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.     

MAPK Pathway Activation Delays G2/M Progression by Destabilizing Cdc25B

Activation of the mitogen-activated protein kinase (MAPK) pathway by growth factors or phorbol esters during G₂ phase delays entry into mitosis; however, the role of the MAPK pathway during G₂/M progression remains controversial. Here, we demonstrate that activation of the MAPK pathway with either epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate induces a G₂ phase delay independent of known G₂ phase checkpoint pathways but was specifically dependent on MAPK/extracellular signal-regulated kinase kinase (MEK1). Activation of MAPK signaling also blocked exit from a G₂ phase checkpoint arrest. Both the G₂ phase delay and blocked exit from the G₂ checkpoint arrest were mediated by the MEK1-dependent destabilization of the critical G₂/M regulator cdc25B. Reintroduction of cdc25B overcame the MEK1-dependent G₂ phase delay. Thus, we have demonstrated a new function for MEK1 that controls G₂/M progression by regulating the stability of cdc25B. This represents a novel mechanism by which factors that activate MAPK signaling can influence the timing of entry into mitosis, particularly exit from a G₂ phase checkpoint arrest.     

Oxidative stress-induced DNA damage of mouse zygotes triggers G2/M checkpoint and phosphorylates Cdc25 and Cdc2

In vitro fertilized (IVF) embryos show both cell cycle and developmental arrest. We previously showed oxidative damage activates the ATM?→?Chk1?→?Cdc25B/Cdc25C cascade to mediate G2/M cell cycle arrest for repair of hydrogen peroxide (H₂O₂)-induced oxidative damage in sperm. However, the mechanisms underlying the developmental delay of zygotes are unknown. To develop a model of oxidative-damaged zygotes, we treated mouse zygotes with different concentrations of H₂O₂ (0, 0.01, 0.02, 0.03, 0.04, 0.05 mM), and evaluated in vitro zygote development, BrdU incorporation to detect the duration of S phase. We also examined reactive oxygen species level and used immunofluorescence to detect activation of γH2AX, Cdc2, and Cdc25. Oxidatively damaged zygotes showed a delay in G2/M phase and produced a higher level of ROS. At the same time, γH2AX was detected in oxidatively damaged zygotes as well as phospho-Cdc25B (Ser323), phospho-Cdc25C (Ser216), and phospho-Cdc2 (Tyr15). Our study indicates that oxidative stress-induced DNA damage of mouse zygotes triggers the cell cycle checkpoint, which results in G2/M cell cycle arrest, and that phospho-Cdc25B (Ser323), phospho-Cdc25C (Ser216), and phospho-Cdc2 (Tyr15) participate in activating the G2/M checkpoint.     

The oncogenic serine/threonine kinase Pim-1 directly phosphorylates and activates the G2/M specific phosphatase Cdc25C

The proto-oncogene Pim-1 encodes a serine-threonine kinase which is a downstream effector of cytokine signaling and can enhance cell cycle progression by altering the activity of several cell cycle regulators among them the G1 specific inhibitor p21(Waf), the phosphatase Cdc25A and the kinase C-TAK1. Here, we demonstrate by using biochemical assays that Pim-1 can interact with the phosphatase Cdc25C and is able to directly phosphorylate the N-terminal region of the protein. Cdc25C is functionally related to Cdc25A but acts specifically at the G2/M cell cycle transition point and can be inactivated by C-TAK1-mediated phosphorylation. Immuno-fluorescence experiments showed that Pim-1 and Cdc25C co-localize in the cytoplasm of both epithelial and myeloid cells. We find that phosphorylation by Pim-1 enhances the phosphatase activity of Cdc25C and in transfected cells that are arrested in G2/M by bleomycin, Pim-1 can enhance progression into G1. Therefore, we propose that Pim-1 activates Cdc25C by a direct phosphorylation and can thereby assume the function of a positive cell cycle regulator at the G2/M transition.     

Regulation of G(2)/M events by Cdc25A through phosphorylation-dependent modulation of its stability

DNA replication in higher eukaryotes requires activation of a Cdk2 kinase by Cdc25A, a labile phosphatase subject to further destabilization upon genotoxic stress. We describe a distinct, markedly stable form of Cdc25A, which plays a previously unrecognized role in mitosis. Mitotic stabilization of Cdc25A reflects its phosphorylation on Ser17 and Ser115 by cyclin B-Cdk1, modifications required to uncouple Cdc25A from its ubiquitin-proteasome-mediated turnover. Cdc25A binds and activates cyclin B-Cdk1, accelerates cell division when overexpressed, and its downregulation by RNA interference (RNAi) delays mitotic entry. DNA damage-induced G(2) arrest, in contrast, is accompanied by proteasome-dependent destruction of Cdc25A, and ectopic Cdc25A abrogates the G(2) checkpoint. Thus, phosphorylation-mediated switches among three differentially stable forms ensure distinct thresholds, and thereby distinct roles for Cdc25A in multiple cell cycle transitions and checkpoints.     

Cdc2-cyclin B-induced G2 to M transition in perch oocyte is dependent on Cdc25

The G2 to M phase transition in perch oocytes is regulated by maturation promoting factor (MPF), a complex of Cdc2 and cyclin B. In Anabas testudineus, a fresh water perch, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, the maturation inducing hormone (MIH), induced complete germinal vesicle breakdown (GVBD) of oocytes at 21 h. An unusual cyclin, p30 cyclin B, has been identified in oocyte extract using both monoclonal and polyclonal antibodies. Surprisingly, Cdc2 could not be identified, although a Northern blot with Cdc2 cDNA demonstrated expression of the gene. Purification of MPF through an immunoaffinity column followed by SDS-PAGE showed three proteins, Cdc2, cyclin B, and a 20 kDa fragment, indicating earlier failure in immunodetection may be due to the interference by this fragment. In uninduced oocytes, p30 cyclin B was present, and its expression was increased by MIH. MIH increased p30 cyclin B accumulation at 3 h, a high level which was maintained between 9 and 21 h, but an effective increase in GVBD and H1 kinase activation could only be observed between 15 and 21 h. This delay in active MPF formation was found to be related to the activation of Cdc25, phosphorylation of which was detected at 12 h, and a substantial increase occurred during 15-18 h. Sodium orthovanadate, a tyrosine phosphatase inhibitor, inhibited H1 kinase activity and GVBD, suggesting the requirement of Cdc25 activity in MPF activation. Our results show occurrence of pre-MPF in uninduced oocytes and its conversion to active MPF requires dephosphorylation by Cdc25, the existence of which has not yet been shown in fish.     

Gossypin induces G2/M arrest in human malignant glioma U251 cells by the activation of Chk1/Cdc25C pathway

Gossypin is a flavone that was originally isolated from Hibiscus vitifolius and has traditionally been used for the treatment of diabetes, jaundice, and inflammation. Recently, gossypin was found to have potent anticancer properties; however, its effect on human gliomas still remain unknown. To investigate the potential anticancer effects of gossypin on malignant gliomas and analyze the associated molecular mechanisms, we treated human glioma U251 cells with gossypin. Our study showed that the treatment of U251 cells with gossypin inhibited cell proliferation in a dose- and time-dependent manner and was observed to be minimally toxic to normal human astrocytes. Gossypin's effect on cell cycle distribution was observed, and we found that it induced G2/M-phase arrest in U251 cells. An analysis of cell-cycle regulatory proteins indicated that the arresting effect of gossypin on the cell cycle at G2/M phase was involved in the phosphorylation of cell division cycle 25C (Cdc25C) tyrosine phosphatase via the activation of checkpoint kinase 1 (Chk1). These findings indicate that gossypin is a potential treatment of gliomas because of gossypin's potential to regulate the proliferation of U251 cells via the cell-cycle regulatory proteins Chk1 and Cdc25C.     

Ultrasensitivity in the Regulation of Cdc25C by Cdk1

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Highlights? The response of Cdc25C to Cdk1 in Xenopus egg extracts is ultrasensitive ? The Hill coefficient for the response is astronomical (～11–32) ? Multisite phosphorylation accounts for some of the ultrasensitivity ? The Cdc25C and Wee1A responses account for the bistability of the mitotic trigger     

Xp38gamma/SAPK3 promotes meiotic G(2)/M transition in Xenopus oocytes and activates Cdc25C

We have studied the role of p38 mitogen-activated protein kinases (MAPKs) in the meiotic maturation of Xenopus oocytes. Overexpression of a constitutively active mutant of the p38 activator MKK6 accelerates progesterone-induced maturation. Immunoprecipit ation experiments indicate that p38gamma/SAPK3 is the major p38 activated by MKK6 in the oocytes. We have cloned Xenopus p38gamma (Xp38gamma) and show that co-expression of active MKK6 with Xp38gamma induces oocyte maturation in the absence of progesterone. The maturation induced by Xp38gamma requires neither protein synthesis nor activation of the p42 MAPK-p90Rsk pathway, but it is blocked by cAMP-dependent protein kinase. A role for the endogenous Xp38gamma in progesterone-induced maturation is supported by the inhibitory effect of kinase-dead mutants of MKK6 and Xp38gamma. Furthermore, MKK6 can rescue the inhibition of oocyte maturation by anthrax lethal factor, a protease that inactivates MAPK kinases. We also show that Xp38gamma can activate the phosphatase XCdc25C, and we identified Ser205 of XCdc25C as a major phosphorylation site for Xp38gamma. Our results indicate that phosphorylation of XCdc25C by Xp38gamma/SAPK3 is important for the meiotic G(2)/M progression of Xenopus oocytes.     

Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells

The human tyrosine phosphatase (p54(cdc25-c)) is activated by phosphorylation at mitosis entry. The phosphorylated p54(cdc25-c) in turn activates the p34-cyclin B protein kinase and triggers mitosis. Although the active p34-cyclin B protein kinase can itself phosphorylate and activate p54(cdc25-c), we have investigated the possibility that other kinases may initially trigger the phosphorylation and activation of p54(cdc25-c). We have examined the effects of the calcium/calmodulin-dependent protein kinase (CaM kinase II) on p54(cdc25-c). Our in vitro experiments show that CaM kinase II can phosphorylate p54(cdc25-c) and increase its phosphatase activity by 2.5-3-fold. Treatment of a synchronous population of HeLa cells with KN-93 (a water-soluble inhibitor of CaM kinase II) or the microinjection of AC3-I (a specific peptide inhibitor of CaM kinase II) results in a cell cycle block in G2 phase. In the KN-93-arrested cells, p54(cdc25-c) is not phosphorylated, p34(cdc2) remains tyrosine phosphorylated, and there is no increase in histone H1 kinase activity. Our data suggest that a calcium-calmodulin-dependent step may be involved in the initial activation of p54(cdc25-c).     

Crystal structure of the catalytic subunit of Cdc25B required for G2/M phase transition of the cell cycle.

Cdc25B is a dual specificity phosphatase involved in the control of cyclin-dependent kinases and the progression of cells through the cell cycle. A series of minimal domain Cdc25B constructs maintaining catalytic activity have been expressed. The structure of a minimum domain construct binding sulfate was determined at 1.9 A resolution and a temperature of 100 K. Other forms of the same co?nstruct were determined at lower resolution and room temperature. The overall folding and structure of the domain is similar to that found for Cdc25A. An important difference between the two is that the Cdc25B domain binds oxyanions in the catalytic site while that of Cdc25A appears unable to bind oxyanions. There are also important conformational differences in the C-terminal region. In Cdc25B, both sulfate and tungstate anions are shown to bind in the catalytic site containing the signature motif (HCxxxxxR) in a conformation similar to that of other protein tyrosine phosphatases and dual specificity phosphatases, with the exception of the Cdc25A. The Cdc25B constructs, with various truncations of the C-terminal residues, are shown to have potent catalytic activity. When cut back to the site at which the Cdc25A structure begins to deviate from the Cdc25B structure, the activity is considerably less. There is a pocket extending from the catalytic site to an anion-binding site containing a chloride about 14 A away. The catalytic cysteine residue, Cys473, can be oxidized to form a disulfide linkage to Cys426. A readily modifiable cysteine residue, Cys484, resides in another pocket that binds a sulfate but not in the signature motif conformation. This region of the structure is highly conserved between the Cdc25 molecules and could serve some unknown function.     

Fission yeast Clp1p phosphatase affects G2/M transition and mitotic exit through Cdc25p inactivation

The Cdc14 family of phosphatases specifically reverses proline-directed phosphorylation events. In Saccharomyces cerevisiae, Cdc14p promotes Cdk1p inactivation at mitotic exit by reversing Cdk1p-dependent phosphorylations. Cdk1p is a proline-directed kinase whose activity is required in all eukaryotes for the transit into mitosis. At mitotic commitment, Cdk1p participates in its own regulation by activating the mitotic inducing phosphatase, Cdc25p, and inhibiting the opposing kinase, Wee1p. We have investigated the ability of Schizosaccharomyces pombe Clp1p, a Cdc14p homolog, to disrupt this auto-amplification loop. We show here that Clp1p is required to dephosphorylate, destabilize, and inactivate Cdc25p at the end of mitosis. Clp1p promotes recognition of Cdc25p by the anaphase-promoting complex/cyclosome, an E3 ubiquitin ligase. Failure to inactivate and destabilize Cdc25p in late mitosis delays progression through anaphase, interferes with septation initiation network signaling, and additionally advances the commitment to mitotic entry in the next cycle. This may be a widely conserved mechanism whereby Cdc14 proteins contribute to Cdk1p inactivation.     

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.     

Spatial Regulation of Cdc42 During Cytokinesis

Cdc42 GTPase plays a critical role in the establishment of cell polarity in most eukaryotic organisms. Cdc42 active state, as that of other GTPases, depends on the bound nucleotide. The protein with GTP is active, and only in this state can it interact with different target effector proteins. The spatio-temporal control of Cdc42 activity is therefore necessary to generate growth polarity. In fission yeast cells, Cdc42 mainly localizes to the division area, and also to the growing tips and to some internal membranes. While the role of Cdc42 in apical growth is well defined, no role has been described for Cdc42 in the process of cell division. Fission yeast Cdc42 activity is regulated by two specific guanidine nucleotide exchange factors (GEFs), Scd1, and Gef1. We discuss here how Hob3, a BAR domain containing protein similar to human BIN3 and S. cerevisiae Rsv161, may be required to recruit Cdc42 to the cell division site as well as for the activation of this GTPase mediated by Gef1. We also discuss the possible role of Cdc42 in the contraction of the actomyosin ring necessary for cytokinesis.     

Ser149 is another potential PKA phosphorylation target of Cdc25B in G2/M transition of fertilized mouse eggs

It is well documented that protein kinase A (PKA) acts as a negative regulator of M phase promoting factor (MPF) by phosphorylating cell division cycle 25 homolog B (Cdc25B) in mammals. However, the molecular mechanism remains unclear. In this study, we identified PKA phosphorylation sites in vitro by LC-MS/MS analysis, including Ser(149), Ser(229), and Ser(321) of Cdc25B, and explored the role of Ser(149) in G(2)/M transition of fertilized mouse eggs. The results showed that the overexpressed Cdc25B-S149A mutant initiated efficient MPF activation by direct dephosphorylation of Cdc2-Tyr(15), resulting in triggering mitosis prior to Cdc25B-WT. Conversely, overexpression of the phosphomimic Cdc25B-S149D mutant showed no significant difference in comparison with the control groups. Furthermore, we found that Cdc25B-Ser(149) was phosphorylated at G(1) and S phases, whereas dephosphorylated at G(2) and M phases, and the phosphorylation of Cdc25B-Ser(149) was modulated by PKA in vivo. In addition, we examined endogenous and exogenous Cdc25B, which were expressed mostly in the cytoplasm at the G(1) and S phases and translocated to the nucleus at the G(2) phase. Collectively, our findings provide evidence that Ser(149) may be another potential PKA phosphorylation target of Cdc25B in G(2)/M transition of fertilized mouse eggs and Cdc25B as a direct downstream substrate of PKA in mammals, which plays important roles in the regulation of early development of mouse embryos.     

CDC25B Phosphorylation by Aurora A Occurs at the G2/M Transition and is Inhibited by DNA Damage

CDC25B is one of the three human dual-specificity phosphatases involved in the activation ofcyclin-dependent kinases at key stages of the cell division cycle. CDC25B that is responsiblefor the activation of CDK1-cyclin B1 is regulated by phosphorylation. The STK15/Aurora-Akinase locally phosphorylates CDC25B on serine 353 at the centrosome during the G2/Mtransition. Here we have investigated this phosphorylation event during the cell cycle, and inresponse to activation of the G2 DNA damage checkpoint. We show that accumulation of theS353-phosphorylated form of CDC25B at the centrosome correlates with the relocalisation ofcyclin B1 to the nucleus and the activation of CDK1 at entry into mitosis. Upon activation ofthe G2/M checkpoint by DNA damage, we demonstrate that Aurora-A is not activated andconsequently CDC25B is not phosphorylated. We show that ectopic expression of Aurora-Aresults in a bypass of the checkpoint that partially overcome by a S353A mutant of CDC25B.Finally, we show that bypass of the G2/M checkpoint by the CHK1 kinase inhibitor UCN-01results in the activation of Aurora-A and phosphorylation of CDC25B on S353. These resultsstrongly suggest that Aurora-A-mediated phosphorylation of CDC25B at the centrosome is animportant step contributing to the earliest events inducing mitosis, upstream of CDK1-cyclinB1 activation.     

Regulation of Mih1/Cdc25 by protein phosphatase 2A and casein kinase 1

The Cdc25 phosphatase promotes entry into mitosis by removing cyclin-dependent kinase 1 (Cdk1) inhibitory phosphorylation. Previous work suggested that Cdc25 is activated by Cdk1 in a positive feedback loop promoting entry into mitosis; however, it has remained unclear how the feedback loop is initiated. To learn more about the mechanisms that regulate entry into mitosis, we have characterized the function and regulation of Mih1, the budding yeast homologue of Cdc25. We found that Mih1 is hyperphosphorylated early in the cell cycle and is dephosphorylated as cells enter mitosis. Casein kinase 1 is responsible for most of the hyperphosphorylation of Mih1, whereas protein phosphatase 2A associated with Cdc55 dephosphorylates Mih1. Cdk1 appears to directly phosphorylate Mih1 and is required for initiation of Mih1 dephosphorylation as cells enter mitosis. Collectively, these observations suggest that Mih1 regulation is achieved by a balance of opposing kinase and phosphatase activities. Because casein kinase 1 is associated with sites of polar growth, it may regulate Mih1 as part of a signaling mechanism that links successful completion of growth-related events to cell cycle progression.