Coordinated regulation of M phase exit and S phase entry by the Cdc2 activity level in the early embryonic cell cycle

In the early embryonic cell cycle, exit from M phase is immediately followed by entry into S phase without an intervening gap phase. To understand the regulatory mechanisms for the cell cycle transition from M to S phase, we examined dependence on Cdc2 inactivation of cell-cycle events occurring during the M-S transition period, using Xenopus egg extracts in which the extent of Cdc2 inactivation at M phase exit was quantitatively controlled. The result demonstrated that MCM binding to and the initiation of DNA replication of nuclear chromatin occurred depending on the decrease of Cdc2 activity to critical levels. Similarly, we found that Cdc2 inhibitory phosphorylation and cyclin B degradation were turned on and off, respectively, depending on the decrease in Cdc2 activity. However, their sensitivity to Cdc2 activity was different, with the turning-on of Cdc2 inhibitory phosphorylation occurring at higher Cdc2 activity levels than the turning-off of cyclin B degradation. This means that, when cyclin B degradation ceases at M phase exit, Cdc2 inhibitory phosphorylation is necessarily activated. In the presence of constitutive synthesis of cyclin B, this condition favors the occurrence of the Cdc2 inactivation period after M phase exit, thereby ensuring progression through S phase. Thus, M phase exit and S phase entry are coordinately regulated by the Cdc2 activity level in the early embryonic cell cycle.     

The cdc25 phosphatase is essential for the G2/M phase transition in the basidiomycete yeast Ustilago maydis

Cdc25-related phosphatases reverse the inhibitory phosphorylation of mitotic Cyclin-dependent kinases mediated by Wee1-related kinases, thereby promoting entry into mitosis. In the fission yeast, Schizosaccharomyces pombe, Cdc25 is required for entry into mitosis, while in the budding yeast Saccharomyces cerevisiae, Mih1 (the homologue of Cdc25) is not required for entry into mitosis or for viability. As these differences were linked to the different cell division and growth mechanism of these species, we sought to analyse the roles of Cdc25 in Ustilago maydis, which as S. cerevisiae divides by budding, but relies in a polar growth. This basidiomycete yeast is perfectly suited to analyse the relationships between cell cycle and morphogenesis. We show that U. maydis contains a single Cdc25-related protein, which is essential for growth. Loss of Cdc25 function results in a specific G2 arrest that correlated with high level of Tyr15 phosphorylation of Cdk1. Moreover, we show genetic interactions of cdc25 with wee1 and clb2 that support the notion that in U. maydis Cdc25 counteracts the Wee1-mediated inhibitory phosphorylation of Cdk1-Clb2 complex. Our results supports a model in which inhibitory phosphorylation of Cdk1 is a primary mechanism operating at G2/M transition in this fungus.     

Cdc2 and Mos regulate Emi2 stability to promote the meiosis I-meiosis II transition

The transition of oocytes from meiosis I (MI) to meiosis II (MII) requires partial cyclin B degradation to allow MI exit without S phase entry. Rapid reaccumulation of cyclin B allows direct progression into MII, producing a cytostatic factor (CSF)-arrested egg. It has been reported that dampened translation of the anaphase-promoting complex (APC) inhibitor Emi2 at MI allows partial APC activation and MI exit. We have detected active Emi2 translation at MI and show that Emi2 levels in MI are mainly controlled by regulated degradation. Emi2 degradation in MI depends not on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), but on Cdc2-mediated phosphorylation of multiple sites within Emi2. As in MII, this phosphorylation is antagonized by Mos-mediated recruitment of PP2A to Emi2. Higher Cdc2 kinase activity in MI than MII allows sufficient Emi2 phosphorylation to destabilize Emi2 in MI. At MI anaphase, APC-mediated degradation of cyclin B decreases Cdc2 activity, enabling Cdc2-mediated Emi2 phosphorylation to be successfully antagonized by Mos-mediated PP2A recruitment. These data suggest a model of APC autoinhibition mediated by stabilization of Emi2; Emi2 proteins accumulate at MI exit and inhibit APC activity sufficiently to prevent complete degradation of cyclin B, allowing MI exit while preventing interphase before MII entry.     

PKN Activation via Transforming Growth Factor-β1 (TGF-β1) Receptor Signaling Delays G2/M Phase Transition in Vascular Smooth Muscle Cells

The transition of vascular smooth muscle cells (VSMCs) from G2 phase into the M (mitosis) phase of the cell cycle is a tightly controlled process. As an arterial SMC prepares for a G2/M transition, the cell has primed the Cdc2/cyclinB1 complex for activation by the phosphorylation of threonine-161 residue on Cdc2. This phosphorylation is necessary but not sufficient for the VSMC to enter into the M phase. In order to enter into mitosis, a phosphatase, Cdc25C, must first dephosphorylate two other critical residues: tyrosine-15 and threonine-14. If Cdc25C phosphatase activity is blocked, VSMC entry into mitosis is delayed. However, how the activity of Cdc25C is regulated has not been fully illustrated.In an earlier published study we have demonstrated that exposure of the VSMC line, PAC-1, to Transforming growth factor-β1 (TGF-β1), activated PKN (a RhoA-dependent kinase). Here we show that exposure to TGF-β1 delays the G2/M transition by 2 hrs in G1/S synchronized and released PAC-1 culture. This delay is abolished by the RhoA kinase inhibitors, HA1077 or Y-27632. More importantly, RNAi knockdown of PKN expression prevents the G2/M transition delay induced by TGF-β1. Changes in PKN activity temporally correlates to the G2/M transition timing. Moreover, Cdc25C is phosphorylated by the TGF-β1-activated PKN. PKN and Cdc25C coimmunoprecipitate with each other. Finally, PKN and Cdc25C co-localize to the nuclear region only during the critical period of time prior to entry into the M phase. Our data demonstrate that Cdc25C activity is negatively regulated by TGF-β1-stimulated PKN. Once activated through TGF-β1 signaling, PKN binds to and phosphorylates Cdc25C. The physical interaction and phosphorylation result in an inactivation of Cdc25C and delay the VSMC entry into the M stage of the cell cycle.     

The Zds proteins control entry into mitosis and target protein phosphatase 2A to the Cdc25 phosphatase

The Wee1 kinase restrains entry into mitosis by phosphorylating and inhibiting cyclin-dependent kinase 1 (Cdk1). The Cdc25 phosphatase promotes entry into mitosis by removing Cdk1 inhibitory phosphorylation. Experiments in diverse systems have established that Wee1 and Cdc25 are regulated by protein phosphatase 2A (PP2A), but a full understanding of the function and regulation of PP2A in entry into mitosis has remained elusive. In budding yeast, entry into mitosis is controlled by a specific form of PP2A that is associated with the Cdc55 regulatory subunit (PP2A(Cdc55)). We show here that related proteins called Zds1 and Zds2 form a tight stoichiometric complex with PP2A(Cdc55) and target its activity to Cdc25 but not to Wee1. Conditional inactivation of the Zds proteins revealed that their function is required primarily at entry into mitosis. In addition, Zds1 undergoes cell cycle-dependent changes in phosphorylation. Together, these observations define a role for the Zds proteins in controlling specific functions of PP2A(Cdc55) and suggest that upstream signals that regulate PP2A(Cdc55) may play an important role in controlling entry into mitosis.     

Critical effect of pigWee1B on the regulation of meiotic resumption in porcine immature oocytes

Porcine immature oocytes require protein synthesis for meiotic resumption, thus the importance of Cdc2 inhibitory phosphorylation in their meiotic arrest remains controversial. We examined the involvement of Cdc2 phosphorylation in the meiotic arrest of porcine oocytes with a special focus on Wee1B, an oocyte-specific Wee1 family member recently reported in mouse oocytes. We cloned a Wee1B homologue of pig by RT-PCR followed by 5’- and 3’-RACE. Overexpression of pigWee1B in porcine immature oocytes by the injection of pigWee1B mRNA almost completely blocked the germinal vesicle breakdown (GVBD) under the low cAMP concentration, which could not block their spontaneous meiotic resumption by itself. The MPF activation and cyclin B synthesis were inhibited in these oocytes. Conversely, downregulation of pigWee1B expression by the injection of specific antisense mRNA induced GVBD in the oocytes, the spontaneous meiotic resumption of which was blocked by the high concentration of cAMP (dbcAMP). In these oocytes, the MPF activity was elevated and cyclin B was accumulated. Downregulation of pigMyt1, another Wee1 family member, could not induce the GVBD under the same condition. The inhibition of tyrosine phosphatase by vanadate blocked the GVBD even in the pigWee1B-downregulated oocytes. These results suggest that the inhibitory phosphorylation of CDC2, which is catalyzed by pigWee1B, but not pigMyt1, is involved in the meiotic arrest of porcine oocytes, and that the inactivation of Wee1B in combination with the phosphatase activation induces the conversion of pre-MPF to the active MPF and starts the cyclin B synthesis, follwed by a further increase of MPF and meiotic resumption.     

Na-H exchange-dependent increase in intracellular pH times G2/M entry and transition

It is well established that activation of the Na-H exchanger NHE1 and increases in intracellular pH (pHi) are early and universal responses to mitogens and have permissive effects in promoting cell proliferation. Despite this evidence, a specific role for NHE1 or pHi in cell cycle progression remains undetermined. We now show that NHE1 activity and pHi regulate the timing of G2/M entry and transition. Prior to G2/M entry there is a rapid and transient increase in NHE1 activity and pHi, but in fibroblasts expressing a mutant NHE1 that lacks ion translocation activity, this increase in pHi is attenuated, S phase is delayed, and G2/M transition is impaired. In the absence of ion translocation by NHE1, expression of cyclin B1 and the kinase activity of Cdc2 are decreased and Wee1 kinase expression increases. Increasing pHi in the absence of NHE1 activity, however, is sufficient to restore Cdc2 activity and cyclin B1 expression and to promote G2/M entry and transition. These data indicate that a transient increase in pHi induced by NHE1 promotes the timing of G2/M, and they suggest that increases in pHi at the completion of S phase may constitute a previously unrecognized checkpoint for progression to G2 and mitosis.     

Regulation of Cdc25C by ERK-MAP kinases during the G2/M transition

Induction of G(2)/M phase transition in mitotic and meiotic cell cycles requires activation by phosphorylation of the protein phosphatase Cdc25. Although Cdc2/cyclin B and polo-like kinase (PLK) can phosphorylate and activate Cdc25 in vitro, phosphorylation by these two kinases is insufficient to account for Cdc25 activation during M phase induction. Here we demonstrate that p42 MAP kinase (MAPK), the Xenopus ortholog of ERK2, is a major Cdc25 phosphorylating kinase in extracts of M phase-arrested Xenopus eggs. In Xenopus oocytes, p42 MAPK interacts with hypophosphorylated Cdc25 before meiotic induction. During meiotic induction, p42 MAPK phosphorylates Cdc25 at T48, T138, and S205, increasing Cdc25's phosphatase activity. In a mammalian cell line, ERK1/2 interacts with Cdc25C in interphase and phosphorylates Cdc25C at T48 in mitosis. Inhibition of ERK activation partially inhibits T48 phosphorylation, Cdc25C activation, and mitotic induction. These findings demonstrate that ERK-MAP kinases are directly involved in activating Cdc25 during the G(2)/M transition.     

The xenopus Suc1/Cks protein promotes the phosphorylation of G(2)/M regulators

The entry into mitosis is controlled by Cdc2/cyclin B, also known as maturation or M-phase promoting factor (MPF). In Xenopus egg extracts, the inhibitory phosphorylations of Cdc2 on Tyr-15 and Thr-14 are controlled by the phosphatase Cdc25 and the kinases Myt1 and Wee1. At mitosis, Cdc25 is activated and Myt1 and Wee1 are inactivated through phosphorylation by multiple kinases, including Cdc2 itself. The Cdc2-associated Suc1/Cks1 protein (p9) is also essential for entry of egg extracts into mitosis, but the molecular basis of this requirement has been unknown. We find that p9 strongly stimulates the regulatory phosphorylations of Cdc25, Myt1, and Wee1 that are carried out by the Cdc2/cyclin B complex. Overexpression of the prolyl isomerase Pin1, which binds to the hyperphosphorylated forms of Cdc25, Myt1, and Wee1 found at M-phase, is known to block the initiation of mitosis in egg extracts. We have observed that Pin1 specifically antagonizes the stimulatory effect of p9 on phosphorylation of Cdc25 by Cdc2/cyclin B. This observation could explain why overexpression of Pin1 inhibits mitotic initiation. These findings suggest that p9 promotes the entry into mitosis by facilitating phosphorylation of the key upstream regulators of Cdc2.     

Two distinct mechanisms for negative regulation of the Wee1 protein kinase.

The Wee1 protein kinase negatively regulates the entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of the Cdc2 protein. To examine the potential mechanisms for Wee1 regulation during the cell cycle, we have introduced a recombinant form of the fission yeast Wee1 protein kinase into Xenopus egg extracts. We find that the Wee1 protein undergoes dramatic changes in its phosphorylation state and kinase activity during the cell cycle. The Wee1 protein oscillates between an underphosphorylated 107 kDa form during interphase and a hyperphosphorylated 170 kDa version at mitosis. The mitosis-specific hyperphosphorylation of the Wee1 protein results in a substantial reduction in its activity as a Cdc2-specific tyrosine kinase. This phosphorylation occurs in the N-terminal region of the protein that lies outside the C-terminal catalytic domain, which was recently shown to be a substrate for the fission yeast Nim1 protein kinase. These experiments demonstrate the existence of a Wee1 regulatory system, consisting of both a Wee1-inhibitory kinase and a Wee1-stimulatory phosphatase, which controls the phosphorylation of the N-terminal region of the Wee1 protein. Moreover, these findings indicate that there are apparently two potential mechanisms for negative regulation of the Wee1 protein, one involving phosphorylation of its C-terminal domain by the Nim1 protein and the other involving phosphorylation of its N-terminal region by a different kinase.     

The Wee1 protein kinase regulates T14 phosphorylation of fission yeast Cdc2.

The Cdc2 protein kinase is a key regulator of the G1-S and G2-M cell cycle transitions in the fission yeast Schizosaccharomyces pombe. The activation of Cdc2 at the G2-M transition is triggered by dephosphorylation at a conserved tyrosine residue Y15. The level of Y15 phosphorylation is controlled by the Wee1 and Mik1 protein kinases acting in opposition to the Cdc25 protein phosphatase. Here, we demonstrate that Wee1 overexpression leads to a high stoichiometry of phosphorylation at a previously undetected site in S. pombe Cdc2, T14. T14 phosphorylation was also detected in certain cell cycle mutants blocked in progression through S phase, indicating that T14 phosphorylation might normally occur at low stoichiometry during DNA replication or early G2. Strains in which the chromosomal copy of cdc2 was replaced with either a T14A or a T14S mutant allele were generated and the phenotypes of these strains are consistent with T14 phosphorylation playing an inhibitory role in the activation of Cdc2 as it does in higher eukaryotes. We have also obtained evidence that Wee1 but not Mik1 or Chk1 is required for phosphorylation at this site, that the Mik1 and Chk1 protein kinases are unable to drive T14 phosphorylation in vivo, that residue 14 phosphorylation requires previous phosphorylation at Y15, and that the T14A mutant, unlike Y15F, is recessive to wild-type Cdc2 activity. Finally, the normal duration of G2 delay after irradiation or hydroxyurea treatment in a T14A mutant strain indicates that T14 phosphorylation is not required for the DNA damage or replication checkpoint controls.     

Mitotic effects of a constitutively active mutant of the Xenopus polo-like kinase Plx1

During mitosis the Xenopus polo-like kinase 1 (Plx1) plays key roles in the activation of Cdc25C, in spindle assembly, and in cyclin B degradation. Previous work has shown that the activation of Plx1 requires phosphorylation on serine and threonine residues. In the present work, we demonstrate that replacement of Ser-128 or Thr-201 with a negatively charged aspartic acid residue (S128D or T201D) elevates Plx1 activity severalfold and that replacement of both Ser-128 and Thr-201 with Asp residues (S128D/T201D) increases Plx1 activity approximately 40-fold. Microinjection of mRNA encoding S128D/T201D Plx1 into Xenopus oocytes induced directly the activation of both Cdc25C and cyclin B-Cdc2. In egg extracts T201D Plx1 delayed the timing of deactivation of Cdc25C during exit from M phase and accelerated Cdc25C activation during entry into M phase. This supports the concept that Plx1 is a "trigger" kinase for the activation of Cdc25C during the G(2)/M transition. In addition, during anaphase T201D Plx1 reduced preferentially the degradation of cyclin B2 and delayed the reduction in Cdc2 histone H1 kinase activity. In early embryos S128D/T201D Plx1 resulted in arrest of cleavage and formation of multiple interphase nuclei. Consistent with these results, Plx1 was found to be localized on centrosomes at prophase, on spindles at metaphase, and at the midbody during cytokinesis. These results demonstrate that in Xenopus laevis activation of Plx1 is sufficient for the activation of Cdc25C at the initiation of mitosis and that inactivation of Plx1 is required for complete degradation of cyclin B2 after anaphase and completion of cytokinesis.     

HIV-1 Vpr induces G2 cell cycle arrest in fission yeast associated with Rad24/14-3-3-dependent, Chk1/Cds1-independent Wee1 upregulation

Viral protein R (Vpr), an accessory protein of human immunodeficiency virus type 1 (HIV-1), induces the G2 cell cycle arrest in fission yeast for which host factors, such as Wee1 and Rad24, are required. Catalyzing the inhibitory phosphorylation of Cdc2, Wee1 is known to serve as a major regulator of G2/M transition in the eukaryotic cell cycle. It has been reported that the G2 checkpoint induced by DNA damage or incomplete DNA replication is associated with phosphorylation and upregulation of Wee1 for which Chk1 and Cds1 kinase is required. In this study, we demonstrate that the G2 arrest induced by HIV-1 Vpr in fission yeast is also associated with increase in the phosphorylation and amount of Wee1, but in a Chk1/Cds1-independent manner. Rad24 and human 14-3-3 appear to contribute to Vpr-induced G2 arrest by elevating the level of Wee1 expression. It appears that Vpr could cause the G2 arrest through a mechanism similar to, but distinct from, the physiological G2 checkpoint controls. The results may provide useful insights into the mechanism by which HIV-1 Vpr causes the G2 arrest in eukaryotic cells. Vpr may also serve as a useful molecular tool for exploring novel cell cycle control mechanisms.     

Differential susceptibility of yeast S and M phase CDK complexes to inhibitory tyrosine phosphorylation

BACKGROUND: Several checkpoint pathways employ Wee1-mediated inhibitory tyrosine phosphorylation of cyclin-dependent kinases (CDKs) to restrain cell-cycle progression. Whereas in vertebrates this strategy can delay both DNA replication and mitosis, in yeast cells only mitosis is delayed. This is particularly surprising because yeasts, unlike vertebrates, employ a single family of cyclins (B type) and the same CDK to promote both S phase and mitosis. The G2-specific arrest could be explained in two fundamentally different ways: tyrosine phosphorylation of cyclin/CDK complexes could leave sufficient residual activity to promote S phase, or S phase-promoting cyclin/CDK complexes could somehow be protected from checkpoint-induced tyrosine phosphorylation. RESULTS: We demonstrate that in Saccharomyces cerevisiae, several cyclin/CDK complexes are protected from inhibitory tyrosine phosphorylation, allowing Clb5,6p to promote DNA replication and Clb3,4p to promote spindle assembly, even under checkpoint-inducing conditions that block nuclear division. In vivo, S phase-promoting Clb5p/Cdc28p complexes were phosphorylated more slowly and dephosphorylated more effectively than were mitosis-promoting Clb2p/Cdc28p complexes. Moreover, we show that the CDK inhibitor (CKI) Sic1p protects bound Clb5p/Cdc28p complexes from tyrosine phosphorylation, allowing the accumulation of unphosphorylated complexes that are unleashed when Sic1p is degraded to promote S phase. The vertebrate CKI p27(Kip1) similarly protects Cyclin A/Cdk2 complexes from Wee1, suggesting that the antagonism between CKIs and Wee1 is evolutionarily conserved. CONCLUSIONS: In yeast cells, the combination of CKI binding and preferential phosphorylation/dephosphorylation of different B cyclin/CDK complexes renders S phase progression immune from checkpoints acting via CDK tyrosine phosphorylation.     

Wee1B is an oocyte-specific kinase involved in the control of meiotic arrest in the mouse

In most species, the meiotic cell cycle is arrested at the transition between prophase and metaphase through unclear somatic signals. Activation of the Cdc2-kinase component of maturation promoting factor (MPF) triggers germinal vesicle breakdown after the luteinizing hormone (LH) surge and reentry into the meiotic cell cycle. Although high levels of cAMP and activation of protein kinase A (PKA) play a critical role in maintaining an inactive Cdc2, the steps downstream of PKA in the oocyte remain unknown. Using a small-pool expression-screening strategy, we have isolated several putative PKA substrates from a mouse oocyte cDNA library. One of these clones encodes a Wee1-like kinase that prevents progesterone-induced oocyte maturation when expressed in Xenopus oocytes. Unlike the widely expressed Wee1 and Myt1, mWee1B mRNA and its protein are expressed only in oocytes, and mRNA downregulation by RNAi injection in vitro or transgenic overexpression of RNAi in vivo causes a leaky meiotic arrest. Ser15 residue of mWee1B is the major PKA phosphorylation site in vitro, and the inhibitory effects of the kinase are enhanced when this residue is phosphorylated. Thus, mWee1B is a key MPF inhibitory kinase in mouse oocytes, functions downstream of PKA, and is required for maintaining meiotic arrest.     

A role for cyclin A1 in the activation of MPF and G2-M transition during meiosis of male germ cells in mice

Cell-cycle transition at G2-M is controlled by MPF (M-phase-promoting factor), a complex consisting of the Cdc2 kinase and a B-type cyclin. We have shown that in mice, targeted disruption of an A-type cyclin gene, cyclin A1, results in a block of spermatogenesis prior to the entry into metaphase I. The meiotic arrest is accompanied by a defect in Cdc2 kinase activation at the G2--M transition, raising the possibility that a cyclin A1-dependent process dictates the activation of MPF. Here we show that like Cdc2, the expression of B-type cyclins is retained in cyclin A1-deficient spermatocytes, while their associated kinases are kept at inactive states. Treatment of arrested germ cells with the protein phosphatase type-1 and -2A inhibitor okadaic acid restores the MPF activity and induces entry into M phase and the formation of normally condensed chromosome bivalents, concomitant with hyperphosphorylation of Cdc25 proteins. Conversely, inhibition of tyrosine phosphatases, including Cdc25s, by vanadate suppresses the okadaic acid-induced metaphase induction. The highest levels of Cdc25A and Cdc25C expression and their subcellular localization during meiotic prophase coincide with that of cyclin A1, and when overexpressed in HeLa cells, cyclin A1 coimmunoprecipitates with Cdc25A. Furthermore, the protein kinase complexes consisting of cyclin A1 and either Cdc2 or Cdk2 phosphorylate both Cdc25A and Cdc25C in vitro. These results suggest that in normal meiotic male germ cells, cyclin A1 participates in the regulation of other protein kinases or phosphatases critical for the G2-M transition. In particular, it may be directly involved in the initial amplification of MPF through the activating phosphorylation on Cdc25 phosphatases.     

Cell cycle regulation of a Xenopus Wee1-like kinase.

Using a polymerase chain reaction-based strategy, we have isolated a gene encoding a Wee1-like kinase from Xenopus eggs. The recombinant Xenopus Wee1 protein efficiently phosphorylates Cdc2 exclusively on Tyr-15 in a cyclin-dependent manner. The addition of exogenous Wee1 protein to Xenopus cell cycle extracts results in a dose-dependent delay of mitotic initiation that is accompanied by enhanced tyrosine phosphorylation of Cdc2. The activity of the Wee1 protein is highly regulated during the cell cycle: the interphase, underphosphorylated form of Wee1 (68 kDa) phosphorylates Cdc2 very efficiently, whereas the mitotic, hyperphosphorylated version (75 kDa) is weakly active as a Cdc2-specific tyrosine kinase. The down-modulation of Wee1 at mitosis is directly attributable to phosphorylation, since dephosphorylation with protein phosphatase 2A restores its kinase activity. During interphase, the activity of this Wee1 homolog does not vary in response to the presence of unreplicated DNA. The mitosis-specific phosphorylation of Wee1 is due to at least two distinct kinases: the Cdc2 protein and another activity (kinase X) that may correspond to an MPM-2 epitope kinase. These studies indicate that the down-regulation of Wee1-like kinase activity at mitosis is a multistep process that occurs after other biochemical reactions have signaled the successful completion of S phase.     

Regulation of the human WEE1Hu CDK tyrosine 15-kinase during the cell cycle.

In higher eukaryotes, the cyclin-dependent kinases (CDKs) are negatively regulated by phosphorylation on threonine 14 (T14) and tyrosine 15 (Y15). In fission yeast, the Wee1 and mitosis inhibitory kinase 1 (Mik1) protein kinases phosphorylate Y15 in Cdc2. WEE1Hu is the only known protein kinase that can carry out this inhibitory phosphorylation on Y15 in higher eukaryotes. In the present study, we examined the endogenous products of WEE1Hu in human cells and found that the original WEE1Hu cDNA lacked 214 amino acids at the N-terminus. The predicted full-length protein has weak, but significant, similarity over its entire length with Mik1. Thus, we suggest that 'WEE1Hu' is a Mik1-related protein rather than a Wee1 homologue. When isolated in immunoprecipitates, the endogenous WEE1Hu phosphorylated several cyclin-associated CDKs on Y15. WEE1Hu activity increased during S and G2 phases in parallel with the level of protein. Its activity decreased at M phase when WEE1Hu became transiently hyperphosphorylated. In addition, a decrease in WEE1Hu protein level was observed at M/G1 phase. Apparently, the hyperphosphorylation and degradation in combination caused inactivation of WEE1Hu at M phase and the following G1 phase. These results suggest that the activity of WEE1Hu is regulated by phosphorylation and proteolytic degradation, and that WEE1Hu plays a role in inhibiting mitosis before M phase by phosphorylating cyclin B1-Cdc2.     

Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms. By inhibiting Cdk chemically, we showed that, in prometaphase, when Cdk1 substrates approach the peak of their phosphorylation, cells become capable of proper M-to-G1 transition. We interfered with the molecular components of the Cdk1-activating feedback system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25 phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid Cdk1 activation and morphologically normal mitotic entry, even in the absence of G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1 inhibition prevented Cdk1/cyclin B kinase activation and full substrate phosphorylation and induced a mitotic "collapse," a terminal state characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These findings suggest that the positive feedback in Cdk activation serves to overcome the activity of Cdk-opposing phosphatases and thus sustains forward progression in mitosis.     

Human Cdc14A regulates Wee1 stability by counteracting CDK-mediated phosphorylation

The activity of Cdk1–cyclin B1 mitotic complexes is regulated by the balance between the counteracting activities of Wee1/Myt1 kinases and Cdc25 phosphatases. These kinases and phosphatases must be strictly regulated to ensure proper mitotic timing. One masterpiece of this regulatory network is Cdk1, which promotes Cdc25 activity and suppresses inhibitory Wee1/Myt1 kinases through direct phosphorylation. The Cdk1-dependent phosphorylation of Wee1 primes phosphorylation by additional kinases such as Plk1, triggering Wee1 degradation at the onset of mitosis. Here we report that Cdc14A plays an important role in the regulation of Wee1 stability. Depletion of Cdc14A results in a significant reduction in Wee1 protein levels. Cdc14A binds to Wee1 at its amino-terminal domain and reverses CDK-mediated Wee1 phosphorylation. In particular, we found that Cdc14A inhibits Wee1 degradation through the dephosphorylation of Ser-123 and Ser-139 residues. Thus the lack of phosphorylation of these two residues prevents the interaction with Plk1 and the consequent efficient Wee1 degradation at the onset of mitosis. These data support the hypothesis that Cdc14A counteracts Cdk1–cyclin B1 activity through Wee1 dephosphorylation.