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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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The FHA/BRCT domains of Nbs1 are important for ATM-mediated phosphorylation of CtIP.A. The CtIP-7A-CDK mutant does not show defects in ATM and checkpoint activation. U2OS cells stably expressing HA-CtIP WT or 7A-CDK mutant, with endogenous CtIP silenced, were treated with or without IR (10 Gy) and allowed to recover for 1 h. Cells were then collected and lysed for Western blot analysis with indicated antibodies. B. U2OS cells stably expressing control vector MKO, sh-Mre11 or sh-Nbs1 were treated with IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. C. Schematic drawing of human Nbs1 protein domains [FHA, BRCT and Mre11- and ATM interacting domains (i.d.)], with indicated phospho-binding sites on the FHA/BRCT domains [31], [32]. D. U2OS cells stably expressing C-terminus Myc-tagged Nbs1 alleles, Nbs1-WT and Nbs1-RRHK (R28A, R43A, H45A and K160M) or empty vector with endogenous Nbs1 silenced by shRNAs were treated with or without IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. E. Purified GST-CtIP WT and GST-CtIP-12A-CDK from Sf9 insect cells were analyzed by SDS-PAGE with Coomassie blue staining. F. CtIP expressed in insect cells is phosphorylated by CDKs. Purified GST-CtIP WT and 12A-CDK mutant protein from insect cells were treated without or with lambda phosphatase (ptase), and anti-CtIP Western blot analysis was performed. G and H. Baculovirus-expressed and purified CtIP-WT and CtIP-12A-CDK mutant proteins (100 ng) were used as substrates for in vitro ATM kinase assays with or without addition of purified Nbs1-WT or Nbs1-RRHK mutant proteins (100 ng and 500 ng). The radiolabeled CtIP was visualized following SDS-PAGE.
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pgen-1003277-g004: The FHA/BRCT domains of Nbs1 are important for ATM-mediated phosphorylation of CtIP.A. The CtIP-7A-CDK mutant does not show defects in ATM and checkpoint activation. U2OS cells stably expressing HA-CtIP WT or 7A-CDK mutant, with endogenous CtIP silenced, were treated with or without IR (10 Gy) and allowed to recover for 1 h. Cells were then collected and lysed for Western blot analysis with indicated antibodies. B. U2OS cells stably expressing control vector MKO, sh-Mre11 or sh-Nbs1 were treated with IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. C. Schematic drawing of human Nbs1 protein domains [FHA, BRCT and Mre11- and ATM interacting domains (i.d.)], with indicated phospho-binding sites on the FHA/BRCT domains [31], [32]. D. U2OS cells stably expressing C-terminus Myc-tagged Nbs1 alleles, Nbs1-WT and Nbs1-RRHK (R28A, R43A, H45A and K160M) or empty vector with endogenous Nbs1 silenced by shRNAs were treated with or without IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. E. Purified GST-CtIP WT and GST-CtIP-12A-CDK from Sf9 insect cells were analyzed by SDS-PAGE with Coomassie blue staining. F. CtIP expressed in insect cells is phosphorylated by CDKs. Purified GST-CtIP WT and 12A-CDK mutant protein from insect cells were treated without or with lambda phosphatase (ptase), and anti-CtIP Western blot analysis was performed. G and H. Baculovirus-expressed and purified CtIP-WT and CtIP-12A-CDK mutant proteins (100 ng) were used as substrates for in vitro ATM kinase assays with or without addition of purified Nbs1-WT or Nbs1-RRHK mutant proteins (100 ng and 500 ng). The radiolabeled CtIP was visualized following SDS-PAGE.

Mentions: Our studies suggest that CDK-mediated phosphorylation of CtIP directly or indirectly modulates damage-induced phosphorylation of CtIP by ATM. Consistent with the previous findings that CtIP is dispensable for ATM activation [16], [29], no defects of ATM activation were detected in the CtIP-7A-CDK-mutant as revealed by damage-induced ATM auto-phosphorylation and Chk2 phosphorylation (Figure 4A). As with many other ATM substrates, damage-induced CtIP phophorylation depends on Nbs1 and Mre11 (Figure 4B), likely due to the role of MRN in promoting ATM activation [30]. Interestingly, however, when we used an Nbs1 FHA/BRCT mutant, Nbs1-RRHK (R28A/R43A/H45A/K160A) carrying mutations at the critical sites for binding to the phospho-motifs in the Nbs1 FHA/BRCT domains [31], [32], we found that damage-induced CtIP phosphorylation was impaired, but ATM auto-phosphorylation and ATM-mediated phosphorylation of Chk2 was normal (Figure 4D). This suggests that the FHA/BRCT domains of Nbs1 are not required for ATM activation per se but are specifically required for ATM to phosphorylate CtIP in response to DNA damage.


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)

The FHA/BRCT domains of Nbs1 are important for ATM-mediated phosphorylation of CtIP.A. The CtIP-7A-CDK mutant does not show defects in ATM and checkpoint activation. U2OS cells stably expressing HA-CtIP WT or 7A-CDK mutant, with endogenous CtIP silenced, were treated with or without IR (10 Gy) and allowed to recover for 1 h. Cells were then collected and lysed for Western blot analysis with indicated antibodies. B. U2OS cells stably expressing control vector MKO, sh-Mre11 or sh-Nbs1 were treated with IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. C. Schematic drawing of human Nbs1 protein domains [FHA, BRCT and Mre11- and ATM interacting domains (i.d.)], with indicated phospho-binding sites on the FHA/BRCT domains [31], [32]. D. U2OS cells stably expressing C-terminus Myc-tagged Nbs1 alleles, Nbs1-WT and Nbs1-RRHK (R28A, R43A, H45A and K160M) or empty vector with endogenous Nbs1 silenced by shRNAs were treated with or without IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. E. Purified GST-CtIP WT and GST-CtIP-12A-CDK from Sf9 insect cells were analyzed by SDS-PAGE with Coomassie blue staining. F. CtIP expressed in insect cells is phosphorylated by CDKs. Purified GST-CtIP WT and 12A-CDK mutant protein from insect cells were treated without or with lambda phosphatase (ptase), and anti-CtIP Western blot analysis was performed. G and H. Baculovirus-expressed and purified CtIP-WT and CtIP-12A-CDK mutant proteins (100 ng) were used as substrates for in vitro ATM kinase assays with or without addition of purified Nbs1-WT or Nbs1-RRHK mutant proteins (100 ng and 500 ng). The radiolabeled CtIP was visualized following SDS-PAGE.
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Related In: Results  -  Collection

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pgen-1003277-g004: The FHA/BRCT domains of Nbs1 are important for ATM-mediated phosphorylation of CtIP.A. The CtIP-7A-CDK mutant does not show defects in ATM and checkpoint activation. U2OS cells stably expressing HA-CtIP WT or 7A-CDK mutant, with endogenous CtIP silenced, were treated with or without IR (10 Gy) and allowed to recover for 1 h. Cells were then collected and lysed for Western blot analysis with indicated antibodies. B. U2OS cells stably expressing control vector MKO, sh-Mre11 or sh-Nbs1 were treated with IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. C. Schematic drawing of human Nbs1 protein domains [FHA, BRCT and Mre11- and ATM interacting domains (i.d.)], with indicated phospho-binding sites on the FHA/BRCT domains [31], [32]. D. U2OS cells stably expressing C-terminus Myc-tagged Nbs1 alleles, Nbs1-WT and Nbs1-RRHK (R28A, R43A, H45A and K160M) or empty vector with endogenous Nbs1 silenced by shRNAs were treated with or without IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. E. Purified GST-CtIP WT and GST-CtIP-12A-CDK from Sf9 insect cells were analyzed by SDS-PAGE with Coomassie blue staining. F. CtIP expressed in insect cells is phosphorylated by CDKs. Purified GST-CtIP WT and 12A-CDK mutant protein from insect cells were treated without or with lambda phosphatase (ptase), and anti-CtIP Western blot analysis was performed. G and H. Baculovirus-expressed and purified CtIP-WT and CtIP-12A-CDK mutant proteins (100 ng) were used as substrates for in vitro ATM kinase assays with or without addition of purified Nbs1-WT or Nbs1-RRHK mutant proteins (100 ng and 500 ng). The radiolabeled CtIP was visualized following SDS-PAGE.
Mentions: Our studies suggest that CDK-mediated phosphorylation of CtIP directly or indirectly modulates damage-induced phosphorylation of CtIP by ATM. Consistent with the previous findings that CtIP is dispensable for ATM activation [16], [29], no defects of ATM activation were detected in the CtIP-7A-CDK-mutant as revealed by damage-induced ATM auto-phosphorylation and Chk2 phosphorylation (Figure 4A). As with many other ATM substrates, damage-induced CtIP phophorylation depends on Nbs1 and Mre11 (Figure 4B), likely due to the role of MRN in promoting ATM activation [30]. Interestingly, however, when we used an Nbs1 FHA/BRCT mutant, Nbs1-RRHK (R28A/R43A/H45A/K160A) carrying mutations at the critical sites for binding to the phospho-motifs in the Nbs1 FHA/BRCT domains [31], [32], we found that damage-induced CtIP phosphorylation was impaired, but ATM auto-phosphorylation and ATM-mediated phosphorylation of Chk2 was normal (Figure 4D). This suggests that the FHA/BRCT domains of Nbs1 are not required for ATM activation per se but are specifically required for ATM to phosphorylate CtIP in response to DNA damage.

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