Functional interaction of BRCA1/ATM-associated BAAT1 with the DNA-PK catalytic subunit

Abstract

Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) play a crucial role in the initial stages of cell response, when cells are exposed to DNA insult such as ionizing radiation (IR) and chemical agents. We previously demonstrated that ATM requires BAAT1 for its activation in response to IR. In the present study, BAAT1 was found to bind to the DNA-PK catalytic subunit (DNA-PKcs) and SMC1. Biochemical analysis indicated that several regions of BAAT1 were responsible for the interaction with these proteins, and their binding affinity was altered after treatment with the IR mimetic, neocarzinostatin (NCS). Phosphorylation of the DNA-PKcs at Ser2056 and SMC1 at Ser966 was induced by NCS, while phosphorylation was reduced when BAAT1 was depleted by siRNA. These results indicate that BAAT1 globally regulates DNA damage signaling during the early stages of apoptosis.

Figure 1.

Tissue-specific expression of BAAT1. Samples from mice (6–7 weeks old) were lysed and immunoblotting was performed to detect the expression of BAAT1 and SMC1. Total lysates (35 μg) for each tissue were studied. Actin was used as a loading control.

Figure 2.

BAAT1 interactions with molecules related to DNA damage response. (A) BAAT1 structural features. Two SNP sites (G20R and R644Q), one putative phosphorylation site (S742) and two HEAT repeat domains (H) are shown. (B) Map of the regions of interaction. To determine the binding region of BAAT1, 293T cells were transfected with the pEBG2 vector (V) or plasmids containing GST-fusion BAAT1 fragments. After 48 h, cells were treated with neocarzinostatin (NCS) or without. Total lysates were isolated and subjected to immunoprecipitation with GSH-sepharose beads as described previously (7). After immunoprecipitation, samples were immunoblotted with anti-DNA-PKcs, and protein levels were confirmed with anti-GST antibodies.

Figure 3.

Time-dependent interaction between BAAT1 and ATM/DNA-PKcs after treatment with NCS. After 48 h, cells were treated with NCS for the indicated times. Total lysates were prepared and immunoprecipitated with the anti-FLAG antibody. After immunoprecipitation, samples were blotted with the indicated protein antibodies.

Figure 4.

Down-regulation of BAAT via siRNA. MCF10A normal human mammary epithelial cells were transfected with siRNA oligomer for BAAT1 knockdown as previously described (7). After 72 h, cells were treated with NCS for the indicated times. Total lysates were prepared and subjected to Western blotting. The level of total protein and phosphorylation by NCS were detected with the indicated antibodies. Actin protein was used as a loading control for immunoblotting.