[Cell Cycle 3:5, 601-603; May 2004]; ©2004 Landes Bioscience
Inactivating Cdc25, Mitotic Style
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ABSTRACT
Benjamin A. Wolfe1 Kathleen L. Gould1,2,*
KEY WORDS
INTRODUCTION
Cdc25, mitotic entry, mitotic exit, Cdk, phosphorylation
The activity of the major mitotic regulatory kinase, the Cdk1-cyclin B complex, oscillates during the cell cycle, being low in interphase and peaking during mitotic progression. Its activity is balanced by the antagonistic actions of the inhibitory Wee1 and Myt1 kinases and the activating phosphatase family of Cdc25 proteins, which together regulate the Thr-14/Tyr-15 phosphorylation state of Cdk1. The relative balance in activities of these regulators determines the cell cycle state, with Wee1 and Myt1 being active in interphase, and inactive during mitosis. Conversely, Cdc25 is activated in mitosis, creating a simple positive feedback loop whereby Cdk1 stimulates Cdc25 and inhibits Wee1 and Myt1 at the G2/M transition.1 This scenario has been used to explain the rapid and irreversible commitment to mitosis. During the past decade, much has been learned about the reactions catalyzed at the G2/M transition, including those that allow for Cdc25 activation. However, fewer studies have focused on the coordinated events in late mitosis that render Cdc25 inactive. Integrating biochemical data from Xenopus and mammalian cells concerning mitotic initiation with our recent analysis of Cdc25 inactivation during mitotic exit in S. pombe, we can now describe in some detail the oscillations in Cdc25 function that contribute to the abrupt activation and inactivation of Cdk1-cyclin B at mitotic initiation and exit, respectively.
1Department of Cell and Developmental Biology and 2Howard Hughes Medical Institute; Vanderbilt University; Nashville, Tennessee USA
*Correspondence to: Kathleen L. Gould; Department of Cell and Developmental Biology and HHMI; Vanderbilt University; 1161 21st Avenue South; Nashville, Tennessee 37232 USA; Tel: 615.343.9502; Fax: 615.343.0723; Email:
[email protected]
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We apologize to those authors whose papers have not been cited due to space limitations. B.A.W. is supported by National Institutes of Health Grant, GM068786. K.L.G is an Investigator of the Howard Hughes Medical Institute.
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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=891
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Received 03/31/04; Accepted 03/31/04
Mitotic entry and exit require activation and inactivation of the Cdk1-cyclin B kinase complex, respectively. The Cdc25 protein phosphatase family activates Cdk1-cyclin B at the G2/M transition by removing inhibitory phosphate groups. Cdc25 family members, held inactive during interphase, are activated during mitotic progression in an amplification loop involving Cdk1-cyclin B. While Cdc25 activation at the G2/M transition is required for the timely initiation of mitosis, recent evidence suggests that the inactivation of Cdc25 in late mitosis may play a role in supporting Cdk1-cyclin B inactivation. Here, we discuss the mechanisms of Cdc25 regulation and how they pertain to both mitotic entry and exit.
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ACTIVATION AT MITOTIC ENTRY Cdc25 proteins are conserved from yeast to mammals, with many higher eukaryotes possessing a number of Cdc25 isoforms (i.e., mammalian A, B and C) playing both overlapping and nonoverlapping roles.2 A shared feature among different Cdc25 isoforms and orthologs is their hyperphosphorylation during mitotic commitment. Depending on which isoform of Cdc25 is involved, hyperphosphorylation can have varying effects: hyperactivation of its phosphatase activity (as occurs with Cdc25C) and/or a stabilizing effect by preventing its recognition by the ubiquitination machinery (as occurs with Cdc25A). Dephosphorylation at mitotic exit either returns Cdc25C phosphatase activity to basal levels or promotes Cdc25A recognition by the E3 ubiquitin ligase, the Anaphase-Promoting Complex (APC/C). S. pombe contains a single Cdc25 protein which is responsible for all phosphatase-dependent activation of Cdk1-cyclin B.3 SpCdc25 appears to possess qualities of both Cdc25C and A as it becomes hyperactive and stabilized during mitotic commitment.4,5 Although it is destabilized during mitotic exit via the APC/C, at this point it is not clear if its mitotic phosphorylation plays a protective role, preventing its recognition by the ubiquitination machinery as occurs with hCdc25A. Many previous investigations of the involvement of Cdc25 proteins in G2/M regulation focused on Cdc25C and the kinases involved in its activation, although recent evidence Cell Cycle
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INACTIVATION AT MITOTIC EXIT
Both Cdc25C and A isoforms are dephosphorylated in late mitosis.6 SpCdc25 is similarly dephosphorylated as cells exit mitosis, and we have recently shown that this requires the S. pombe Cdc14 family member, Clp1. Because both Cdc25C and A are phosphorylated in vitro and in vivo by Cdk1-cyclin B, these two isoforms likely represent targets of one or both of the Cdc14 orthologs in higher eukaryotes. Although this has yet to be demonstrated, we speculate that dephosphorylation of Cdc25C and SpCdc25 proteins in late mitosis by Cdc14 family members might play 2 roles: first, it would inactivate the hyperactive phosphatase, and second, it would free the phosphatase to be phosphorylated yet again by one of the response site kinases, ultimately leading to its nuclear exclusion via 14-3-3 binding. Consistent with this idea, Cdc25 is not properly removed from the nucleus in clp1 mutants during late mitosis,5 potentially because rephosphorylation of the response site is blocked. A similar defect is observed in mutants of the S. pombe 14-3-3 protein, Rad24.16 Do the Cdc25C inactivating phosphatases function with 14-3-3 proteins to promote nuclear exclusion of Cdc25C in late mitosis, and if so, which kinase(s) is the late mitotic response site kinase? While considerable information could be gained from experiments in the genetically tractable S. pombe, these are difficult questions to address as there are 9 sites which can interact with 14-3-3 molecules when a checkpoint response is elicited,17 instead of just one critical site as in Xenopus and mammals. Experiments from Xenopus and mammalian cells closely examining the regulation of the dephosphorylation and phosphorylation reactions at the Cdc25 response site will greatly aid our knowledge of these signals. As for a potential late mitotic response site kinase, PKA may be an excellent candidate as its activity is decreased upon mitotic commitment, but then resurges just prior to cyclin destruction in Xenopus early embryonic cycles.18 Does preventing Cdc25 inactivation in late mitosis have an effect on the timing of Cdk1-cyclin B inactivation? We have found that cells lacking clp1 do delay mitotic Cdk1 inactivation.5 We asked whether it was the presence of hyperactive, stable Cdc25 in the nucleus of these cells that was responsible for sustained Cdk1 activity. Indeed, when Cdc25 was inactivated in clp1 mutant cells, the kinetics of Cdk1 inactivation occurred with only a slight delay, suggesting that failure to disrupt the Cdk1 auto-feedback loop can maintain the balance in favor of the mitotic state. In much the same way that the positive feedback loop rapidly stimulates mitotic entry, inactivation of it can quickly turn the tables on Cdk1 activity.
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suggests that Cdc25A may also play an important role in dephosphorylation of Cdk1-cyclin B during this transition.6 Izumi and Maller first identified phosphorylation sites in the N-terminus critical for XCdc25C mitotic hyperactivation. Because many of these sites corresponded to Cdk1 consensus sites (S/T-P-X-K/R),7 and because in vitro recombinant Cdk1-cyclin B could stimulate hCdc25C activity,8 it was hypothesized that Cdk1-cyclin B positively regulated its own activity through a feed-forward loop. Another variable was added when an additional mitotic kinase, the Xenopus Polo-like kinase Plx1, was purified as a Cdc25C activating kinase.9 More recently, Polo boxes, the signature motif found in all Polo-like kinases, have been shown to be a phosphoepitope binding module, targeted to Cdc25C after prior phosphorylation by Cdk1.10 Hence, Cdk1cyclin B may not only stimulate some degree of mitotic Cdc25C activity, but also recruit Polo kinases to the phosphatase and allow further activation. Although it has become increasingly clear that both Cdk1 and Polo kinases are involved in Cdc25 activation at the G2/M transition, the chain of events leading to the initiation of the positive feedback loop have been less clear. During interphase growth and in times of DNA damage or stress, Cdc25 proteins are prevented from entering the nucleus through interaction with 14-3-3 proteins.11 These small proteins recognize a phosphoepitope on Cdc25 resulting from phosphorylation at the checkpoint response site Ser-216 (Ser-287 in Xenopus). This site appears to integrate many different cellular cues, each one producing the same outcome: cell cycle delay through Cdc25 inhibition. A number of kinases are reported to phosphorylate this site in vivo including Chk1/2, C-Tak1,12 and the cAMP dependent protein kinase A (PKA).13 In addition to retaining Cdc25 in the cytoplasm, 14-3-3 binding blocks dephosphorylation at the response site. How then is this interaction disrupted temporally and spatially to allow for mitotic initiation with the proper kinetics? The Kornbluth lab has identified Cdk2 as a “primer” kinase which can disrupt the interaction between 14-3-3 and Cdc25 by phosphorylating one of the N-terminal mitotic phosphosites in Cdc25.14 14-3-3 removal then allows dephosphorylation of the response site by the phosphatase PP1 in Xenopus, ultimately leading to nuclear accumulation and activation of Cdc25 through mitotic phosphorylations. When coupled with evidence that mitotic phosphorylations at critical -2 Ser sites by Cdk1 in Xenopus (Ser-285) and mammals (Ser-214) prevents phosphorylation at the checkpoint response site,15 these results help us to build a new model of how mitotic commitment is not only initiated but sustained (Fig. 1).
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Figure 1. Mitotic Regulation of Cdc25 proteins. During interphase and in times of checkpoint activation, Cdc25 is tethered in the cytoplasm through its phosphorylation-dependent interaction with 14-3-3 molecules. At the G2/M transition, Cdk2, acting as a mitotic initiation trigger, phosphorylates Cdc25 and frees it from 14-3-3, in turn, allowing the response site to be dephosphorylated by PP1. Cdc25 activity is further stimulated by the mitotic kinases, Cdk1 and Polo, which together either stimulate Cdc25 activity (Cdc25C) or protect it from proteolysis (Cdc25A). These mitotic phosphorylations are reversed by phosphatases (Cdc14, PP2A), turning off the positive amplification loop and resetting Cdc25 activity and levels to those seen in interphase.
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16. Lopez-Girona A, Furnari B, Mondesert O, Russell P. Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein. Nature 1999; 397:172-5. 17. Zeng Y, Piwnica-Worms H. DNA damage and replication checkpoints in fission yeast require nuclear exclusion of the Cdc25 phosphatase via 14-3-3 binding. Mol Cell Biol 1999; 19:7410-9. 18. Grieco D, Porcellini A, Avvedimento EV, Gottesman ME. Requirement for cAMP-PKA pathway activation by M phase-promoting factor in the transition from mitosis to interphase. Science 1996; 271:1718-23. 19. Tang Z, Coleman TR, Dunphy WG. Two distinct mechanisms for negative regulation of the Wee1 protein kinase. EMBO J 1993; 12:3427-36. 20. Harvey SL, Kellogg DR. Conservation of mechanisms controlling entry into mitosis: Budding yeast wee1 delays entry into mitosis and is required for cell size control. Curr Biol 2003; 13:264-75.
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There are two plausible mechanisms that could account for the disruption of the Cdk1 positive feedback loop in late mitosis. Downregulation of Cdk1-cyclin B by degradation of small pools of cyclin B could reduce Cdk1 activity to levels such that more cyclin B is targeted for degradation. Disruption of the positive feedback loop would therefore occur passively as the amount of Cdk1 activity required to sustain it is eliminated. Second, active disruption of the positive feedback loop through activation of specific phosphatase(s) could reduce Cdk1 activity to levels that are competent to stimulate APC activity. This would subsequently lead to more cyclin B destruction and a concomitant drop in kinase activity. We believe that disruption of this feedback loop plays an active role in lowering Cdk1 activity, and we speculate that the concerted efforts of protein phosphatases (i.e., Cdc14 family members) and response site kinases play important roles in turning off this amplification loop. The latter model also suggests that the inhibitory Thr-14/Tyr-15 kinases are similarly dephosphorylated and activated during late mitosis to aid in the inactivation of Cdk1-cyclin B. Wee1 proteins in Xenopus are inactivated through phosphorylation in mitosis,19 possibly due to the concerted efforts of Cdk1-cyclin B and Polo kinases. Although it is not clear if Wee1 proteins are reactivated by dephosphorylation in late mitosis, S. cerevisiae Swe1p is transiently dephosphorylated during the exit from mitosis, just prior to its degradation and degradation of the major cyclin B, Clb2.20 While activity assays have not been reported for Swe1p, its mitotic hyperphosphorylation may inhibit its catalytic activity. Therefore it is possible that the dephosphorylated form of Swe1p in late mitosis plays a role in inactivating Cdk1-cyclin B. Should this be the case, then the feed-forward amplification loop which rapidly stimulates activation of Cdk1-cyclin B at mitotic onset, additionally provides a rapid switch to inactivate mitotic Cdk1-cyclin B at mitotic exit. References
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1. Coleman TR, Dunphy WG. Cdc2 regulatory factors. Curr Opin Cell Biol 1994; 6:877-82. 2. Donzelli M, Draetta GF. Regulating mammalian checkpoints through Cdc25 inactivation. EMBO Rep 2003; 4:671-7. 3. Russell P, Nurse P. cdc25+ functions as an inducer in the mitotic control of fission yeast. Cell 1986; 45:145-53. 4. Kovelman R, Russell P. Stockpiling of Cdc25 during a DNA replication checkpoint arrest in Schizosaccharomyces pombe. Mol Cell Biol 1996; 16:86-93. 5. Wolfe BA, Gould KL. Fission yeast Clp1p phosphatase affects G2/M transition and mitotic exit through Cdc25p inactivation. Embo J 2004; 23:919-29. 6. Mailand N, Podtelejnikov AV, Groth A, Mann M, Bartek J, Lukas J. Regulation of G2/M events by Cdc25A through phosphorylation-dependent modulation of its stability. EMBO J 2002; 21:5911-20. 7. Izumi T, Maller JL. Elimination of cdc2 phosphorylation sites in the cdc25 phosphatase blocks initiation of M-phase. Mol Biol Cell 1993; 4:1337-50. 8. Hoffmann I, Clarke PR, Marcote MJ, Karsenti E, Draetta G. Phosphorylation and activation of human cdc25-C by cdc2—cyclin B and its involvement in the self-amplification of MPF at mitosis. EMBO J 1993; 12:53-63. 9. Kumagai A, Dunphy WG. Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts. Science 1996; 273:1377-80. 10. Elia AE, Cantley LC, Yaffe MB. Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates. Science 2003; 299:1228-31. 11. Takizawa CG, Morgan DO. Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C. Curr Opin Cell Biol 2000; 12:658-65. 12. Peng CY, Graves PR, Ogg S, Thoma RS, Byrnes 3rd MJ, Wu Z, et al. C-TAK1 protein kinase phosphorylates human Cdc25C on serine 216 and promotes 14-3-3 protein binding. Cell Growth Differ 1998; 9:197-208. 13. Duckworth BC, Weaver JS, Ruderman JV. G2 arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A. Proc Natl Acad Sci USA 2002; 99:16794-9. 14. Margolis SS, Walsh S, Weiser DC, Yoshida M, Shenolikar S, Kornbluth S. PP1 control of M phase entry exerted through 14-3-3-regulated Cdc25 dephosphorylation. EMBO J 2003; 22:5734-45. 15. Bulavin DV, Higashimoto Y, Demidenko ZN, Meek S, Graves P, Phillips C, et al. Dual phosphorylation controls Cdc25 phosphatases and mitotic entry. Nat Cell Biol 2003; 5:545-51.
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