Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition

Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr¹⁶¹ by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr¹⁴ and Tyr¹⁵ phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G₂/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr¹⁵. In addition, Tyr¹⁵-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G₂ and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr¹⁶¹ phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G₂/M is tightly coupled and regulated by Cdc25 phosphatases.     

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.     

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.     

Induction of G2/M phase arrest by squamocin in chronic myeloid leukemia (K562) cells

Squamocin is one of the annonaceous acetogenins and has been reported to have anticancer activity. Squamocin was found to inhibit the growth of K562 cells in a time- and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest in K562 cells following 24 h exposure to squamocin. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a dose-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that squamocin did not change the steady-state levels of Cdk2, Cdk4, cyclin A, cyclin B1, cyclin D3 and cyclin E, but decreased the protein levels of Cdk1 and Cdc25C. These results suggest that squamocin inhibits the proliferation of K562 cells via G2/M arrest in association with the induction of p21, p27 and the reduction of Cdk1 and Cdc25C kinase activities.     

Human Cdc14A becomes a cell cycle gene in controlling Cdk1 activity at the G2/M transition

Cdc14 belongs to a dual-specificity phosphatase family highly conserved through evolution that preferentially reverses CDK (Cyclin dependent kinases) –dependent phosphorylation events. In the yeast Saccharomyces cerevisiae, Cdc14 is an essential regulator of late mitotic events and exit from mitosis by counteracting CDK activity at the end of mitosis. However, many studies have shown that Cdc14 is dispensable for exiting mitosis in all other model systems analyzed. In fission yeast, the Cdc14 homologue Flp1/Clp1 regulates the stability of the mitotic inducer Cdc25 at the end of mitosis to ensure Cdk1 inactivation before cytokinesis. We have recently reported that human Cdc14A, the Cdc14 isoform located at the centrosomes during interphase, down-regulates Cdc25 activity at the G2/M transition to prevent premature activation of Cdk1-Cyclin B1 complexes and untimely entry into mitosis. Here we speculate about new molecular mechanisms for Cdc14A and discuss the current evidence suggesting that Cdc14 phosphatase plays a role in cell cycle control in higher eukaryotes.     

An unexpected role for PI4,5P2 in EGF receptor endosomal trafficking

In mammalian cells, three Cdc25 phosphatases A, B, C coordinate cell cycle progression through activating dephosphorylation of Cyclin-dependent kinases. Whereas Cdc25B is believed to trigger entry into mitosis, Cdc25C is thought to act at a later stage of mitosis and in the nucleus. We report that a fraction of Cdc25C localises to centrosomes in a cell cycle-dependent fashion, as of late S phase and throughout G2 and mitosis. Moreover, Cdc25C colocalises with Cyclin B1 at centrosomes in G2 and in prophase and Fluorescence Recovery after Photobleaching experiments reveal that they are both in dynamic exchange between the centrosome and the cytoplasm. The centrosomal localisation of Cdc25C is essentially mediated by its catalytic C-terminal domain, but does not require catalytic activity. In fact phosphatase-dead and substrate-binding hotspot mutants of Cdc25C accumulate at centrosomes together with phosphoTyr15-Cdk1 and behave as dominant negative forms that impair entry into mitosis. Taken together, our data suggest an unexpected function for Cdc25C at the G2/M transition, in dephosphorylation of Cdk1. We propose that Cdc25C may participate in amplification of Cdk1-Cyclin B1 activity following initial activation by Cdc25B, and that this process is initiated at the centrosome, then further propagated throughout the cytoplasm thanks to the dynamic behavior of both Cdc25C and Cyclin B1.     

Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro.

p33cdk2 is a serine-threonine protein kinase that associates with cyclins A, D, and E and has been implicated in the control of the G1/S transition in mammalian cells. Recent evidence indicates that cyclin-dependent kinase 2 (Cdk2), like its homolog Cdc2, requires cyclin binding and phosphorylation (of threonine-160) for activation in vivo. However, the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown. We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts. Recombinant Cdk2 is essentially inactive as a histone H1 kinase (< 4 x 10(-5) pmol phosphate transferred.min-1 x microgram-1 Cdk2). However, in the presence of equimolar cyclin A, the specific activity is approximately 16 pmol.mon-1 x microgram-1, 4 x 10(5)-fold higher than Cdk2 alone. Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex: the activity of Cdk2T160E was indistinguishable from Cdk2, whereas that of Cdk2T160A was reduced by five-fold. To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160, complexes were treated with Cdc2 activating kinase (CAK), purified approximately 12,000-fold from Xenopus eggs. This treatment resulted in an 80-fold increase in specific activity. This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK (approximately 1600 pmol.mon-1 x microgram-1). Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK. In striking contrast with cyclin A, cyclin B did not directly activate Cdk2. However, both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK. For the Cdk2/cyclin A complex, both cyclin binding and phosphorylation contribute significantly to activation, although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol. The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed.     

FOXM1c is activated by cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1, but repressed by GSK-3alpha

Two different inhibitory domains, N-terminus and central domain, keep FOXM1c almost inactive despite its strong transactivation domain. Here, we demonstrate that cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1 activate FOXM1c. Cyclin E/Cdk2 does not target its transactivation domain or its DNA-binding domain. Instead, its activating effect strictly depends on the presence of either the central domain or the N-terminus of FOXM1c and thus is completely lost if both inhibitory domains are deleted. Cyclin E/Cdk2 activates FOXM1c by releasing its transactivation domain from the repression by these two inhibitory domains. However, it does not directly increase the transactivation potential of the TAD. The DNA-binding is not affected by cyclin E/Cdk2, neither directly nor indirectly. These two activating effects of cyclin E/Cdk2 via central domain and N-terminus are additive. Cyclin A/Cdk2 and cyclin A/Cdk1 show similar characteristics. GSK-3alpha, another proliferation-associated kinase, represses FOXM1c.     

Regulation of Cdc25A half-life in interphase by cyclin-dependent kinase 2 activity

Cdc25A regulates cell cycle progression, has oncogenic and anti-apoptotic activity, and is over-expressed in many human tumors. Phosphorylation by Chk1 and Cds1/Chk2 down-regulates Cdc25A levels in response to genotoxic stresses. Nevertheless, it remains unclear whether Chk1 and Cds1/Chk2 are uniquely responsible for regulating Cdc25A stability during interphase or if other kinase activities contribute. Here we report that treatment of HeLa cells with the cyclin-dependent kinase inhibitor roscovitine caused a concentration- and time-dependent increase in Cdc25A protein levels. Transfection with dominant-negative Cdk mutants demonstrated that only a Cdk2 mutant increased Cdc25A protein levels; Cdk1 and Cdk3 mutants had no effect. The increased Cdc25A protein levels were the result of an increase in the half-life of the protein; no increase in Cdc25A mRNA levels was observed. These results demonstrate Cdk2 kinase activity contributes to the labile nature of Cdc25A during interphase and redefine the nature of the Cdc25A-Cdk2 autoamplification feedback loop.     

The overlooked greatwall: a new perspective on mitotic control

The role of the dual specificity protein phosphatase, Cdc25, in activating the cyclin-dependent kinase-cyclin B complex (Cdk1-CycB) by overcoming the inhibitory Wee1 kinase is a long-established principle for mitotic entry. Recently, however, evidence has emerged of a regulatory network that facilitates Cdk1-CycB activity by inhibiting the form of protein phosphatase 2A having a B55 regulatory subunit (PP2A-B55). Here, I review the genetic and biochemical evidence for Greatwall kinase and its substrate Endosulphine as the key components of this previously obscure regulatory network. Not only is the inhibition of PP2A-B55 by phospho-endosulphine required to prevent dephosphorylation of Cdk1-CycB substrates until mitotic exit, but it is also required to promote Cdc25 activity and inhibit Wee1 at mitotic entry. I discuss how these alternating states of preferential PP2A-B55 or Cdk1-CycB activity can have an impact upon the regulation of Polo kinase and its ability to bind different partner proteins as mitosis progresses.     

Cdc2-cyclin E complexes regulate the G1/S phase transition

The cyclin-dependent kinase inhibitor p27(Kip1) is known as a negative regulator of cell-cycle progression and as a tumour suppressor. Cdk2 is the main target of p27 (refs 2, 3) and therefore we hypothesized that loss of Cdk2 activity should modify the p27(-/-) mouse phenotype. Here, we show that although p27(-/-) Cdk2(-/-) mice developed ovary tumours and tumours in the anterior lobe of the pituitary, we failed to detect any functional complementation in p27(-/-) Cdk2(-/-) double-knockout mice, indicating a parallel pathway regulated by p27. We observed elevated levels of S phase and mitosis in tissues of p27(-/-) Cdk2(-/-) mice concomitantly with elevated Cdc2 activity in p27(-/-) Cdk2(-/-) extracts. p27 binds to Cdc2, cyclin B1, cyclin A2, or suc1 complexes in wild-type and Cdk2(-/-) extracts. In addition, cyclin E binds to and activates Cdc2. Our in vivo results provide strong evidence that Cdc2 may compensate the loss of Cdk2 function.     

When the Checkpoints Have Gone: Insights into Cdc25 Functional Activation

DNA-responsive checkpoints operate at the G2/M transition to prevent premature mitosis in the presence of incompletely replicated or damaged DNA. These pathways prevent mitotic entry, at least in part, by suppressing Cdc25, the phosphatase that activates Cdc2/Cyclin B. To gain insight into how checkpoint signaling controls Cdc25 function, we have carefully examined the individual steps in Cdc25 activation. We found that removal of the regulatory protein, 14-3-3, that binds to phosphorylated Cdc25 during interphase is one of the early steps in mitotic activation. Moreover, our studies unexpectedly implicated the phosphatase PP1 and the G1/S kinase Cdk2 in the process of Cdc25 activation. Here we integrate our findings and those of others to propose a model for Cdc25 activation in an effort to provide insight into novel loci of DNA-responsive checkpoint control of mitotic entry.     

Inhibition of CDC2/Cyclin B1 in response to selenium-induced oxidative stress during spermatogenesis: potential role of Cdc25c and p21

Various cell cycle regulators control and coordinate the process of cell cycle. Because of the crucial involvement of CDC2, Cyclin B1, Cdc25c, and p21 in cell cycle regulation, the present study was aimed to investigate the possibility that selenium (Se)-induced oxidative stress mediated alterations in Cdc25c and p21 may cause modulations in the CDC2/Cyclin B1 complex responsible for G2/M phase checkpoint during meiosis I of spermatogenesis. To create different Se status-deficient, adequate and excess Se, male Balb/c mice were fed yeast based Se deficient diet (group I) and deficient diet supplemented with Se as sodium selenite at 0.2 and 1 ppm Se (group II and III) for a period of 8 weeks. After completion of the diet feeding schedule, a significant decrease in the Se and glutathione peroxidase levels were observed in the Se deficient group (I), whereas Se excess group (III) demonstrated an increase in Se levels. Increased levels of lipid peroxidation (LPO) were seen in both group I and group III when compared to group II, thus indicating oxidative stressed conditions. The mRNA and protein expression of CDC2, Cyclin B1, and Cdc25c were found to be significantly decreased in groups I and III. However, the expression of p21, a kinase inhibitor, was found to be elevated in Se deficient and Se excess fed groups. A statistically significant decrease in the CDC2 kinase activity was also seen in the Se deficient and excess groups. These findings suggest that under the influence of Se-induced oxidative stress, the down regulation of CDC2/Cyclin B1 complex is mediated through changes in Cdc25c and p21 leading to the cell cycle arrest and thus providing new dimensions to the molecular mechanisms underlying male infertility.     

When the checkpoints have gone: insights into Cdc25 functional activation

DNA-responsive checkpoints operate at the G(2)/M transition to prevent premature mitosis in the presence of incompletely replicated or damaged DNA. These pathways prevent mitotic entry, at least in part, by suppressing Cdc25, the phosphatase that activates Cdc2/Cyclin B. To gain insight into how checkpoint signaling controls Cdc25 function, we have carefully examined the individual steps in Cdc25 activation. We found that removal of the regulatory protein, 14-3-3, that binds to phosphorylated Cdc25 during interphase is one of the early steps in mitotic activation. Moreover, our studies unexpectedly implicated the phosphatase PP1 and the G(1)/S kinase Cdk2 in the process of Cdc25 activation. Here we integrate our findings and those of others to propose a model for Cdc25 activation in an effort to provide insight into novel loci of DNA-responsive checkpoint control of mitotic entry.     

Multifaceted regulation of cell cycle progression by estrogen: regulation of Cdk inhibitors and Cdc25A independent of cyclin D1-Cdk4 function

Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G(1)/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16(INK4a) to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16(INK4a) inhibited G(1)/S transition induced in MCF-7 cells by 17-beta-estradiol (E(2)) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G(1) and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21(Cip1) and p27(Kip1) was decreased, however, in both control and p16(INK4a)-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E(2) in control and p16(INK4a)-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16(INK4a). Inhibition of Cdc25A activity in p16(INK4a)-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E(2)-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisense CDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16(INK4a)-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21(Cip1) and p27(Kip1).     

Specificity of natural and artificial substrates for human Cdc25A

Cdc25A is a dual-specific protein phosphatase involved in the regulation of the kinase activity of Cdk-cyclin complexes in the eukaryotic cell cycle. To understand the mechanism of this important regulator, we have generated highly purified biochemical reagents to determine the kinetic constants for human Cdc25A with respect to a set of peptidic, artificial, and natural substrates. Cdc25A and its catalytic domain (dN25A) demonstrate very similar kinetics toward the artificial substrates p-nitrophenyl phosphate (k(cat)/K(m) = 15-25 M(-1) s(-1)) and 3-O-methylfluorescein phosphate (k(cat)/K(m) = 1.1-1.3 x 10(4) M(-1) s(-1)). Phospho-peptide substrates exhibit extremely low second-order rate constants and a flat specificity profile toward Cdc25A and dN25A (k(cat)/K(m) = 1 to 10 M(-1) s(-1)). In contrast to peptidic substrates, Cdc25A and dN25A are highly active phosphatases toward the natural substrate, T14- and Y15-bis-phosphorylated Cdk2/CycA complex (Cdk2-pTpY/CycA) with k(cat)/K(m) values of 1.0-1.1 x 10(6) M(-1) s(-1). In the context of the Cdk2-pTpY/CycA complex, phospho-threonine is preferred over phospho-tyrosine by more than 10-fold. The highly homologous catalytic domain of Cdc25c is essentially inactive toward Cdk2-pTpY/CycA. Taken together these data indicate that a significant degree of the specificity of Cdc25 toward its Cdk substrate resides within the catalytic domain itself and yet is in a region(s) that is outside the phosphate binding site of the enzyme.     

A critical balance between Cyclin B synthesis and Myt1 activity controls meiosis entry in Xenopus oocytes

In fully grown oocytes, meiosis is arrested at first prophase until species-specific initiation signals trigger maturation. Meiotic resumption universally involves early activation of M phase-promoting factor (Cdc2 kinase-Cyclin B complex, MPF) by dephosphorylation of the inhibitory Thr14/Tyr15 sites of Cdc2. However, underlying mechanisms vary. In Xenopus oocytes, deciphering the intervening chain of events has been hampered by a sensitive amplification loop involving Cdc2-Cyclin B, the inhibitory kinase Myt1 and the activating phosphatase Cdc25. In this study we provide evidence that the critical event in meiotic resumption is a change in the balance between inhibitory Myt1 activity and Cyclin B neosynthesis. First, we show that in fully grown oocytes Myt1 is essential for maintaining prophase I arrest. Second, we demonstrate that, upon upregulation of Cyclin B synthesis in response to progesterone, rapid inactivating phosphorylation of Myt1 occurs, mediated by Cdc2 and without any significant contribution of Mos/MAPK or Plx1. We propose a model in which the appearance of active MPF complexes following increased Cyclin B synthesis causes Myt1 inhibition, upstream of the MPF/Cdc25 amplification loop.