Structural basis for recruitment of the CHK1 DNA damage kinase by the CLASPIN scaffold protein.
Ontology highlight
ABSTRACT:
SUBMITTER: Day M
PROVIDER: S-EPMC8204404 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Glycogen synthase kinase 3beta (GSK3beta) is a serine/threonine kinase involved in insulin, growth factor and Wnt signalling. In Wnt signalling, GSK3beta is recruited to a multiprotein complex via interaction with axin, where it hyperphosphorylates beta-catenin, marking it for ubiquitylation and destruction. We have now determined the crystal structure of GSK3beta in complex with a minimal GSK3beta-binding segment of axin, at 2.4 A resolution. The structure confirms the co-localization of the binding sites for axin and FRAT in the C-terminal domain of GSK3beta, but reveals significant differences in the interactions made by axin and FRAT, mediated by conformational plasticity of the 285-299 loop in GSK3beta. Detailed comparison of the axin and FRAT GSK3beta complexes allows the generation of highly specific mutations, which abrogate binding of one or the other. Quantitative analysis suggests that the interaction of GSK3beta with the axin scaffold enhances phosphorylation of beta-catenin by >20 000-fold.
| S-EPMC140752 | biostudies-literature
Project description:BackgroundAberrant activation of DNA damage response (DDR) is a major cause of chemoresistance in colorectal cancer (CRC). CHK1 is upregulated in CRC and contributes to therapeutic resistance. We investigated the upstream signaling pathways governing CHK1 activation in CRC.MethodsWe identified CHK1-binding proteins by mass spectrometry analysis. We analyzed the biologic consequences of knockout or overexpression of TRAF4 using immunoblotting, immunoprecipitation, and immunofluorescence. CHK1 and TRAF4 ubiquitination was studied in vitro and in vivo. We tested the functions of TRAF4 in CHK1 phosphorylation and CRC chemoresistance by measuring cell viability and proliferation, anchorage-dependent and -independent cell growth, and mouse xenograft tumorigenesis. We analyzed human CRC specimens by immunohistochemistry.ResultsTRAF4 catalyzed the ubiquitination of CHK1 in multiple CRC cell lines. Following DNA damage, ubiquitination of CHK1 at K132 by TRAF4 is required for CHK1 phosphorylation and activation mediated by ATR. Notably, TRAF4 was highly expressed in chemotherapy-resistant CRC specimens and positively correlated with phosphorylated CHK1. Furthermore, depletion of TRAF4 impaired CHK1 activity and sensitized CRC cells to fluorouracil and other chemotherapeutic agents in vitro and in vivo.ConclusionsThese data reveal two novel steps required for CHK1 activation in which TRAF4 serves as a critical intermediary and suggest that inhibition of the ATR-TRAF4-CHK1 signaling may overcome CRC chemoresistance.
| S-EPMC7193419 | biostudies-literature
Project description:SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1 (SMARCAL1) is a recently identified DNA damage response protein involved in remodeling stalled replication forks. The eukaryotic single-strand DNA binding protein replication protein A (RPA) recruits SMARCAL1 to stalled forks in vivo and facilitates regression of forks containing leading strand gaps. Both activities are mediated by a direct interaction between an RPA binding motif (RBM) at the N-terminus of SMARCAL1 and the C-terminal winged-helix domain of the RPA 32 kDa subunit (RPA32C). Here we report a biophysical and structural characterization of the SMARCAL1-RPA interaction. Isothermal titration calorimetry and circular dichroism spectroscopy revealed that RPA32C binds SMARCAL1-RBM with a Kd of 2.5 μM and induces a disorder-to-helix transition. The crystal structure of RPA32C was refined to 1.4 Å resolution, and the SMARCAL1-RBM binding site was mapped on the structure on the basis of nuclear magnetic resonance chemical shift perturbations. Conservation of the interaction surface to other RBM-containing proteins allowed construction of a model for the RPA32C/SMARCAL1-RBM complex. The implications of our results are discussed with respect to the recruitment of SMARCAL1 and other DNA damage response and repair proteins to stalled replication forks.
| S-EPMC4020579 | biostudies-literature
Project description:Cell-cycle checkpoints induced by DNA damage or replication play critical roles in the maintenance of genomic integrity during cell proliferation. Biochemical analysis of checkpoint pathways has been greatly facilitated by the use of cell-free systems made from Xenopus eggs. In the present study, we describe a human cell-free system that reproduces a DNA-dependent checkpoint pathway acting on the Chk1 protein kinase. In this system, double-stranded DNA oligonucleotides induce the phosphorylation of Chk1 at activating sites targeted by ATR [ATM (ataxia telangiectasia mutated)- and Rad3-related] and ATM kinases. Phosphorylation of Chk1 is dependent on the interaction of Claspin, a protein first identified in Xenopus as a Chk1-binding protein. We show that the DNA-dependent binding of Chk1 to Claspin requires two phosphorylation sites, Thr916 and Ser945, which lie within the Chk1-binding domain of Claspin. Using a phosphopeptide derived from the consensus motif of these sites, we show that the interaction of Claspin with Chk1 is required for the ATR/ATM-dependent phosphorylation of Chk1. Using a panel of protein kinase inhibitors, we provide evidence that Chk1 is phosphorylated at an additional site in response to activation of the checkpoint response, probably by autophosphorylation. Claspin is phosphorylated in the Chk1-binding domain in an ATR/ATM-dependent manner and is also targeted by additional kinases in response to double-stranded DNA oligonucleotides. This cell-free system will facilitate further biochemical analysis of the Chk1 pathway in humans.
| S-EPMC1138979 | biostudies-literature
Project description:Rad4/Cut5 is a scaffold protein in the Chk1-mediated DNA damage checkpoint in S. pombe. However, whether it contains a robust ATR-activation domain (AAD) required for checkpoint signaling like its orthologs TopBP1 in humans and Dpb11 in budding yeast has been incompletely clear. To identify the putative AAD in Rad4, we carried out an extensive genetic screen looking for novel mutants with an enhanced sensitivity to replication stress or DNA damage in which the function of the AAD can be eliminated by the mutations. Two new mutations near the N-terminus were identified that caused significantly higher sensitivities to DNA damage or chronic replication stress than all previously reported mutants, suggesting that most of the checkpoint function of the protein is eliminated. However, these mutations did not affect the activation of Rad3 (ATR in humans) yet eliminated the scaffolding function of the protein required for the activation of Chk1. Several mutations were also identified in or near the recently reported AAD in the C-terminus of Rad4. However, all mutations in the C-terminus only slightly sensitized the cells to DNA damage. Interestingly, a mutant lacking the whole C-terminus was found resistant to DNA damage and replication stress almost like the wild type cells. Consistent with the resistance, all known Rad3 dependent phosphorylations of checkpoint proteins remained intact in the C-terminal deletion mutant, indicating that unlike that in Dpb11, the C-terminus of Rad4 does not contain a robust AAD. These results, together with those from the biochemical studies, show that Rad4 mainly functions as a scaffold protein in the Chk1, not the Cds1(CHK2 in humans), checkpoint pathway. It plays a minor role or is functionally redundant with an unknown factor in Rad3 activation.
| S-EPMC3963969 | biostudies-literature
Project description:The DNA damage response triggers cell-cycle checkpoints, DNA repair and apoptosis using multiple post-translational modifications as molecular switches. However, how ubiquitination regulates ATR signaling in response to replication stress and single-strand break is still unclear. Here, we identified the deubiquitination enzyme (DUB) USP20 as a pivotal regulator of ATR-related DDR pathway. Through screening a panel of DUBs, we identified USP20 as critical for replication stress response. USP20 is phosphorylated by ATR, resulting in disassociation of the E3 ubiquitin ligase HERC2 from USP20 and USP20 stabilization. USP20 in turn deubiquitinates and stabilizes Claspin and enhances the activation of ATR-Chk1 signaling. These findings reveal USP20 to be a novel regulator of ATR-dependent DNA damage signaling.
| S-EPMC4245938 | biostudies-literature
Project description:CLASPIN is an essential mediator in the DNA replication checkpoint, responsible for ATR (ataxia telangiectasia and Rad3-related protein)-dependent activation of CHK1 (checkpoint kinase 1). Here we found a dynamic signaling pathway that regulates CLASPIN turn over. Under unperturbed conditions, the E3 ubiquitin ligase HERC2 regulates the stability of the deubiquitinating enzyme USP20 by promoting ubiquitination-mediated proteasomal degradation. Under replication stress, ATR-mediated phosphorylation of USP20 results in the disassociation of HERC2 from USP20. USP20 in turn deubiquitinates K48-linked-polyubiquitinated CLASPIN, stabilizing CLASPIN and ultimately promoting CHK1 phosphorylation and CHK1-directed checkpoint activation. Inhibition of USP20 expression promotes chromosome instability and xenograft tumor growth. Taken together, our findings demonstrated a novel function of HERC2/USP20 in coordinating CHK1 activation by modulating CLASPIN stability, which ultimately promotes genome stability and suppresses tumor growth.
| S-EPMC4245974 | biostudies-literature
Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Although SARS-CoV-2 was reported to alter several cellular pathways, its impact on DNA integrity and the mechanisms involved remain unknown. Here we show that SARS-CoV-2 causes DNA damage and elicits an altered DNA damage response. Mechanistically, SARS-CoV-2 proteins ORF6 and NSP13 cause degradation of the DNA damage response kinase CHK1 through proteasome and autophagy, respectively. CHK1 loss leads to deoxynucleoside triphosphate (dNTP) shortage, causing impaired S-phase progression, DNA damage, pro-inflammatory pathways activation and cellular senescence. Supplementation of deoxynucleosides reduces that. Furthermore, SARS-CoV-2 N-protein impairs 53BP1 focal recruitment by interfering with damage-induced long non-coding RNAs, thus reducing DNA repair. Key observations are recapitulated in SARS-CoV-2-infected mice and patients with COVID-19. We propose that SARS-CoV-2, by boosting ribonucleoside triphosphate levels to promote its replication at the expense of dNTPs and by hijacking damage-induced long non-coding RNAs' biology, threatens genome integrity and causes altered DNA damage response activation, induction of inflammation and cellular senescence.
| S-EPMC10104783 | biostudies-literature
Project description:The ataxia telangiectasia mutated and Rad3-related (ATR)-checkpoint kinase 1 (Chk1) axis is the major signaling pathway activated in response to replication stress and is essential for the intra-S checkpoint. ATR phosphorylates and activates a number of molecules to coordinate cell cycle progression. Chk1 is the major effector downstream from ATR and plays a critical role in intra-S checkpoint on replication stress. Activation of Chk1 kinase also requires its association with Claspin, an adaptor protein essential for Chk1 protein stability, recruitment and ATR-dependent Chk1 phosphorylation. We have previously reported that, on replication stress, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is rapidly phosphorylated by ATR at the stalled replication forks and is required for cellular resistance to replication stresses although the impact of DNA-PKcs onto the ATR signaling pathway remains elusive. Here we report that ATR-dependent Chk1 phosphorylation and Chk1 signaling are compromised in the absence of DNA-PKcs. Our investigation reveals that DNA-PKcs is required to maintain Chk1-Claspin complex stability and transcriptional regulation of Claspin expression. The impaired Chk1 activity results in a defective intra-S checkpoint response in DNA-PKcs-deficient cells. Taken together, these results suggest that DNA-PKcs, in addition to its direct role in DNA damage repair, facilitates ATR-Chk1 signaling pathway in response to replication stress.
| S-EPMC3985680 | biostudies-literature
Project description:Centrosomal abnormalities are frequently observed in cancers and in cells with defective DNA repair. Here, we used light and electron microscopy to show that DNA damage induces centrosome amplification, not fragmentation, in human cells. Caffeine abrogated this amplification in both ATM (ataxia telangiectasia, mutated)- and ATR (ATM and Rad3-related)-defective cells, indicating a complementary role for these DNA-damage-responsive kinases in promoting centrosome amplification. Inhibition of checkpoint kinase 1 (Chk1) by RNA-mediated interference or drug treatment suppressed DNA-damage-induced centrosome amplification. Radiation-induced centrosome amplification was abrogated in Chk1(-/-) DT40 cells, but occurred at normal levels in Chk1(-/-) cells transgenically expressing Chk1. Expression of kinase-dead Chk1, or Chk1S345A, through which the phosphatidylinositol-3-kinase cannot signal, failed to restore centrosome amplification, showing that signalling to Chk1 and Chk1 catalytic activity are necessary to promote centrosome overduplication after DNA damage.
| S-EPMC2002539 | biostudies-literature