Inactivating Cdc25, mitotic style

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.     

Chk1-Dependent Regulation of Cdc25B Functions to Coordinate Mitotic Events

The coordination of mitotic spindle formation and chromatin condensation is an essential prerequisite for successful mitosis. Both events are thought to be initiated by cyclin B/Cdk1, whose initial activation occurs in late prophase at the centrosomes. Recently, we have shown that Chk1 localizes to interphase centrosomes and thereby negatively regulates entry into mitosis by preventing premature activation of cyclin B/Cdk1. Here, we demonstrate that inhibition of Chk1 kinase induces mitotic entry with regular spindle assembly but aberrant and mislocalized chromatin. This effect, which we have termed the ‘paraspindle’ phenotype, was reverted by downregulation of Cdc25B phosphatase using siRNA, which restored normal mitosis with regular chromatin. Analogous to Chk1 inhibition, the ‘paraspindle’ phenotype was induced by overexpression of Cdc25B but not Cdc25A. Our results suggest that Chk1 functions to coordinate mitotic events through regulation of Cdc25B.     

Many Fingers on the Mitotic Trigger: Post-Translational Regulation of the Cdc25C Phosphatase

AbstractThe mitotic inducer Cdc25 phosphatase controls the activation of Cdc2/cyclin B protein kinase and entry into mitosis in eukaryotic cells. Cdc25C is highly regulated by multiple post-translational modifications within its N-terminal regulatory domain and site-specific protein interactions. Phosphorylation of one inhibitory site targeted by multiple kinases determines the timing of Cdc25C activation and arrests cells in G2 in response to checkpoint, stress, developmental and extracellular signals. In mitosis, phosphorylation of several Ser/Thr residues and Pin1-catalysed peptidyl-proline isomerisation produces activation. Phosphorylation of one activating site is antagonistic to the proximal inhibitory site and maintains Cdc25C activity during mitosis. Phosphorylation and interacting proteins also modulate the nuclear import and export signals on Cdc25C, inducing dramatic changes in its localisation within the cell. Thus, the regulation of Cdc25C activity and localisation integrates multiple signals that govern the decision to enter mitosis.     

Cdc25 phosphatases

Comment on: Thomas Y, et al. Cell Cycle 2010; 9:4338–50.     

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.     

Mitotic exit: the Cdc14 double cross

The release of Cdc14 from the nucleolus occurs in two waves in early and late anaphase, controlled by the FEAR and MEN pathways, respectively. Two new papers report the localisation at the spindle pole body of the Cdc14 released in early anaphase and, surprisingly, show that the two pulses of released Cdc14 have opposite effects on MEN activation.     

Catalytic mechanism of Cdc25

Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases, thus causing initiation and progression of successive phases of the cell cycle. Although it is not significantly homologous in sequence or structure to other dual-specificity phosphatases, Cdc25 belongs to the class of well-studied cysteine phosphatases as it contains their active site signature motif. Like other dual-specificity phosphatases, Cdc25 contains an active site cysteine whose pK(a) of 5.9 can be measured in pH-dependent kinetics using both small molecule and protein substrates such as Cdk2-pTpY/CycA. We have previously shown that the catalytic acid expected in phosphatases of this family and apparent in kinetics with the natural protein substrate does not appear to lie within the known structure of Cdc25 [Chen, W., et al. (2000) Biochemistry 39, 10781]. Here we provide experimental evidence for a novel mechanism wherein Cdc25 uses as its substrate a monoprotonated phosphate in contrast to the more typical bisanionic phosphate. Our pH-dependent studies, including one-turnover kinetics, solvent kinetic isotope effects, equilibrium perturbation, substrate depletion, and viscosity measurements, show that the monoprotonated phosphate of the protein substrate Cdk2-pTpY/CycA provides the critical proton to the leaving group. Additionally, we provide evidence that Glu474 on the Cdc25 enzyme serves an important role as a base in the transfer of the proton from the phosphate to the leaving group. Because of its greater intrinsic reactivity, the use of a monoprotonated phosphate as a phosphatase substrate is a chemically attractive solution and suggests the possibility of designing inhibitors specific for the Cdc25 dual-specificity phosphatase, an important anticancer target.     

Cdc25 phosphatases: structure, specificity, and mechanism

Cdc25 phosphatases, as activators of the Cdk/cyclins, play critical roles in the regulation of the eukaryotic cell cycle. Because of their overexpression and correlation with poor prognosis in many diverse cancers, Cdc25 phosphatases are attractive targets for anticancer drug development. Over the past few years, much knowledge of the basic enzymology of the Cdc25 phosphatases that may aid in the development of specific inhibitors has been gained. We review herein the structure, specificity, and mechanism of the Cdc25 phosphatases with a special focus on the activity of Cdc25 phosphatases with native protein substrates.     

Human Cdc14A phosphatase modulates the G2/M transition through Cdc25A and Cdc25B

The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division. However, the functions of human Cdc14 homologues remain poorly understood. Here we have tested the hypothesis that Cdc14A might regulate Cdc25 mitotic inducers in human cells. We found that increasing levels of Cdc14A delay entry into mitosis by inhibiting Cdk1-cyclin B1 activity. By contrast, lowering the levels of Cdc14A accelerates mitotic entry. Biochemical analyses revealed that Cdc14A acts through key Cdk1-cyclin B1 regulators. We observed that Cdc14A directly bound to and dephosphorylated Cdc25B, inhibiting its catalytic activity. Cdc14A also regulated the activity of Cdc25A at the G2/M transition. Our results indicate that Cdc14A phosphatase prevents premature activation of Cdk1 regulating Cdc25A and Cdc25B at the entry into mitosis.     

Mitotic Cdc6 stabilizes anaphase-promoting complex substrates by a partially Cdc28-independent mechanism, and this stabilization is suppressed by deletion of Cdc55

Ectopic expression of Cdc6p results in mitotic delay, and this has been attributed to Cdc6p-mediated inhibition of Cdc28 protein kinase and failure to activate the anaphase-promoting complex (APC). Here we show that endogenous Cdc6p delays a specific subset of mitotic events and that Cdc28 inhibition is not sufficient to account for it. The depletion of Cdc6p in G(2)/M cells reveals that Cdc6p is rate limiting for the degradation of the APC/Cdc20 substrates Pds1p and Clb2p. Conversely, the premature expression of Cdc6p delays the degradation of APC/Cdc20 substrates. Abolishing Cdc6p/Cdc28p interaction does not eliminate the Cdc6-dependent delay of these anaphase events. To identify additional Cdc6-mediated, APC-inhibitory mechanisms, we looked for mutants that reversed the mitotic delay. The deletion of SWE1, RAD24, MAD2, or BUB2 had no effect. However, disrupting CDC55, a PP2A regulatory subunit, suppressed the Cdc6p-dependent delay of Pds1 and Clb2 destruction. A specific role for CDC55 was supported by demonstrating that the lethality of Cdc6 ectopic expression in a cdc16-264 mutant is suppressed by the deletion of CDC55, that endogenous Cdc6p coimmunoprecipitates with the Cdc55 and Tpd3 subunits of PP2A, that Cdc6p/Cdc55p/Tpd3 interaction occurs only during mitosis, and that Cdc6 affects PP2A-Cdc55 activity during anaphase. This demonstrates that the levels and timing of accumulation of Cdc6p in mitosis are appropriate for mediating the modulation of APC/Cdc20.     

14-3-3 Proteins Act as Negative Regulators of the Mitotic Inducer Cdc25 in Xenopus Egg Extracts

Cdc25, the dual-specificity phosphatase that dephosphorylates the Cdc2–cyclin B complex at mitosis, is highly regulated during the cell cycle. In Xenopus egg extracts, Cdc25 is associated with two isoforms of the 14-3-3 protein. Cdc25 is complexed primarily with 14-3-3ε and to a lesser extent with 14-3-3ζ. The association of these 14-3-3 proteins with Cdc25 varies dramatically during the cell cycle: binding is high during interphase but virtually absent at mitosis. Interaction with 14-3-3 is mediated by phosphorylation of Xenopus Cdc25 at Ser-287, which resides in a consensus 14-3-3 binding site. Recombinant Cdc25 with a point mutation at this residue (Cdc25-S287A) is incapable of binding to 14-3-3. Addition of the Cdc25-S287A mutant to Xenopus egg extracts accelerates mitosis and overrides checkpoint-mediated arrests of mitotic entry due to the presence of unreplicated and damaged DNA. These findings indicate that 14-3-3 proteins act as negative regulators of Cdc25 in controlling the G₂–M transition.     

Mitotic Transmission of Artificial Chromosomes in Cdc Mutants of the Yeast, Saccharomyces Cerevisiae

In the yeast, Saccharomyces cerevisiae, cell division cycle (CDC) genes have been identified whose products are required for the execution of different steps in the cell cycle. In this study, the fidelity of transmission of a 14-kb circular minichromosome and a 155-kb linear chromosome fragment was examined in cell divisions where specific CDC products were temporarily inactivated with either inhibitors, or temperature sensitive mutations in the appropriate CDC gene. All of the cdc mutants previously shown to induce loss of endogenous linear chromosomes also induced loss of a circular minichromosome and a large linear chromosome fragment in our study (either 1:0 or 2:0 loss events). Therefore, the efficient transmission of these artificial chromosomes depends upon the same trans factors that are required for the efficient transmission of endogenous chromosomes. In a subset of cdc mutants (cdc6, cdc7 and cdc16), the rate of minichromosome loss was significantly greater than the rate of loss of the linear chromosome fragment, suggesting that a structural feature of the minichromosome (nucleotide content, length or topology) makes the minichromosome hypersensitive to the level of function of these CDC gene products. In another subset of cdc mutants (cdc7 and cdc17), the relative rate of 1:0 events to 2:0 events differed for the minichromosome and chromosome fragment, suggesting that the type of chromosome loss event observed in these mutants was dependent upon chromosome structure. Finally, we show that 2:0 events for the minichromosome can occur by both a RAD52 dependent and RAD52 independent mechanism. These results are discussed in the context of the molecular functions of the CDC products.     

Cdc25A and ERK interaction: EGFR-independent ERK activation by a protein phosphatase Cdc25A inhibitor, compound 5

Extracellular signal-regulated kinase (ERK) plays a central role in regulating cell growth, differentiation, and apoptosis. We previously found that 2-(2-mercaptoethanol)-3-methyl-1,4-napthoquinone or Compound 5 (Cpd 5), is a Cdc25A protein phosphatase inhibitor and causes prolonged, strong ERK phosphorylation which is triggered by epidermal growth factor receptor (EGFR) activation. We now report that Cpd 5 can directly cause ERK phosphorylation by inhibiting Cdc25A activity independently of the EGFR pathway. We found that Cdc25A physically interacted with and de-phosphorylated phospho-ERK both in vitro and in cell culture. Inhibition of Cdc25A activity by Cpd 5 resulted in ERK hyper-phosphorylation. Transfection of Hep3B human hepatoma cells with inactive Cdc25A mutant enhanced Cpd 5 action on ERK phosphorylation, whereas over-expression of Cdc25A attenuated this Cpd 5 action. Furthermore, endogenous Cdc25A knock-down by Cdc25A siRNA resulted in a constitutive-like ERK phosphorylation and Cpd 5 treatment further enhanced it. In EGFR-devoid NR6 fibroblasts and MEK (ERK kinase) mutated MCF7 cells, Cpd 5 treatment also resulted in ERK phosphorylation, providing support for the idea that Cpd 5 can directly act on ERK phosphorylation by inhibiting Cdc25A activity. These data suggest that phospho-ERK is likely another Cdc25A substrate, and Cpd 5-caused ERK phosphorylation is probably regulated by both EGFR-dependent and EGFR-independent pathways.     

Arylstibonic acids are potent and isoform-selective inhibitors of Cdc25a and Cdc25b phosphatases

Arylstibonates structurally resemble phosphotyrosine side chains in proteins and here we addressed the ability of such compounds to act as inhibitors of a panel of mammalian tyrosine and dual-specificity phosphatases. Two arylstibonates both possessing a carboxylate side chain were identified as potent inhibitors of the protein tyrosine phosphatase PTP-ß. In addition, they inhibited the dual-specificity, cell cycle regulatory phosphatases Cdc25a and Cdc25b with sub-micromolar potency. However, the Cdc25c phosphatase was not affected demonstrating that arylstibonates may be viable leads from which to develop isoform specific Cdc25 inhibitors.     

Redox regulation of Cdc25C

The Cdc25 family of dual specific phosphatases are critical components of cell cycle progression and checkpoint control. Certain stresses such as ultraviolet light stimulate the rapid and selective destruction of Cdc25A protein through a Chk1 protein kinase-dependent pathway. We demonstrate that in contrast to cellular stresses previously examined, hydrogen peroxide exposure affects Cdc25C but not Cdc25A levels. Pharmacological inhibition of Chk1 activity or a mutant of Cdc25C that lacks the Chk1 phosphorylation site still undergoes degradation in response to oxidants. We also demonstrate that in vitro hydrogen peroxide stimulates an intramolecular disulfide bond between the active site cysteine at position 377 and another invariant cysteine at position 330. The in vivo stability of Cdc25C is substantially reduced by the mutation of either of these two cysteine residues. In contrast, a double (C2) mutant of both cysteine 330 and cysteine 377 results in a protein that is more stable than wild type Cdc25C and is resistant to oxidative stress-induced degradation. In addition, the C2 mutant, which is unable to form an intramolecular disulfide bond, has reduced binding to 14-3-3 in vitro and in vivo. These results suggest that oxidative stress may induce cell cycle arrest in part through the degradation of Cdc25C.     

黄牛、牦牛和犏牛睾丸组织中Cdc2、Cdc25A基因mRNA表达水平

黄牛和牦牛远缘杂交后代犏牛雄性不育是牦牛杂交改良中的一大难题。Cdc2和Cdc25A是减数分裂的两个关键基因, 其表达水平的下降将使精子发生不能正常进行, 导致雄性不育。为了探讨Cdc2、Cdc25A基因mRNA表达水平与犏牛雄性不育的关系, 文章采用荧光定量PCR技术对Cdc2和Cdc25A基因的组织表达特征以及在黄牛、牦牛和犏牛睾丸组织中的表达水平进行了分析。结果表明: Cdc2和Cdc25A基因在牦牛各种组织中广泛表达, 说明Cdc2和Cdc25A基因在各种组织细胞分裂和细胞周期运行中均发挥作用; 黄牛和牦牛睾丸组织中Cdc2、Cdc25A基因表达水平均显著高于犏牛(P<0.05), 说明睾丸组织中Cdc2和Cdc25A基因的低表达可能与犏牛雄性不育相关。     

Pro-apoptotic Role of Cdc25A: ACTIVATION OF CYCLIN B1/Cdc2 BY THE Cdc25A C-TERMINAL DOMAIN*

Cdc25A is a dual specificity protein phosphatase that activates cyclin/cyclin-dependent protein kinase (Cdk) complexes by removing inhibitory phosphates from conserved threonine and tyrosine in Cdks. To address how Cdc25A promotes apoptosis, Jurkat cells were treated with staurosporine, an apoptosis inducer. Upon staurosporine treatment, a Cdc25A C-terminal 37-kDa fragment, designated C37, was generated by caspase cleavage at Asp-223. Thr-507 in C37 became dephosphorylated, which prevented 14-3-3 binding, as shown previously. C37 exhibited higher phosphatase activity than full-length Cdc25A. C37 with alanine substitution for Thr-507 (C37/T507A) that imitated the cleavage product during staurosporine treatment interacted with Cdc2, Cdk2, cyclin A, and cyclin B1 and markedly activated cyclin B1/Cdc2. The dephosphorylation of Thr-507 might expose the Cdc2/Cdk2-docking site in C37. C37/T507A also induced apoptosis in Jurkat and K562 cells, resulting from activating cyclin B1/Cdc2 but not Cdk2. Thus, this study reveals that Cdc25A is a pro-apoptotic protein that amplifies staurosporine-induced apoptosis through the activation of cyclin B1/Cdc2 by its C-terminal domain.     

Cdc25B and Cdc25C differ markedly in their properties as initiators of mitosis.

We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.