation. In addition to this global shift in protein phosphorylation, careful spatial control of protein (de)phosphorylation is equally important for spindle assembly, chromosome disjunction and chromosome alignment. In this review, we discuss the underlying mechanisms that enforce the dramatic global shift in protein phosphorylation as well as the mechanisms that allow for highly localized substrate phosphorylation in mitosis.
The transient mitotic histone H3 phosphorylation by various protein kinases regulates chromosome condensation and segregation, but the counteracting phosphatases have been poorly characterized [1-8]. We show here that PP1γ is the major histone H3 phosphatase acting on the mitotically phosphorylated (ph) residues H3T3ph, H3S10ph, H3T11ph, and H3S28ph. In addition, we identify Repo-Man, a chromosome-bound interactor of PP1γ [9], as a selective regulator of H3T3ph and H3T11ph dephosphorylation. Repo-Man promotes H3T11ph dephosphorylation by an indirect mechanism but directly and specifically targets H3T3ph for dephosphorylation by associated PP1γ. The PP1γ/Repo-Man complex opposes the protein kinase Haspin-mediated spreading of H3T3ph to the chromosome arms until metaphase and catalyzes the net dephosphorylation of H3T3ph at the end of mitosis. Consistent with these findings, Repo-Man modulates in a PP1-dependent manner the H3T3ph-regulated chromosomal targeting of Aurora kinase B and its substrate MCAK. Our study defines a novel mechanism by which PP1 counteracts Aurora B.
Initiation and maintenance of mitosis require the activation of protein kinase cyclin B–Cdc2 and the inhibition of protein
phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall
(Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition.
We identified cyclic adenosine monophosphate–regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl
that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the
absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although
both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry
in Xenopus egg extracts.
Here we show that the functional human ortholog of Greatwall protein kinase (Gwl) is the microtubule-associated serine/threonine kinase-like protein, MAST-L. This kinase promotes mitotic entry and maintenance in human cells by inhibiting protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates cyclin B-Cdc2 substrates. The complete depletion of Gwl by siRNA arrests human cells in G2. When the levels of this kinase are only partially depleted, however, cells enter into mitosis with multiple defects and fail to inactivate the spindle assembly checkpoint (SAC). The ability of cells to remain arrested in mitosis by the SAC appears to be directly proportional to the amount of Gwl remaining. Thus, when Gwl is only slightly reduced, cells arrest at prometaphase. More complete depletion correlates with the premature dephosphorylation of cyclin B-Cdc2 substrates, inactivation of the SAC, and subsequent exit from mitosis with severe cytokinesis defects. These phenotypes appear to be mediated by PP2A, as they could be rescued by either a double Gwl/PP2A knockdown or by the inhibition of this phosphatase with okadaic acid. These results suggest that the balance between cyclin B-Cdc2 and PP2A must be tightly regulated for correct mitotic entry and exit and that Gwl is crucial for mediating this regulation in somatic human cells.
The CyclinB1-Cdk1 kinase is the catalytic activity at the heart of mitosis-promoting factor (MPF), yet fundamental questions concerning its role in mitosis remained unresolved. It is not known when and how rapidly CyclinB1-Cdk1 is activated in mammalian cells, nor how its activation coordinates the substantial changes in the cell at mitosis. Here, we have developed a FRET biosensor specific for CyclinB1-Cdk1 that enables us to assay its activity with very high temporal precision in living human cells. We show that CyclinB1-Cdk1 is inactive in G2 phase and activated at a set time before nuclear envelope breakdown, thereby initiating the events of prophase. CyclinB1-Cdk1 levels rise to their maximum extent over the course of approximately 30 min, and we demonstrate that different levels of CyclinB1-Cdk1 kinase activity trigger different mitotic events, thus revealing how the remarkable reorganization of the cell is coordinated at mitotic entry.
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.
Reversible phosphorylation on serine, threonine and tyrosine is the most widely studied posttranslational modification of proteins. The number of phosphorylated sites on a protein (n) shows a significant increase from prokaryotes, with n </= 7 sites, to eukaryotes, with examples having n >/= 150 sites. Multisite phosphorylation has many roles and site conservation indicates that increasing numbers of sites cannot be due merely to promiscuous phosphorylation. A substrate with n sites has an exponential number (2(n)) of phospho-forms and individual phospho-forms may have distinct biological effects. The distribution of these phospho-forms and how this distribution is regulated have remained unknown. Here we show that, when kinase and phosphatase act in opposition on a multisite substrate, the system can exhibit distinct stable phospho-form distributions at steady state and that the maximum number of such distributions increases with n. Whereas some stable distributions are focused on a single phospho-form, others are more diffuse, giving the phospho-proteome the potential to behave as a fluid regulatory network able to encode information and flexibly respond to varying demands. Such plasticity may underlie complex information processing in eukaryotic cells and suggests a functional advantage in having many sites. Our results follow from the unusual geometry of the steady-state phospho-form concentrations, which we show to constitute a rational algebraic curve, irrespective of n. We thereby reduce the complexity of calculating steady states from simulating 3 x 2(n) differential equations to solving two algebraic equations, while treating parameters symbolically. We anticipate that these methods can be extended to systems with multiple substrates and multiple enzymes catalysing different modifications, as found in posttranslational modification 'codes' such as the histone code. Whereas simulations struggle with exponentially increasing molecular complexity, mathematical methods of the kind developed here can provide a new language in which to articulate the principles of cellular information processing.
Protein phosphatase 1 (PP-1) is known to be a critical component of eukaryotic cell cycle progression. In vitro, our previous studies showed that cdc2 kinase phosphorylates Thr-320 (T320) in PP-1, and that this leads to inhibition of enzyme activity. To examine directly the phosphorylation of PP-1 in intact mammalian cells, an antibody has been prepared that specifically recognizes PP-1C alpha phosphorylated at T320. Cell synchronization studies revealed in a variety of cell types that T320 of PP-1 was phosphorylated to high levels only during early to mid-mitosis. The phosphorylation of T320 of PP-1 was reduced by the cyclin-dependent protein kinase inhibitor, olomoucine, and increased by the PP-1/PP-2A inhibitor, calyculin A. Immunofluorescence microscopy using phospho-T320 antibody indicated that in NIH 3T3 cells the phosphorylation of PP-1 began to increase from basal levels in prophase and to peak at metaphase. Immunostaining indicated that phospho-PP-1 was localized exclusively to nonchromosomal regions. Furthermore, in cell fractionation studies of mitotic cells, phospho-PP-1 was detectable only in the soluble fraction. These observations suggest that phosphorylation by cdc2 kinase in early to mid-mitosis and inhibition of PP-1 activity is likely to contribute to the increased state of phosphorylation of proteins that is critical to the initiation of normal cell division.
It was determined that the myosin phosphatase (MP) activity and content of myosin phosphatase target subunit 1 (MYPT1) were correlated in subcellular fractions of human hepatocarcinoma (HepG2) cells. In control cells MYPT1 was localized in the cytoplasm and in the nucleus, as determined by confocal microscopy. Treatment of HepG2 cells with 50 nM okadaic acid (OA), a cell-permeable phosphatase inhibitor, induced several changes: 1) a marked redistribution of MYPT1 to the plasma membrane associated with an increased level of phosphorylation of MYPT1 at Thr695. Both effects showed only a slight influence with the Rho-kinase inhibitor, Y-27632; 2) an increase in phosphorylation of MYPT1 at Thr850 associated with its accumulation in the perinuclear region and nucleus. These effects were markedly reduced by Y-27632; 3) an increased phosphorylation of the 20 kDa myosin II light chain at Ser19 associated with an increased location of myosin II at the cell center. These effects were partially counteracted by Y-27632; 4) an increase in stress fiber formation and a decrease in cell migration, both OA-induced effects were blocked by Y-27632. In HepG2 lysates, OA (5-100 nM) did not affect MP activity but inhibited PP2A activity. These results indicate that OA induces differential phosphorylation and translocation of MYPT1, dependent on PP2A and, to varying extents, on ROK. These changes are associated with an increased level of myosin II phosphorylation and attenuation of hepatic cell migration.
Natural product extracts have proven to be a rich source of small molecules that potently inhibit the catalytic activity of certain PPP-family ser/thr protein phosphatases. To date, the list of inhibitors includes okadaic acid (produced by marine dinoflagelates, Prorocentrum sp. and Dinophysis sp.), calyculin A, dragmacidins (isolated from marine sponges), microcystins, nodularins (cyanobacteria, Microcystis sp. and Nodularia sp.), tautomycin, tautomycetin, cytostatins, phospholine, leustroducsins, phoslactomycins, fostriecin (soil bacteria, Streptomyces sp.), and cantharidin (blister beetles, approx 1500 species). Many of these compounds share structural similarities, and several have become readily available for research purposes. Here we will review the specificity of available inhibitors and present methods for their use in studying sensitive phosphatases. Common mistakes in the employment of these compounds will also be addressed briefly, notably the widespread misconception that they only inhibit the activity of PP1 and PP2A. Inhibitors of PP2B (calcineurin) will only be mentioned in passing, except to state that, in our hands, cypermethrin, deltamethrin, and fenvalerate, which are sold as potent inhibitors of PP2B, do not inhibit the catalytic activity of PP2B.