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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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CtIP is phosphorylated by CDKs at multiple sites.A. Lysates from indicated cell lines were collected and treated with (+) or without (−) lambda phosphatase (ptase), followed by immunoblotting for endogenous CtIP. B. Schematic drawing of twelve putative CDK consensus sites (SP/TP motifs) on CtIP, with the middle cluster of sites 7A-CDK and 5A-CDK indicated. Myc-CtIP WT or indicated mutants were expressed in 293T cells, and cell lysates were treated with or without lambda phosphatase, followed by anti-Myc immunoblotting. C. and D. U2OS cells stably expressing HA-CtIP WT or indicated mutants with endogenous CtIP silenced, were treated with or without CPT (2 µM, 2 h) or IR (10 Gy, recovered for 1 h), followed by immunoblotting for anti-HA. E. Myc-BRCA1 and/or HA-CtIP WT or indicated mutants were expressed in 293T cells and co-immunoprecipitation was performed.
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pgen-1003277-g002: CtIP is phosphorylated by CDKs at multiple sites.A. Lysates from indicated cell lines were collected and treated with (+) or without (−) lambda phosphatase (ptase), followed by immunoblotting for endogenous CtIP. B. Schematic drawing of twelve putative CDK consensus sites (SP/TP motifs) on CtIP, with the middle cluster of sites 7A-CDK and 5A-CDK indicated. Myc-CtIP WT or indicated mutants were expressed in 293T cells, and cell lysates were treated with or without lambda phosphatase, followed by anti-Myc immunoblotting. C. and D. U2OS cells stably expressing HA-CtIP WT or indicated mutants with endogenous CtIP silenced, were treated with or without CPT (2 µM, 2 h) or IR (10 Gy, recovered for 1 h), followed by immunoblotting for anti-HA. E. Myc-BRCA1 and/or HA-CtIP WT or indicated mutants were expressed in 293T cells and co-immunoprecipitation was performed.

Mentions: Treating cell lysates with phosphatase or mutating 12 putative CDK sites (SP/TP sites) increased CtIP mobility on SDS-PAGE to a similar level (Figure 2A and Figure 2B, middle panel), suggesting that CtIP is mainly phosphorylated by CDKs during the cell cycle. However, the CtIP mutants S327A or T847A/S889A only slightly reduced the phosphorylation-dependent shift (Figure 2B, middle and bottom panels), suggesting that CDK sites other than previously identified S327 and T847 [17], [20] are also phosphorylated in vivo. Indeed, mutating a middle cluster of seven CDK sites (CtIP-7A-CDK: S233A, T245A, S276A, T315A, S347A, S549A and S568A, excluding S327) almost completely abolished the CtIP shift (Figure 2B, bottom panel).


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)

CtIP is phosphorylated by CDKs at multiple sites.A. Lysates from indicated cell lines were collected and treated with (+) or without (−) lambda phosphatase (ptase), followed by immunoblotting for endogenous CtIP. B. Schematic drawing of twelve putative CDK consensus sites (SP/TP motifs) on CtIP, with the middle cluster of sites 7A-CDK and 5A-CDK indicated. Myc-CtIP WT or indicated mutants were expressed in 293T cells, and cell lysates were treated with or without lambda phosphatase, followed by anti-Myc immunoblotting. C. and D. U2OS cells stably expressing HA-CtIP WT or indicated mutants with endogenous CtIP silenced, were treated with or without CPT (2 µM, 2 h) or IR (10 Gy, recovered for 1 h), followed by immunoblotting for anti-HA. E. Myc-BRCA1 and/or HA-CtIP WT or indicated mutants were expressed in 293T cells and co-immunoprecipitation was performed.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585124&req=5

pgen-1003277-g002: CtIP is phosphorylated by CDKs at multiple sites.A. Lysates from indicated cell lines were collected and treated with (+) or without (−) lambda phosphatase (ptase), followed by immunoblotting for endogenous CtIP. B. Schematic drawing of twelve putative CDK consensus sites (SP/TP motifs) on CtIP, with the middle cluster of sites 7A-CDK and 5A-CDK indicated. Myc-CtIP WT or indicated mutants were expressed in 293T cells, and cell lysates were treated with or without lambda phosphatase, followed by anti-Myc immunoblotting. C. and D. U2OS cells stably expressing HA-CtIP WT or indicated mutants with endogenous CtIP silenced, were treated with or without CPT (2 µM, 2 h) or IR (10 Gy, recovered for 1 h), followed by immunoblotting for anti-HA. E. Myc-BRCA1 and/or HA-CtIP WT or indicated mutants were expressed in 293T cells and co-immunoprecipitation was performed.
Mentions: Treating cell lysates with phosphatase or mutating 12 putative CDK sites (SP/TP sites) increased CtIP mobility on SDS-PAGE to a similar level (Figure 2A and Figure 2B, middle panel), suggesting that CtIP is mainly phosphorylated by CDKs during the cell cycle. However, the CtIP mutants S327A or T847A/S889A only slightly reduced the phosphorylation-dependent shift (Figure 2B, middle and bottom panels), suggesting that CDK sites other than previously identified S327 and T847 [17], [20] are also phosphorylated in vivo. Indeed, mutating a middle cluster of seven CDK sites (CtIP-7A-CDK: S233A, T245A, S276A, T315A, S347A, S549A and S568A, excluding S327) almost completely abolished the CtIP shift (Figure 2B, bottom panel).

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