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The interaction of CtIP and Nbs1 connects CDK and ATM to regulate HR-mediated double-strand break repair.

Wang H, Shi LZ, Wong CC, Han X, Hwang PY, Truong LN, Zhu Q, Shao Z, Chen DJ, Berns MW, Yates JR, Chen L, Wu X - PLoS Genet. (2013)

Bottom Line: We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1.We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs.Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
CtIP plays an important role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.

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ATM is important for CtIP hyper-phosphorylation after DNA damage.A. Indicated cell lines were treated with IR (10 Gy) or CPT (2 µM, 2 h) or No and lysed. Western blotting of CtIP was performed using cell lysates incubated with lambda phosphatase (ptase) or without ptase (-). B. U2OS and/or T98G cells were treated with ATM inhibitor (ATMi) KU-55933 (20 µM, 1 h) or infected with retroviruses encoding shRNAs for ATM or vector control MKO, treated with IR (10 Gy) and lysed 1 h later. Western blotting was performed with indicated antibodies. C. Schematic drawing of eight putative ATM consensus sites (SQ/TQ motifs) on CtIP. U2OS cells stably expressing HA-CtIP wild-type (WT) or HA-CtIP 8A-ATM mutant (all SQ/TQ sites mutated to AQ) with endogenous CtIP silenced by shRNAs and siRNA, were treated with CPT (2 µM, 2 h) followed by anti-HA Western blotting. D–F. EGFP-based HR assay, clonogenic survival and RPA foci formation assays were performed with U2OS cells stably expressing indicated CtIP variants, with endogenous CtIP silenced. Data shown represents the mean of three independent experiments; error bars, s.d. In panel F, indicated cells were treated with DMSO (No) or CPT (1 µM, 2 h) and fixed for immunostaining. The red and white arrows indicate representative RPA2 and γH2AX foci-positive and foci-negative cells, respectively, with RPA2-immunostained cells enlarged in right-side panels. The percentage of RPA2 foci-positive cells among γH2AX-positive cells for each sample is shown. G. Western blot of U2OS cells expressing indicated CtIP variants with endogenous CtIP silenced and treated with CPT (2 µM, 2 h). H. The C-terminus protein sequence of human CtIP was aligned with CtIP homologues from other indicated species, with newly identified conserved T859 site shown. I. ATM in vitro kinase assay was performed with purified GST-tagged CtIP fragments (residues 750–897), containing either WT or the T859A mutation. Coomassie Blue staining indicates protein loading.
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pgen-1003277-g001: ATM is important for CtIP hyper-phosphorylation after DNA damage.A. Indicated cell lines were treated with IR (10 Gy) or CPT (2 µM, 2 h) or No and lysed. Western blotting of CtIP was performed using cell lysates incubated with lambda phosphatase (ptase) or without ptase (-). B. U2OS and/or T98G cells were treated with ATM inhibitor (ATMi) KU-55933 (20 µM, 1 h) or infected with retroviruses encoding shRNAs for ATM or vector control MKO, treated with IR (10 Gy) and lysed 1 h later. Western blotting was performed with indicated antibodies. C. Schematic drawing of eight putative ATM consensus sites (SQ/TQ motifs) on CtIP. U2OS cells stably expressing HA-CtIP wild-type (WT) or HA-CtIP 8A-ATM mutant (all SQ/TQ sites mutated to AQ) with endogenous CtIP silenced by shRNAs and siRNA, were treated with CPT (2 µM, 2 h) followed by anti-HA Western blotting. D–F. EGFP-based HR assay, clonogenic survival and RPA foci formation assays were performed with U2OS cells stably expressing indicated CtIP variants, with endogenous CtIP silenced. Data shown represents the mean of three independent experiments; error bars, s.d. In panel F, indicated cells were treated with DMSO (No) or CPT (1 µM, 2 h) and fixed for immunostaining. The red and white arrows indicate representative RPA2 and γH2AX foci-positive and foci-negative cells, respectively, with RPA2-immunostained cells enlarged in right-side panels. The percentage of RPA2 foci-positive cells among γH2AX-positive cells for each sample is shown. G. Western blot of U2OS cells expressing indicated CtIP variants with endogenous CtIP silenced and treated with CPT (2 µM, 2 h). H. The C-terminus protein sequence of human CtIP was aligned with CtIP homologues from other indicated species, with newly identified conserved T859 site shown. I. ATM in vitro kinase assay was performed with purified GST-tagged CtIP fragments (residues 750–897), containing either WT or the T859A mutation. Coomassie Blue staining indicates protein loading.

Mentions: DNA damage induces CtIP phosphorylation, as revealed by phosphatase-sensitive mobility shift of CtIP on SDS-PAGE after ionization radiation (IR) or camptothecin (CPT) treatment (Figure 1A and data not shown). By using ATM inhibitor (ATMi) KU-55933 or expressing ATM shRNAs, we found that IR- and CPT-induced CtIP phosphorylation is dependent on ATM (Figure 1B and data not shown). In vitro kinase assays showed that purified CtIP can be phosphorylated by ATM (Figure S1A). Mutating all eight putative ATM kinase sites (SQ/TQ) on CtIP (CtIP-8A-ATM) completely abolished damage-induced CtIP phosphorylation shift (Figure 1C), further supporting that damage-induced phosphorylation is mediated by ATM.


The interaction of CtIP and Nbs1 connects CDK and ATM to regulate HR-mediated double-strand break repair.

Wang H, Shi LZ, Wong CC, Han X, Hwang PY, Truong LN, Zhu Q, Shao Z, Chen DJ, Berns MW, Yates JR, Chen L, Wu X - PLoS Genet. (2013)

ATM is important for CtIP hyper-phosphorylation after DNA damage.A. Indicated cell lines were treated with IR (10 Gy) or CPT (2 µM, 2 h) or No and lysed. Western blotting of CtIP was performed using cell lysates incubated with lambda phosphatase (ptase) or without ptase (-). B. U2OS and/or T98G cells were treated with ATM inhibitor (ATMi) KU-55933 (20 µM, 1 h) or infected with retroviruses encoding shRNAs for ATM or vector control MKO, treated with IR (10 Gy) and lysed 1 h later. Western blotting was performed with indicated antibodies. C. Schematic drawing of eight putative ATM consensus sites (SQ/TQ motifs) on CtIP. U2OS cells stably expressing HA-CtIP wild-type (WT) or HA-CtIP 8A-ATM mutant (all SQ/TQ sites mutated to AQ) with endogenous CtIP silenced by shRNAs and siRNA, were treated with CPT (2 µM, 2 h) followed by anti-HA Western blotting. D–F. EGFP-based HR assay, clonogenic survival and RPA foci formation assays were performed with U2OS cells stably expressing indicated CtIP variants, with endogenous CtIP silenced. Data shown represents the mean of three independent experiments; error bars, s.d. In panel F, indicated cells were treated with DMSO (No) or CPT (1 µM, 2 h) and fixed for immunostaining. The red and white arrows indicate representative RPA2 and γH2AX foci-positive and foci-negative cells, respectively, with RPA2-immunostained cells enlarged in right-side panels. The percentage of RPA2 foci-positive cells among γH2AX-positive cells for each sample is shown. G. Western blot of U2OS cells expressing indicated CtIP variants with endogenous CtIP silenced and treated with CPT (2 µM, 2 h). H. The C-terminus protein sequence of human CtIP was aligned with CtIP homologues from other indicated species, with newly identified conserved T859 site shown. I. ATM in vitro kinase assay was performed with purified GST-tagged CtIP fragments (residues 750–897), containing either WT or the T859A mutation. Coomassie Blue staining indicates protein loading.
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Related In: Results  -  Collection

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pgen-1003277-g001: ATM is important for CtIP hyper-phosphorylation after DNA damage.A. Indicated cell lines were treated with IR (10 Gy) or CPT (2 µM, 2 h) or No and lysed. Western blotting of CtIP was performed using cell lysates incubated with lambda phosphatase (ptase) or without ptase (-). B. U2OS and/or T98G cells were treated with ATM inhibitor (ATMi) KU-55933 (20 µM, 1 h) or infected with retroviruses encoding shRNAs for ATM or vector control MKO, treated with IR (10 Gy) and lysed 1 h later. Western blotting was performed with indicated antibodies. C. Schematic drawing of eight putative ATM consensus sites (SQ/TQ motifs) on CtIP. U2OS cells stably expressing HA-CtIP wild-type (WT) or HA-CtIP 8A-ATM mutant (all SQ/TQ sites mutated to AQ) with endogenous CtIP silenced by shRNAs and siRNA, were treated with CPT (2 µM, 2 h) followed by anti-HA Western blotting. D–F. EGFP-based HR assay, clonogenic survival and RPA foci formation assays were performed with U2OS cells stably expressing indicated CtIP variants, with endogenous CtIP silenced. Data shown represents the mean of three independent experiments; error bars, s.d. In panel F, indicated cells were treated with DMSO (No) or CPT (1 µM, 2 h) and fixed for immunostaining. The red and white arrows indicate representative RPA2 and γH2AX foci-positive and foci-negative cells, respectively, with RPA2-immunostained cells enlarged in right-side panels. The percentage of RPA2 foci-positive cells among γH2AX-positive cells for each sample is shown. G. Western blot of U2OS cells expressing indicated CtIP variants with endogenous CtIP silenced and treated with CPT (2 µM, 2 h). H. The C-terminus protein sequence of human CtIP was aligned with CtIP homologues from other indicated species, with newly identified conserved T859 site shown. I. ATM in vitro kinase assay was performed with purified GST-tagged CtIP fragments (residues 750–897), containing either WT or the T859A mutation. Coomassie Blue staining indicates protein loading.
Mentions: DNA damage induces CtIP phosphorylation, as revealed by phosphatase-sensitive mobility shift of CtIP on SDS-PAGE after ionization radiation (IR) or camptothecin (CPT) treatment (Figure 1A and data not shown). By using ATM inhibitor (ATMi) KU-55933 or expressing ATM shRNAs, we found that IR- and CPT-induced CtIP phosphorylation is dependent on ATM (Figure 1B and data not shown). In vitro kinase assays showed that purified CtIP can be phosphorylated by ATM (Figure S1A). Mutating all eight putative ATM kinase sites (SQ/TQ) on CtIP (CtIP-8A-ATM) completely abolished damage-induced CtIP phosphorylation shift (Figure 1C), further supporting that damage-induced phosphorylation is mediated by ATM.

Bottom Line: We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1.We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs.Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
CtIP plays an important role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.

Show MeSH
Related in: MedlinePlus