In vitro phosphorylation of BRCA2 by the checkpoint kinase CHEK2.
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ABSTRACT:
SUBMITTER: Kim S
PROVIDER: S-EPMC2570522 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Renal cell carcinoma (RCC) occurs in sporadic and heritable forms. Genetic mutations have been identified as risk factors in 1-2% of RCC. The aim of this study was to evaluate I157T and CHEK2*1100delC mutations of checkpoint kinase 2 (CHEK2) gene in RCC. Medical records of 40 clear cell RCC patients who had genetic tests and consultation at the Genetic Outpatient Clinic, Maria Sk?odowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland, were reviewed retrospectively. Mutation profile was assessed by ASA-PCR and RFLP-PCR techniques. Only three female patients had CHEK2 mutation (I157T). No CHEK2*1100delC was observed in any of the patients. These tumors were N0, and two were Grade 3. One showed capsular infiltration. No blood vessel infiltration or metastases was observed. Overall, RCC from patients with CHEK2 mutation did not display any special characteristics when compared with those without the mutation. While no association between CHEK2 mutation and RCC could be established, all three patients with CHEK2 mutation developed second neoplasms many years after first diagnosis. Further studies, especially regarding CHEK2 mutation as a predictive factor for second neoplasm in RCC patients, are warranted.
| S-EPMC5906733 | biostudies-literature
Project description:Checkpoint kinase 1 (Chk1), a serine/threonine protein kinase, is centrally involved in cell-cycle checkpoints and cellular response to DNA damage. Phosphorylation of Chk1 at 2 Ser/Gln (SQ) sites, Ser-317 and Ser-345, by the upstream kinase ATR is critical for checkpoint activation. However, the precise molecular mechanisms controlling Chk1 phosphorylation and subsequent checkpoint activation are not well understood. Here, we report unique autoregulatory mechanisms that control protein phosphorylation of human Chk1, as well as checkpoint activation and cell viability. Phosphorylation of Ser-317 is required, but not sufficient, for maximal phosphorylation at Ser-345. The N-terminal kinase domain of Chk1 prevents Chk1 phosphorylation at the C-terminus by ATR in the absence of DNA damage. Loss of the inhibitory effect imposed by the N-terminus causes constitutive phosphorylation of Chk1 by ATR under normal growth conditions, which in turn triggers artificial checkpoints that suppress the S-phase progression. Furthermore, two point mutations were identified that rendered Chk1 constitutively active, and expression of the constitutively active mutant form of Chk1 inhibited cancer cell proliferation. Our findings therefore reveal unique regulatory mechanisms of Chk1 phosphorylation and suggest that expression of constitutively active Chk1 may represent a novel strategy to suppress tumor growth. Cancer Res; 72(15); 3786-94. ©2012 AACR.
| S-EPMC3412312 | biostudies-literature
Project description:In recent years, immune checkpoint inhibitors (ICPI) have become widely used for multiple solid malignancies. Reliable predictive biomarkers for selection of patients who would benefit most are lacking. Several tumor types with somatic or germline alterations in genes involved in the DNA damage response (DDR) pathway harbor a higher tumor mutational burden, possibly associated with an increased tumoral neoantigen load. These neoantigens are thought to lead to stronger immune activation and enhanced response to ICPIs. We present a series of seven patients with different malignancies with germline disease-associated variants in DDR genes (BRCA1, BRCA2, CHEK2) responding favorably to ICPIs.
| S-EPMC8395488 | biostudies-literature
Project description:ImportanceApproximately 50% of the risk for the development of testicular germ cell tumors (TGCTs) is estimated to be heritable, but no mendelian TGCT predisposition genes have yet been identified. It is hypothesized that inherited pathogenic DNA repair gene (DRG) alterations may drive susceptibility to TGCTs.ObjectiveTo systematically evaluate the enrichment of germline pathogenic variants in the mendelian cancer predisposition DRGs in patients with TGCTs vs healthy controls.Design, setting, and participantsA case-control enrichment analysis was performed from January 2016 to May 2018 to screen for 48 DRGs in 205 unselected men with TGCT and 27 173 ancestry-matched cancer-free individuals from the Exome Aggregation Consortium cohort in the discovery stage. Significant findings were selectively replicated in independent cohorts of 448 unselected men with TGCTs and 442 population-matched controls, as well as 231 high-risk men with TGCTs and 3090 ancestry-matched controls. Statistical analysis took place from January to May 2018.Main outcomes and measuresGene-level enrichment analysis of germline pathogenic variants in individuals with TGCTs relative to cancer-free controls.ResultsAmong 205 unselected men with TGCTs (mean [SD] age, 33.04 [9.67] years), 22 pathogenic germline DRG variants, one-third of which were in CHEK2 (OMIM 604373), were identified in 20 men (9.8%; 95% CI, 6.1%-14.7%). Unselected men with TGCTs were approximately 4 times more likely to carry germline loss-of-function CHEK2 variants compared with cancer-free individuals from the Exome Aggregation Consortium cohort (odds ratio [OR], 3.87; 95% CI, 1.65-8.86; nominal P = .006; q = 0.018). Similar enrichment was also seen in an independent cohort of 448 unselected Croatian men with TGCTs (mean [SD] age, 31.98 [8.11] years) vs 442 unselected Croatian men without TGCTs (at least 50 years of age at time of sample collection) (OR, >1.4; P = .03) and 231 high-risk men with TGCTs (mean [SD] age, 31.54 [9.24] years) vs 3090 men (all older than 50 years) from the Penn Medicine Biobank (OR, 6.30; 95% CI, 2.34-17.31; P = .001). The low-penetrance CHEK2 variant (p.Ile157Thr) was found to be a Croatian founder TGCT risk variant (OR, 3.93; 95% CI, 1.53-9.95; P = .002). Individuals with the pathogenic CHEK2 loss-of-function variants developed TGCTs 6 years earlier than individuals with CHEK2 wild-type alleles (5.95 years; 95% CI, 1.48-10.42; P = .009).Conclusions and relevanceThis multicenter case-control analysis of men with or without TGCTs provides evidence for CHEK2 as a novel moderate-penetrance TGCT susceptibility gene, with potential clinical utility. In addition to highlighting DNA-repair deficiency as a potential mechanism driving TGCT susceptibility, this analysis also provides new avenues to explore management strategies and biological investigations for high-risk individuals.
| S-EPMC6459214 | biostudies-literature
Project description:We conducted imputation to the 1000 Genomes Project of four genome-wide association studies of lung cancer in populations of European ancestry (11,348 cases and 15,861 controls) and genotyped an additional 10,246 cases and 38,295 controls for follow-up. We identified large-effect genome-wide associations for squamous lung cancer with the rare variants BRCA2 p.Lys3326X (rs11571833, odds ratio (OR) = 2.47, P = 4.74 × 10(-20)) and CHEK2 p.Ile157Thr (rs17879961, OR = 0.38, P = 1.27 × 10(-13)). We also showed an association between common variation at 3q28 (TP63, rs13314271, OR = 1.13, P = 7.22 × 10(-10)) and lung adenocarcinoma that had been previously reported only in Asians. These findings provide further evidence for inherited genetic susceptibility to lung cancer and its biological basis. Additionally, our analysis demonstrates that imputation can identify rare disease-causing variants with substantive effects on cancer risk from preexisting genome-wide association study data.
| S-EPMC4074058 | biostudies-literature
Project description:The spindle checkpoint senses unattached kinetochores and inhibits the Cdc20-bound anaphase-promoting complex or cyclosome (APC/C), to delay anaphase, thereby preventing aneuploidy. A critical checkpoint inhibitor of APC/C(Cdc20) is the mitotic checkpoint complex (MCC). It is unclear whether MCC suffices to inhibit all cellular APC/C. Here we show that human checkpoint kinase Bub1 not only directly phosphorylates Cdc20, but also scaffolds Plk1-mediated phosphorylation of Cdc20. Phosphorylation of Cdc20 by Bub1-Plk1 inhibits APC/C(Cdc20) in vitro and is required for checkpoint signalling in human cells. Bub1-Plk1-dependent Cdc20 phosphorylation is regulated by upstream checkpoint signals and is dispensable for MCC assembly. A phospho-mimicking Cdc20 mutant restores nocodazole-induced mitotic arrest in cells depleted of Mad2 or BubR1. Thus, Bub1-Plk1-mediated phosphorylation of Cdc20 constitutes an APC/C-inhibitory mechanism that is parallel, but not redundant, to MCC formation. Both mechanisms are required to sustain mitotic arrest in response to spindle defects.
| S-EPMC4773433 | biostudies-literature
Project description:The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.
| S-EPMC8387023 | biostudies-literature
Project description:Previous studies have shown that two rare variants, rs11571833 in BRCA2 and rs17879961 in CHEK2 were associated with lung cancer. However, the associations between these two variants and urinary tract cancers risk remain largely unexplored. We applied imputation of three genome-wide association studies published in the database of Genotypes and Phenotypes (dbGaP). Unconditional logistic regression analysis and meta-analysis were performed to assess the association between these two variants and the risk of urinary tract cancers. Our results showed that rs11571833[T] had an effect on urinary tract cancers predisposition (ORmeta = 1.45, Pmeta = 0.013), especially associated with increased the risk of bladder cancer (ORmeta = 1.60, Pmeta = 0.010). Moreover, rs17879961[C] had a protective effect on the urinary tract cancers (ORmeta = 0.67, Pmeta = 1.0 × 10(-3)) and was mostly associated with a lower incidence of renal cell carcinoma (ORmeta = 0.51, Pmeta = 2.0 × 10(-3)). Together, our study indicates that BRCA2 and CHEK2 play an important role in the genetic susceptibility to urinary tract cancers.
| S-EPMC5025839 | biostudies-literature
Project description:Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.
| S-EPMC4424366 | biostudies-literature
Project description:Inherited mutations in the tumor suppressor BRCA2 are predisposed to pancreatic adenocarcinomas, which carry activating mutations in the KRAS oncogene in more than 95% of cases, as well as frequent TP53 inactivation. Here, we have established an RNA interference (RNAi) screen to identify genes whose depletion selectively inhibits the growth of cells lacking BRCA2, and then studied the effects of the genetic depletion or pharmacologic inhibition of 1 candidate, the checkpoint kinase 1 (CHK1), in the context of pancreatic cancer. Pharmacologic inhibition of CHK1 using small-molecule inhibitors (CHK1i) reduced cell growth in several cell lines depleted of BRCA2. Unexpectedly, these drugs did not suppress the growth of BRCA2-deficient pancreatic cancer cell lines from humans or gene-targeted mice expressing active Kras and trans-dominant inhibitory mutant Trp53. Remarkably, the expression of KRAS(G12V) and TP53(G154V) in BRCA2-depleted HEK293 cells was sufficient to render them resistant to CHK1i (but not to mitomycin C or inhibitors of PARP1). CHK1i sensitivity was restored by gemcitabine, an S-phase genotoxin used to treat pancreatic adenocarcinoma. Thus, the growth-suppressive effect of CHK1 inhibition in BRCA2-mutant tumors can be opposed by concurrent KRAS activation and TP53 mutations typical of pancreatic adenocarcinoma, and CHK1i resistance in this setting can be overcome by gemcitabine. Our findings show that approaches that use potential therapeutic targets for cancer identified in synthetic lethal RNAi screens are affected by the genetic context of specific malignancies and combination therapy with other agents. This concept should be taken into account in the ongoing and future development of targeted cancer therapies.
| S-EPMC3087888 | biostudies-literature