CHK1 phosphorylates CDC25B during the cell cycle in the absence of DNA damage (original) (raw)
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Journal of Biological Chemistry, 2003
Inhibition of cyclin-dependent kinases (CDKs) by Thr 14 /Tyr 15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G 2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominantnegative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr 14 /Tyr 15 phosphorylation in normal cell cycle and during the DNA damage checkpoint. Cyclins and cyclin-dependent kinases (CDKs) 1 are key regulators of the eukaryotic cell cycle. In mammalian cells, differ
PLoS ONE, 2011
Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/ cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.
Cdc25 Inhibited In Vivo and In Vitro by Checkpoint Kinases Cds1 and Chk1
Molecular biology of …, 1999
In the fission yeast Schizosaccharomyces pombe, the protein kinase Cds1 is activated by the S-M replication checkpoint that prevents mitosis when DNA is incompletely replicated. Cds1 is proposed to regulate Wee1 and Mik1, two tyrosine kinases that inhibit the mitotic kinase Cdc2. Here, we present evidence from in vivo and in vitro studies, which indicates that Cds1 also inhibits Cdc25, the phosphatase that activates Cdc2. In an in vivo assay that measures the rate at which Cdc25 catalyzes mitosis, Cds1 contributed to a mitotic delay imposed by the S-M replication checkpoint. Cds1 also inhibited Cdc25-dependent activation of Cdc2 in vitro. Chk1, a protein kinase that is required for the G 2 -M damage checkpoint that prevents mitosis while DNA is being repaired, also inhibited Cdc25 in the in vitro assay. In vitro, Cds1 and Chk1 phosphorylated Cdc25 predominantly on serine-99. The Cdc25 alanine-99 mutation partially impaired the S-M replication and G 2 -M damage checkpoints in vivo. Thus, Cds1 and Chk1 seem to act in different checkpoint responses to regulate Cdc25 by similar mechanisms.
The polo-like kinase 1 regulates CDC25B-dependent mitosis entry
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2009
Activation of cyclin-dependent kinase complexes (CDK) at key cell cycle transitions is dependent on their dephosphorylation by CDC25 dual-specificity phosphatases (CDC25A, B and C in human). The CDC25B phosphatase plays an essential role in controlling the activity of CDK1-cyclin B complexes at the entry into mitosis and together with polo-like kinase 1 (PLK1) in regulating the resumption of cell cycle progression after DNA damage-dependent checkpoint arrest in G2. In this study, we analysed the regulation of CDC25Bdependent mitosis entry by PLK1. We demonstrate that PLK1 activity is essential for the relocation of CDC25B from the cytoplasm to the nucleus. By gain and loss of function analyses, we show that PLK1 stimulates CDC25B-induced mitotic entry in both normal conditions and after DNA-damage induced G2/M arrest. Our results support a model in which the relocalisation of CDC25B to the nucleus at the G2-M transition by PLK1 regulates its mitotic inducing activity.
The G2/M checkpoint phosphatase cdc25C is located within centrosomes
The International Journal of Biochemistry & Cell Biology, 2007
cdc25C is a phosphatase which regulates the activity of the mitosis promoting factor cyclin B/cdk1 by dephosphorylation, thus triggering G 2 /M transition. The activity and the sub-cellular localisation of cdc25C are regulated by phosphorylation. It is well accepted that cdc25C has to enter the nucleus to activate the cyclin B/cdk1 complex at G 2 /M transition. Here, we will show that cdc25C is located in the cytoplasm at defined dense structures, which according to immunofluorescence analysis, electron microscopy as well as biochemical subfractionation, are proven to be the centrosomes. Since cyclin B and cdk1 are also located at the centrosomes, this subfraction of cdc25C might participate in the control of the onset of mitosis suggesting a further role for cdc25C at the centrosomes.
Human Cdc14A becomes a cell cycle gene in controlling Cdk1 activity at the G2/M transition
Cell Cycle, 2011
C dc14 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 stages 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 homolog 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, downregulates Cdc25 activity at the G 2 /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.
Journal of Biological Chemistry, 2006
DNA damage induced by the carcinogen benzo[a]pyrene dihydrodiol epoxide (BPDE) induces a Chk1-dependent S-phase checkpoint. Here, we have investigated the molecular basis of BPDE-induced S-phase arrest. Chk1-dependent inhibition of DNA synthesis in BPDE-treated cells occurred without detectable changes in Cdc25A levels, Cdk2 activity, or Cdc7/ Dbf4 interaction. Overexpression studies showed that Cdc25A, cyclin A/Cdk2, and Cdc7/Dbf4 were not rate-limiting for DNA synthesis when the BPDE-induced S-phase checkpoint was active. To investigate other potential targets of the S-phase checkpoint, we tested the effects of BPDE on the chromatin association of DNA replication factors. The levels of chromatinassociated Cdc45 (but not soluble Cdc45) were reduced concomitantly with BPDE-induced Chk1 activation and inhibition of DNA synthesis. The chromatin association of Mcm7, Mcm10, and proliferating cell nuclear antigen was unaffected by BPDE treatment. However, the association between Mcm7 and Cdc45 in the chromatin fraction was inhibited in BPDE-treated cells. Chromatin immunoprecipitation analyses demonstrated reduced association of Cdc45 with the -globin origin of replication in BPDE-treated cells. The inhibitory effects of BPDE on DNA synthesis, Cdc45/Mcm7 associations, and interactions between Cdc45 and the -globin locus were abrogated by the Chk1 inhibitor UCN-01. Taken together, our results show that the association between Cdc45 and Mcm7 at origins of replication is negatively regulated by Chk1 in a Cdk2-independent manner. Therefore, Cdc45 is likely to be an important target of the Chk1-mediated S-phase checkpoint. Cells are continuously exposed to endogenous and exogenous sources of DNA damage. Unrepaired DNA damage can cause mutations and therefore poses a serious threat to genomic stability. Cells have evolved multiple mechanisms to minimize the detrimental effects of DNA damage. Cell cycle checkpoints are signal transduction pathways that respond to DNA damage by eliciting transient delays in the cell cycle (1). The resulting delays integrate DNA repair with cell cycle progression and are thought to be important for maintaining genomic stability. In response to genotoxins, the DNA damage response pathways prevent entry into S-phase (the G 1 /S checkpoint), slow progression through S-phase (the intra-S-phase or S-phase checkpoint), and block entry into mitosis (the G 2 /M checkpoint) (1). Components of checkpoint signaling pathways are highly conserved in eukaryotes. DNA damage is detected by sensors (e.g. ATR/ATRIP, 9-1-1 complex, and ATM) with the aid of mediators (e.g. BRCA1, 53BP1, and Claspin). Sensors act upstream of transducers (e.g. Chk1 and Chk2), which in turn activate effectors (p53 and Cdc25A) that interact with cell cycle machinery to inhibit cell cycle progression (2). The S-phase checkpoint down-regulates initiation of DNA synthesis in response to DNA damage acquired during S-phase (3). We characterized previously an intra-S-phase checkpoint elicited by the environmental carcinogen benzo[a]pyrene (4). Benzo[a]pyrene undergoes intracellular metabolism to generate the reactive species benzo[a]pyrene dihydrodiol epoxide (BPDE) 2 (5). BPDE reacts covalently with DNA to generate bulky adducts, mainly at N-2 of deoxyguanosine residues (6, 7). Different doses of BPDE inhibit DNA synthesis via distinct mechanisms. Low concentrations of BPDE (Ͻ100 nM) inhibit initiation of DNA synthesis transiently, yet do not affect elongation of existing replicons (8). Thus, the transient inhibition of DNA synthesis induced by Ͻ100 nM BPDE is an S-phase checkpoint that inhibits firing of origins of replication in response to DNA damage acquired during S-phase. We have shown that the checkpoint induced by low concentrations of BPDE is mediated via the Chk1 and 9-1-1 pathways (4, 9). Cells eventually recover from the S-phase checkpoint, and low doses of BPDE do not affect cell viability (9). However, higher concentrations of BPDE (200-600 nM) inhibit initiation and elongation steps of DNA replication, resulting in an irreversible block to DNA synthesis and loss of viability (8).