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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activation of Cdc2/cyclin B kinase and entry into mitosis requires dephosphorylation of inhibitory sites on Cdc2 by Cdc25 phosphatase. In vertebrates, Cdc25C is inhibited by phosphorylation at a single site targeted by the checkpoint kinases Chk1 and Cds1/Chk2 in response to DNA damage or replication arrest. In Xenopus early embryos, the inhibitory site on Cdc25C (S287) is also phosphorylated by a distinct protein kinase that may determine the intrinsic timing of the cell cycle. We show that S287-kinase activity is repressed in extracts of unfertilized Xenopus eggs arrested in M phase but is rapidly stimulated upon release into interphase by addition of Ca2+, which mimics fertilization. S287-kinase activity is not dependent on cyclin B degradation or inactivation of Cdc2/cyclin B kinase, indicating a direct mechanism of activation by Ca2+. Indeed, inhibitor studies identify the predominant S287-kinase as Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII phosphorylates Cdc25C efficiently on S287 in vitro and, like Chk1, is inhibited by 7-hydroxystaurosporine (UCN-01) and debromohymenialdisine, compounds that abrogate G2 arrest in somatic cells. CaMKII delays Cdc2/cyclin B activation via phosphorylation of Cdc25C at S287 in egg extracts, indicating that this pathway regulates the timing of mitosis during the early embryonic cell cycle.
Mol Biol Cell 2003 Oct
PMID:Regulation of Cdc2/cyclin B activation in Xenopus egg extracts via inhibitory phosphorylation of Cdc25C phosphatase by Ca(2+)/calmodulin-dependent protein [corrected] kinase II. 1451 14

The replication checkpoint is a dedicated sensor-response system activated by impeded replication forks. It stabilizes stalled forks and arrests division, thereby preserving genome integrity and promoting cell survival. In budding yeast, Tof1 is thought to act as a specific mediator of the replication checkpoint signal that activates the effector kinase Rad53. Here we report studies of fission yeast Swi1, a Tof1-related protein required for a programmed fork-pausing event necessary for mating type switching. Our studies have shown that Swi1 is vital for proficient activation of the Rad53-like checkpoint kinase Cds1. Together they are required to prevent fork collapse in the ribosomal DNA repeats, and they also prevent irreversible fork arrest at a newly identified hydroxyurea pause site. Swi1 also has Cds1-independent functions. Rad22 DNA repair foci form during S phase in swi1 mutants and to a lesser extent in cds1 mutants, indicative of fork collapse. Mus81, a DNA endonuclease required for recovery from collapsed forks, is vital in swi1 but not cds1 mutants. Swi1 is recruited to chromatin during S phase. We propose that Swi1 stabilizes replication forks in a configuration that is recognized by replication checkpoint sensors.
Mol Cell Biol 2003 Nov
PMID:Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1. 1456 29

The checkpoint kinase 1 (Chk1) is an essential component of the DNA damage checkpoint. Previous studies have demonstrated an indispensable role for the p53-related transcription factor p73alpha in DNA damage-induced apoptosis. Here, we provide evidence that p73alpha is a target of Chk1. We found that endogenous p73alpha is serine phosphorylated by endogenous Chk1 upon DNA damage, which is a mechanism required for the apoptotic-inducing function of p73alpha. Consistent with this, we discovered that endogenous p73alpha interacts with Chk1 and is phosphorylated by Chk1 at serine 47 in vitro and in vivo. In contrast, Chk2 does not phosphorylate p73alpha in vitro. Moreover, mutation of serine 47 abolishes both Chk1-dependent phosphorylation of p73alpha upon DNA damage in vivo and the ability of Chk1 to upregulate the transactivation capacity of p73alpha. Our data indicate a novel biochemical pathway through which the p73alpha proapoptotic function requires DNA damage-triggered p73alpha phosphorylation by Chk1.
Mol Cell Biol 2003 Nov
PMID:p73alpha regulation by Chk1 in response to DNA damage. 3130 27

Fission yeast Mrc1 (mediator of replication checkpoint 1) is an adaptor checkpoint protein required for Rad3-dependent activation of the checkpoint kinase Cds1 in response to arrest of replication forks. Here we report studies on the regulation of Mrc1 by phosphorylation. Replication arrest induced by hydroxyurea (HU) induces Mrc1 phosphorylation that is detected by a change in Mrc1 electrophoretic mobility. Phosphorylation is maintained in cds1Delta, rad3Delta, and tel1Delta single mutants but eliminated in a rad3Delta tel1Delta double mutant. Mrc1 has two clusters of S/TQ motifs that are potential Rad3/Tel1 phosphorylation sites. Mutation of six S/TQ motifs in these two clusters strongly impairs Mrc1 phosphorylation. Two motifs located at S604 and T645 are vital for HU resistance. The T645A mutation strongly impairs a Cds1-Mrc1 yeast two-hybrid interaction that is dependent on a functional forkhead-associated (FHA) domain in Cds1, indicating that phosphorylation of T645 mediates Mrc1's association with Cds1. Consistent with this model, the T645 region of Mrc1 effectively substitutes for the T11 region of Cds1 that is thought to be phosphorylated by Rad3 and to mediate FHA-dependent oligomerization of Cds1. The S/TQ cluster that includes S604 is needed for Mrc1's increased association with chromatin in replication-arrested cells. These data indicate that Rad3 and Tel1 regulate Mrc1 through differential phosphorylation to control Cds1.
Mol Cell Biol 2003 Nov
PMID:Replication checkpoint protein Mrc1 is regulated by Rad3 and Tel1 in fission yeast. 1458 96

The pathway determining malignant cellular transformation, which depends upon mutation of the BRCA1 tumor suppressor gene, is poorly defined. A growing body of evidence suggests that promotion of DNA double-strand break repair by homologous recombination (HR) may be the means by which BRCA1 maintains genomic stability, while a role of BRCA1 in error-prone nonhomologous recombination (NHR) processes has just begun to be elucidated. The BRCA1 protein becomes phosphorylated in response to DNA damage, but the effects of phosphorylation on recombinational repair are unknown. In this study, we tested the hypothesis that the BRCA1-mediated regulation of recombination requires the Chk2- and ATM-dependent phosphorylation sites. We studied Rad51-dependent HR and random chromosomal integration of linearized plasmid DNA, a subtype of NHR, which we demonstrate to be dependent on the Mre11-Rad50-Nbs1 complex. Prevention of Chk2-mediated phosphorylation via mutation of the serine 988 residue of BRCA1 disrupted both the BRCA1-dependent promotion of HR and the suppression of NHR. Similar results were obtained when endogenous Chk2 kinase activity was inhibited by expression of a dominant-negative Chk2 mutant. Surprisingly, the opposing regulation of HR and NHR did not require the ATM phosphorylation sites on serines 1423 and 1524. Together, these data suggest a functional link between recombination control and breast cancer predisposition in carriers of Chk2 and BRCA1 germ line mutations. We propose a dual regulatory role for BRCA1 in maintaining genome integrity, whereby BRCA1 phosphorylation status controls the selectivity of repair events dictated by HR and error-prone NHR.
Mol Cell Biol 2004 Jan
PMID:Chk2 phosphorylation of BRCA1 regulates DNA double-strand break repair. 1470 43

We have used genetic and microarray analysis to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila melanogaster. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member EIGER: Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. Our results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways.
Mol Cell Biol 2004 Feb
PMID:Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage. 1472 67

The retinoblastoma protein (Rb)/E2F pathway links cellular proliferation control to apoptosis and is critical for normal development and cancer prevention. Here we define a transcription-mediated pathway in which deregulation of E2F1 by ectopic E2F expression or Rb inactivation by E7 of human papillomavirus type 16 signals apoptosis by inducing the expression of Chk2, a component of the DNA damage response. E2F1- and E7-mediated apoptosis are compromised in cells from patients with the related disorders ataxia telangiectasia and Nijmegen breakage syndrome lacking functional Atm and Nbs1 gene products, respectively. Both Atm and Nbs1 contribute to Chk2 activation and p53 phosphorylation following deregulation of normal Rb growth control. E2F2, a related E2F family member that does not induce apoptosis, also activates Atm, resulting in phosphorylation of p53. However, we found that the key commitment step in apoptosis induction is the ability of E2F1, and not E2F2, to upregulate Chk2 expression. Our results suggest that E2F1 plays a central role in signaling disturbances in the Rb growth control pathway and, by upregulation of Chk2, may sensitize cells to undergo apoptosis.
Mol Cell Biol 2004 Apr
PMID:Apoptosis associated with deregulated E2F activity is dependent on E2F1 and Atm/Nbs1/Chk2. 1502 84

Rad9 is required for the activation of DNA damage checkpoint pathways in budding yeast. Rad9 is phosphorylated after DNA damage in a Mec1- and Tel1-dependent manner and subsequently interacts with Rad53. This Rad9-Rad53 interaction has been suggested to trigger the activation and phosphorylation of Rad53. Here we show that Mec1 controls the Rad9 accumulation at double-strand breaks (DSBs). Rad9 was phosphorylated after DSB induction and associated with DSBs. However, its phosphorylation and association with DSBs were significantly decreased in cells carrying a mec1Delta or kinase-negative mec1 mutation. Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo. In addition, multiple mutations in the Rad9 S/TQ motifs resulted in its defective association with DSBs. Phosphorylation of Rad9 was partially defective in cells carrying a weak mec1 allele (mec1-81), whereas its association with DSBs occurred efficiently in the mec1-81 mutants, as found in wild-type cells. However, the Rad9-Rad53 interaction after DSB induction was significantly decreased in mec1-81 mutants, as it was in mec1Delta mutants. Deletion mutation in RAD53 did not affect the association of Rad9 with DSBs. Our results suggest that Mec1 promotes association of Rad9 with sites of DNA damage, thereby leading to full phosphorylation of Rad9 and its interaction with Rad53.
Mol Cell Biol 2004 Apr
PMID:Association of Rad9 with double-strand breaks through a Mec1-dependent mechanism. 1506 Jan 50

The p53 tumor suppressor protein is phosphorylated and activated by several DNA damage-inducible kinases, such as ATM, and is a key effector of the DNA damage response by promoting cell cycle arrest or apoptosis. Deregulation of the Rb-E2F1 pathway also results in the activation of p53 and the promotion of apoptosis, and this contributes to the suppression of tumor development. Here, we describe a novel connection between E2F1 and the ATM DNA damage response pathway. In primary human fibroblasts lacking functional ATM, the ability of E2F1 to induce the phosphorylation of p53 and apoptosis is impaired. In contrast, ATM status has no effect on transcriptional activation of target genes or the stimulation of DNA synthesis by E2F1. Cells containing mutant Nijmegen breakage syndrome protein (NBS1), a component of the Mre11-Rad50 DNA repair complex, also have attenuated p53 phosphorylation and apoptosis in response to E2F1 expression. Moreover, E2F1 induces ATM- and NBS1-dependent phosphorylation of the checkpoint kinase Chk2 at Thr68, a phosphorylation site that stimulates Chk2 activity. Delayed gammaH2AX phosphorylation and absence of ATM autophosphorylation at Ser1981 suggest that E2F1 stimulates ATM through a unique mechanism that is distinct from agents that cause DNA double-strand breaks. These findings identify new roles for several DNA damage response factors by demonstrating that they also participate in the oncogenic stress signaling pathway between E2F1 and p53.
Mol Cancer Res 2004 Apr
PMID:E2F1 uses the ATM signaling pathway to induce p53 and Chk2 phosphorylation and apoptosis. 1514 Sep 42

The checkpoint kinase Chk2 is activated in response to DNA damage through pathways requiring protein kinases ATM and/or ATR. The means by which Chk2 is activated by these kinases still remains to be addressed. Here we describe a cell-free system to study the activation of Chk2. Chk2 produced by a wheat germ extract in vitro transcription/translation system is inactive and can be activated by incubating with a rabbit reticulocyte lysate. This method will be useful for identification of cofactors required for activation of Chk2.
Methods Mol Biol 2004
PMID:Establishment of a cell-free system to study the activation of Chk2. 1518 52


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