Phosphatidyl inositol 3‐kinase‐like serine/threonine protein kinases (PIKKs) are required for DNA damage‐induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21

摘要

Replication protein A (RPA) is a single‐stranded DNA (ssDNA) binding protein involved in various processes, including nucleotide excision repair and DNA replication. The 32 kDa subunit of RPA (RPA32) is phosphorylated in response to various DNA‐damaging agents, and two protein kinases, ataxia‐telangiectasia mutated (ATM) and the DNA‐dependent protein kinase (DNA‐PK) have been implicated in DNA damage‐induced phosphorylation of RPA32. However, the relative roles of ATM and DNA‐PK in the site‐specific DNA damage‐induced phosphorylation of RPA32 have not been reported. Here we generated a phosphospecific antibody that recognizes Thr21‐phosphorylated RPA32. We show that both DNA‐PK and ATM phosphorylate RPA32 on Thr21 in vitro. Ionizing radiation (IR)‐induced phosphorylation of RPA32 on Thr21 was defective in ATM‐deficient cells, while camptothecin (CPT)‐induced phosphorylation of RPA32 on Thr21 was defective in cells lacking functional DNA‐PK. Neither ATM nor DNA‐PK was required for etoposide (ETOP)‐induced RPA32 Thr21 phosphorylation. However, two inhibitors of the ATM‐ and Rad3‐related (ATR) protein kinase activity prevented ETOP‐induced Thr21 phosphorylation. Inhibition of DNA replication prevented both the IR‐ and CPT‐induced phosphorylation of Thr21, whereas ETOP‐induced Thr21 phosphorylation did not require active DNA replication. Thus, the regulation of RPA32 Thr21 phosphorylation by multiple DNA damage response protein kinases suggests that Thr21 phosphorylation of RPA32 is a crucial step within the DNA damage response.