The end of mitosis from a phosphatase perspective

Abstract

Transition through mitosis, the cell division cycle phase deputed to segregate replicated chromosomes, requires a wave of protein phosphorylation. While in the past decades a wealth of information has been gathered on the major kinase activities responsible for the onset of mitosis, only recently has a picture emerged of how their effects are reversed by protein phosphatases at the end of mitosis. Here, we summarized some recent data on the relevance for protein phosphatases in the reversal of mitotic phosphorylation required to complete mitosis in vertebrate cells.

Keywords: :

• spindle assembly checkpoint
• phosphatase
• mitosis exit
• anaphase
• Fcp1
• APC/C
• Cdc20
• Mad2
• Wee1
• cdk1

Establishment of mitosis is dictated by the activity of the master mitotic kinase cyclin B-dependent kinase 1 (cycB-cdk1; also called M-phase promoting factor, MPF) and requires extensive protein phosphorylation operated by cycB-cdk1 itself and by a number of other kinases.^1-3 Completion of mitosis relies on inactivation of cycB-cdk1, by means of ubiquitin-dependent cyclin B proteolysis and reversal of mitotic phosphorylation.⁴ During mitosis, protein phosphorylation serves structural changes, like nuclear envelope breakdown, chromatin condensation and spindle assembly, and regulatory purposes, like maintenance of high cycB-cdk1 activity until spindle assembly completion.^4-7 Indeed, in mitosis, cycB-cdk1 sustains itself via phosphorylation-dependent feedback loops: cycB-cdk1 phosphorylates and activates its activator, the phosphatase Cdc25C, while it phosphorylates and inactivates its inactivator, the kinase Wee1.⁸ In addition, activities of cycB-cdk1 and other mitotic kinases are needed for a functional spindle assembly checkpoint (SAC), a safeguard mechanism that delays cyclin degradation and anaphase onset until spindle assembly by restraining the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) in association with its coactivator Cdc20 (APC/C^Cdc20).⁵ Indeed, unattached kinetochores, or lack of adequate interkinetochore tension, activate SAC effector proteins like Mad2 and BubR1 that bind Cdc20 in a mitotic checkpoint complex (MCC) and restrain APC/C ativity.^5,9 Although the precise details by which SAC requires phosphorylation are still unclear, it has been suggested that phosphorylation of Cdc20 and of USP44, a SAC-regulating deubiquitinating peptidase, may help to stabilizes the APC/C^MCC complex.^10-13 Upon spindle assembly completion, exit from mitosis and return to the interphase state require cycB-cdk1 inactivation and reversal of mitotic phosphorylation. It is now clear that protein phosphatases like PP1, PP2A and Cdc14, the latter primarily in yeast, are required to reverse most mitotic phosphorylation downstream of MPF inactivation.⁴ A major controller of PP2A activity in mitosis is the Greatwall kinase (Gwl).^14,15 Gwl is activated in an MPF-dependent manner at the onset of mitosis and inhibits PP2A activity by phosphorylating Endosulphine and ARP19 proteins and transforming them into potent PP2A inhibitors.^16,17 At the end of mitosis, loss of MPF activity decreases Gwl potency, allowing PP2A reactivation.^14,15,18 PP1 appears to be even more directly inactivated by MPF while, upon MPF inactivation at the end of mitosis, its reactivation promotes dephosphorylations required to complete mitosis.¹⁹ Indeed, loss of MPF allows formation of the PP1 chromosome-targeting complex, Repo-Man:PP1 that is required for rearrangement of chromosome structure at the end of mitosis.²⁰ Thus, PP1 and PP2A phosphatases play crucial roles in reversing mitotic phosphorylations, allowing the return to the interphase state downstream cycB-cdk1 inactivation. Until recently, however, the possibility that reversal of mitotic phosphorylation was required to initiate MPF inactivation has been poorly explored. Perhaps this is because it was assumed that upon spindle assembly, lack of activation of SAC effector proteins would directly lead to cycB-cdk1 inactivation by cyclin B proteolysis. However, recent evidence has indicated that, in addition to spindle assembly-dependent loss of SAC effector proteins activation, timely mitosis exit requires active dissolution of previously formed APC/C^MCC complex and a transient MCC-free APC/C^Cdc20 complex.^12,13,21-23 APC/C^MCC complex dissolution appears to be an active, energy-dependent process,^{12,13,21,22,24} while spontaneous release of Cdc20 from Mad2 would be too slow to allow rapid APC/C activation at the metaphase-to-anaphase transition.²⁵ Indeed, APC/C^MCC complex dissolution appears to require ubiquitin-dependent proteolysis and to be strongly affected by phosphorylation.^22,25-27 In the absence of proteasome activity, spindle assembly proceeds apparently normally; however, the SAC inhibitory proteins do not dissociate from APC/C.^22,23,27 Nevertheless, inhibiting mitosis kinases like cycB1-cdk1, Aurora B1 or Mps1 leads to MCC dissociation from the APC/C, even under condition of inhibited proteasome.^22,25 These observations suggest that proteolysis may be directly and indirectly involved in the turnover of APC/C-bound MCC required for rapid SAC inactivation. APC/C-dependent Cdc20 proteolysis may disassemble APC/C-bound MCC, then, depending on spindle assembly, other MCC or free Cdc20 is replaced onto the APC/C.^22,25-27 At the same time, proteolysis may be required for activity of a phosphatase that promotes proteolysis-dependent and -independent disassembly of APC/C-bound MCC, then, depending on spindle assembly, other MCC or free Cdc20 is replaced or exchanged onto the APC/C.^22,25 These mechanisms may contribute to the rapid APC/C^Cdc20 activation that ensues when the last replicated chromosome pair has correctly attached to the spindle.^22,25-28 Recently, expanding on previous genetic evidence obtained in Aspergillus Nidulans from the group of Osmani,²⁹ we have provided data indicating that timely SAC and cycB-cdk1 inactivation at the end of mitosis requires activity of the phosphatase Fcp1.³⁰ Fcp1 is an essential gene in several species known as a crucial transcription regulator by modulating the phosphorylation status of the RNA polymerase II-C-terminal domain (RNAP II-CTD).³¹ Depletion of Fcp1 phosphatase in cells and cell extracts delayed mitosis exit, impairing MCC inactivation, namely Mad2-Cdc20 complex dissolution, without significantly affecting spindle assembly.³⁰ Our data suggest that Fcp1 directly targeted, in a transcription-independent manner, three relevant mitotically phosphorylated proteins involved in SAC and cdk1 activity control: the APC/C coactivator Cdc20, the SAC-sustaining deubiquitinase USP44 and the cdk1 inhibitory kinase Wee1. Dephosphorylation of Cdc20 and USP44 has been described to correlate with SAC silencing and APC/C^Cdc20 activation.^5,12 Indeed, mitotic phosphorylation of USP44 has been suggested to stimulate USP44 activity in maintaining SAC-dependent APC/C inhibition.¹² Our data indicated that Fcp1 can directly dephosphorylate USP44 and that Fcp1-dephosphorylated USP44 has substantially reduced ubiquitin peptidase activity. Cdc20 phosphorylation, as well, has been shown to help sustain the SAC, while Cdc20 dephosphorylation has been recently shown to stimulate interaction with APC/C and ubiquitin ligase activity of the complex.^5,32 In addition, the observation that Fcp1 is required to dephosphorylate Wee1-T-239, to control cdk1 inhibitory phosphorylation, and that this dephosphorylation begins very early during SAC resolution (before significant cyclin degradation),³⁰ suggests that also in somatic cells, reversal of inhibitory phosphorylation of cdk1 is an integral pathway of the mitosis exit program, much like in meiosis and early embryo.^33,34 Indeed, blocking cdk1 inhibitory phosphorylation during mitosis exit in somatic cells, albeit it does not indefinitely arrests cells in mitosis, affects the timing and the quality of mitosis completion (our unpublished observations). How Fcp1 activity is regulated during mitosis exit is still unknown at present. We do know that Fcp1-dependent dephosphorylations require APC/C and proteasome activity;³⁰ however, to establish whether these activities are required to remove a proteic inhibitor of Fcp1 or to affect other mechanisms of Fcp1 control will require further work. Together, our findings suggest that Fcp1 activity is required at the end of mitosis to reverse mitotic phosphorylations that hold active cycB-cdk1 until spindle assembly completion. Exit from mitosis and return to the interphase state requires the action of PP2A and PP1 phosphatases. These phosphatases could be activated as a consequence of loss of cycB-cdk1 activity. For instance, activity of Gwl is sustained by cycB-cdk1, thus, upon cycB-cdk1 inactivation, decreased Gwl activity leads to derepression of PP2A. Loss of cycB-cdk1 activity will also lead to reversal of inhibitory phosphorylation of PP1 and Repo-Man, leading to derepression of PP1-dependent dephosphorylation required for mitosis completion. However, loss of cycB-cdk1 activity may lead to these events, provided that a phosphatase reverses previously performed cycB-cdk1-, or other mitotic kinases-, dependent phosphorylations. We have reported that loss of Fcp1 significantly impaired dephosphorylation of several mitotic phosphorylated proteins, identified by the anti MPM-2 antibody, which may depend on the action of PP2A and PP1. In addition, these dephosphorylations were significantly impaired, even in Fcp1-depleted cell extracts that were treated with a cdk1 inhibitor. Thus, it is possible to hypothesize a phosphatase cascade in the control of mitosis exit. Fcp1 may be involved in reversing mitotic phosphorylations to downregulate cycB-cdk1 activity, but at the same time, phosphorylations that control the action of downstream phosphatases, like PP2A and PP1, for instance, by directly targeting Gwl and/or Endosulphine/ARP19, PP1 and/or Rapo-Man. In this regard we have obtained preliminary results that indicate that Fcp1 physically interacted with Gwl (R.V., L.P., R.D.M, A.P. and D.G. unpublished data). Further work will be required to determine whether Fcp1 may reverse activatory phosphorylation of Gwl, controlling this way activation of PP2A. Also to be investigated in the future will be whether crucial regulatory phosphorylations in PP1 and in Rapo-Man are under the control of Fcp1. The dawn of a phosphatase cascade controlling mitosis exit is appearing at the horizon.

Acknowledgments

The authors wish to thank Associazione Italiana per la Ricerca sul Cancro (AIRC) for support. L.P. is recipient of a fellowship from Fondazione Italiana per la Ricerca sul Cancro (FIRC).