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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 interacts with the FHA/BRCT domains of Nbs1 in a CDK phosphorylation-dependent manner.A to D. Characterizing the interaction of CtIP and Nbs1. Indicated Nbs1 and CtIP constructs were co-expressed in Sf21 insect cells by baculovirus infection, followed by anti-HA IP or GST pull-down assays and immunoblotting. A. Full-length CtIP-12A-CDK mutant associates with Nbs1 containing C-terminal GST-tag. B. Both the N-terminus and C-terminus of Nbs1 bind to CtIP. C. Top: The phospho-binding sites in the Nbs1 FHA/BRCT domains are important for mediating the interaction with CtIP. Middle: The CtIP-12A-CDK mutant fails to bind to the Nbs1 FHA/BRCT domains. Bottom: Both CtIP-WT and CtIP-12A-CDK mutant interact with the C-terminus of Nbs1. D. The CtIP 5mCDK sites are important for interaction with Nbs1 FHA/BRCT domains. Top: The CtIP 200–600 fragment containing the 5A-CDK mutations fails to bind to Nbs1 FHA/BRCT domains. Bottom: The 5mCDK sites redundantly mediate the interaction of CtIP with Nbs1 FHA-BRCT domains. E. Purified CtIP (100 ng) was used for in vitro ATM kinase assays in the presence or absence of Nbs1-WT-N (residues 1–335), Nbs1-RRHK-N mutant (residues 1–335) or Nbs1-WT-C (residues 336-end) (100 ng and 500 ng).
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pgen-1003277-g005: CtIP interacts with the FHA/BRCT domains of Nbs1 in a CDK phosphorylation-dependent manner.A to D. Characterizing the interaction of CtIP and Nbs1. Indicated Nbs1 and CtIP constructs were co-expressed in Sf21 insect cells by baculovirus infection, followed by anti-HA IP or GST pull-down assays and immunoblotting. A. Full-length CtIP-12A-CDK mutant associates with Nbs1 containing C-terminal GST-tag. B. Both the N-terminus and C-terminus of Nbs1 bind to CtIP. C. Top: The phospho-binding sites in the Nbs1 FHA/BRCT domains are important for mediating the interaction with CtIP. Middle: The CtIP-12A-CDK mutant fails to bind to the Nbs1 FHA/BRCT domains. Bottom: Both CtIP-WT and CtIP-12A-CDK mutant interact with the C-terminus of Nbs1. D. The CtIP 5mCDK sites are important for interaction with Nbs1 FHA/BRCT domains. Top: The CtIP 200–600 fragment containing the 5A-CDK mutations fails to bind to Nbs1 FHA/BRCT domains. Bottom: The 5mCDK sites redundantly mediate the interaction of CtIP with Nbs1 FHA-BRCT domains. E. Purified CtIP (100 ng) was used for in vitro ATM kinase assays in the presence or absence of Nbs1-WT-N (residues 1–335), Nbs1-RRHK-N mutant (residues 1–335) or Nbs1-WT-C (residues 336-end) (100 ng and 500 ng).

Mentions: FHA and BRCT domains often mediate the binding of phospho-proteins through specific interactions of phosphorylated motifs [33]–[35]. To understand how Nbs1 facilitates CtIP phosphorylation by ATM through its FHA/BRCT domains in a CDK phosphorylation-dependent manner, we characterized the interactions of Nbs1 with CtIP. As shown in Figure 5A, full-length Nbs1 binds to CtIP-WT and CtIP-12A-CDK at similar levels. Further analysis revealed that both the N-terminus of Nbs1 [Nbs1-(1–335), containing the FHA/BRCT domains] and C-terminus of Nbs1 [Nbs1-(336-end)] bind to CtIP (Figure 5B), suggesting that CtIP and Nbs1 interact at more than one site.


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 interacts with the FHA/BRCT domains of Nbs1 in a CDK phosphorylation-dependent manner.A to D. Characterizing the interaction of CtIP and Nbs1. Indicated Nbs1 and CtIP constructs were co-expressed in Sf21 insect cells by baculovirus infection, followed by anti-HA IP or GST pull-down assays and immunoblotting. A. Full-length CtIP-12A-CDK mutant associates with Nbs1 containing C-terminal GST-tag. B. Both the N-terminus and C-terminus of Nbs1 bind to CtIP. C. Top: The phospho-binding sites in the Nbs1 FHA/BRCT domains are important for mediating the interaction with CtIP. Middle: The CtIP-12A-CDK mutant fails to bind to the Nbs1 FHA/BRCT domains. Bottom: Both CtIP-WT and CtIP-12A-CDK mutant interact with the C-terminus of Nbs1. D. The CtIP 5mCDK sites are important for interaction with Nbs1 FHA/BRCT domains. Top: The CtIP 200–600 fragment containing the 5A-CDK mutations fails to bind to Nbs1 FHA/BRCT domains. Bottom: The 5mCDK sites redundantly mediate the interaction of CtIP with Nbs1 FHA-BRCT domains. E. Purified CtIP (100 ng) was used for in vitro ATM kinase assays in the presence or absence of Nbs1-WT-N (residues 1–335), Nbs1-RRHK-N mutant (residues 1–335) or Nbs1-WT-C (residues 336-end) (100 ng and 500 ng).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003277-g005: CtIP interacts with the FHA/BRCT domains of Nbs1 in a CDK phosphorylation-dependent manner.A to D. Characterizing the interaction of CtIP and Nbs1. Indicated Nbs1 and CtIP constructs were co-expressed in Sf21 insect cells by baculovirus infection, followed by anti-HA IP or GST pull-down assays and immunoblotting. A. Full-length CtIP-12A-CDK mutant associates with Nbs1 containing C-terminal GST-tag. B. Both the N-terminus and C-terminus of Nbs1 bind to CtIP. C. Top: The phospho-binding sites in the Nbs1 FHA/BRCT domains are important for mediating the interaction with CtIP. Middle: The CtIP-12A-CDK mutant fails to bind to the Nbs1 FHA/BRCT domains. Bottom: Both CtIP-WT and CtIP-12A-CDK mutant interact with the C-terminus of Nbs1. D. The CtIP 5mCDK sites are important for interaction with Nbs1 FHA/BRCT domains. Top: The CtIP 200–600 fragment containing the 5A-CDK mutations fails to bind to Nbs1 FHA/BRCT domains. Bottom: The 5mCDK sites redundantly mediate the interaction of CtIP with Nbs1 FHA-BRCT domains. E. Purified CtIP (100 ng) was used for in vitro ATM kinase assays in the presence or absence of Nbs1-WT-N (residues 1–335), Nbs1-RRHK-N mutant (residues 1–335) or Nbs1-WT-C (residues 336-end) (100 ng and 500 ng).
Mentions: FHA and BRCT domains often mediate the binding of phospho-proteins through specific interactions of phosphorylated motifs [33]–[35]. To understand how Nbs1 facilitates CtIP phosphorylation by ATM through its FHA/BRCT domains in a CDK phosphorylation-dependent manner, we characterized the interactions of Nbs1 with CtIP. As shown in Figure 5A, full-length Nbs1 binds to CtIP-WT and CtIP-12A-CDK at similar levels. Further analysis revealed that both the N-terminus of Nbs1 [Nbs1-(1–335), containing the FHA/BRCT domains] and C-terminus of Nbs1 [Nbs1-(336-end)] bind to CtIP (Figure 5B), suggesting that CtIP and Nbs1 interact at more than one site.

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