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Title: PP2A/B55 and Fcp1 Regulate Greatwall and Ensa Dephosphorylation during Mitotic Exit}
Article:
Abstract:
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.
Introduction:
Phosphorylation of more than thousand proteins by Cdk1 and other mitotic kinases drives entry into mitosis[1],[2]. As cells exit mitosis, these post-translational modifications have to be removed by phosphatases. Mitotic kinase and phosphatase activity appears to be inversely regulated to avoid futile cycles of phosphorylation and dephosphorylation[3],[4]. Moreover, mitotic kinases themselves are regulated by phosphorylation and dephosphorylation resulting in a complex feedback system of cell cycle control[5]. Cdk1 is negatively regulated by phosphorylation at Thr14/Tyr15 by Wee1 and Myt1 kinases and dephosphorylation of this site by the Cdc25 phosphatase constitutes the decision point to enter mitosis[6]. Cdk1 actively participates in its own activation by negatively regulating its inhibitor Wee1[7]–[9] and positively regulating its activator Cdc25[10]. In Xenopus egg extracts this switch is counteracted by the phosphatase PP2A/B55δ[11],[12] suggesting that inhibition of PP2A/B55δ is an intrinsic element of the G2/M transition. This is achieved by Greatwall kinase( Gwl)[13]–[15] that phosphorylates and activates the PP2A/B55δ inhibitors Endosulfin( Ensa) and ARPP19[16]–[19]. The Gwl phosphorylation motive FDSGDY is identical in Ensa and ARPP19 and thus detectable with the same phospho-specific antibody. For simplicity we will refer in our analysis to Ensa/ARPP19, because it is impossible to distinguish between the phosphorylation of the two proteins with specific antibodies. Depletion of Gwl kinase in Xenopus mitotic extracts results in rapid Cdk1 inactivation and exit from mitosis, while Gwl depletion in interphase extracts blocks Cdk1 Thr14/Tyr15 dephosphorylation and mitotic entry[14],[15]. In human cells Gwl kinase depletion causes a delay in mitotic entry, reduces Cdk substrate phosphorylation and results in chromosome alignment defects and aberrant mitotic exit[20],[21]. Cdk1 phosphorylates Gwl at multiple sites and is required for its activation[22]. Thus, Cdk1 and Gwl activation are locked in a complex feedback loop at the G2/M transition. To gain a more precise understanding of this switch-like transition, the phosphatases that target Gwl itself and Ensa/ARPP19 have to be identified. Inactivation of these phosphatases is likely to play a major role in the initiation of the Cdk1 activation loop. Moreover, the reactivation of these phosphatases is likely to be a crucial element during mitotic exit. The identity of the major Cdk1 counteracting phosphatase in mammalian cells is also still under debate. Current models propose that PP2A/B55δ is not only required for the Cdk1 activation loop, but also to directly dephosphorylate mitotic substrates during mitotic exit[3],[23]. Thus, PP2A/B55δ has been proposed to be the major Cdk1 counteracting phosphatase during mitotic exit in Xenopus egg extracts, equivalent to the function of Cdc14 phosphatase in budding yeast. This hypothesis is based on the observation that co-depletion of Wee1, Myt1 and Gwl causes mitotic exit from Xenopus egg extracts despite persistent high Cdk1 activity[24]. Conversely, B55δ depletion does not block Cdk1 substrate dephosphorylation, when mitotic exit is triggered by Cdk1 inhibition in Xenopus egg extracts[12] and PP1 has also been implicated to act as a major mitotic exit phosphatase[25]. In human cells PP2A/B55α has been implicated in regulating mitotic exit, but B55α depletion shows only a delay but not a block of Cdk substrate dephosphorylation following Cdk inactivation[26]. Thus, the network of phosphatases that counteract Gwl, Ensa/ARPP19 and Cdk phosphorylation during mitotic exit remains to be determined.
Discussion:
The results presented here suggest a complex phosphatase network counteracting the Cdk1 activation loop. These data allow us to update the model of the mammalian mitotic switch by including the new information on PP2A/B55 and Fcp1( Fig. 6). The inclusion of an additional layer of phosphatase regulation does not alter the fundamental characteristic of the mitotic switch model, but it influences the temporal dynamics of mitotic entry and exit. Our mathematical modeling together with experiments indicates that mutual antagonism between Gwl and PP2A/B55 accelerates both mitotic entry and exit with synthesis and degradation of CycB, respectively. Our data also indicate that while PP2A is principally responsible for the inactivation step of Gwl, phosphatases other than PP2A regulate additional phosphorylation sites and also contribute to Gwl inactivation( see Fig. 4C and D). We have also identified Fcp1 as the phosphatase counteracting the Gwl phosphorylation site in Ensa/ARPP19. This implicates that Fcp1 is yet another intrinsic element of the mitotic switch and helps explain the observation that this phosphatase is required for mitotic exit[32]. Fcp1 appears to be subjected to further regulation by Cdk1 and activated during mitotic exit. These novel elements of the mitotic switch remain to be investigated. Strikingly neither PP2A, PP1 nor Fcp1 appear to be sufficient to dephosphorylate the bulk of mitotic Cdk1 substrates, if the Gwl feedback loop is bypassed by Cdk1 inhibition. Dephosphorylation of the majority of Cdk substrates proceeds despite the presence of inhibitors( Fig. 4A), depletion of B55 subunits( Fig. 5A) and Fcp1( Fig. 5B) and persistent activation of Gwl and Ensa( Fig. 4A and 5B), which additionally inhibit PP2A/B55. These results suggest that PP2A/B55 is not the only mitotic exit phosphatase that removes the bulk of mitotic phosphorylation. This notion is also supported by experiments in Xenopus egg extracts, in which Cdk1 inhibition by p27 can trigger mitotic dephosphorylation even in the absence of B55δ[12]. This is further supported by the observation that B55 depleted cells do not show a prolonged arrest in mitosis, as would be expected in the absence of the major Cdk1 antagonizing phosphatase[26]. Thus, the nature of the phosphatases that directly counteracts Cdk1 during the mitotic exit in mammalian cells remains elusive. In yeast Cdc14 covers this role, but single deletions of the mammalian equivalents Cdc14 A and B do not have the expected mitotic phenotypes[33],[34]. However, a double deletion of both orthologues has, to our knowledge, not been reported. Further experiments will be necessary to identify the mammalian version of this crucial mitotic exit phosphatase, and to analyse how it is integrated in the mitotic entry and exit switches.
Greatwall kinase regulates a switch between kinase and phosphatase activity during mitotic entry and constitutes an essential element of mitotic control . This control system is further complicated by the fact that Greatwall itself is regulated via phosphorylation and acts by phosphorylating its substrates ENSA and ARPP19 . A missing link in this central mitotic switch is the phosphatase that counteracts Greatwall and its target Ensa . We have used mathematical modeling and experimental validation to identify these phosphatases . We demonstrate genetic evidence that Greatwall phosphorylation is counteracted by PP2A/B55 , while Fcp1 regulates ENSA dephosphorylation . Based on these findings we present a new model for the regulation of mammalian cell division .