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.
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.
Mesh Terms:
Antibodies, Ataxia Telangiectasia Mutated Proteins, Camptothecin, Cell Cycle Proteins, Cell Line, DNA Damage, DNA Replication, DNA-Activated Protein Kinase, DNA-Binding Proteins, Etoposide, Humans, Nuclear Proteins, Phosphatidylinositol 3-Kinases, Phosphorylation, Phosphothreonine, Protein Serine-Threonine Kinases, Protein Subunits, Radiation, Ionizing, Replication Protein A, Threonine, Tumor Suppressor Proteins
Antibodies, Ataxia Telangiectasia Mutated Proteins, Camptothecin, Cell Cycle Proteins, Cell Line, DNA Damage, DNA Replication, DNA-Activated Protein Kinase, DNA-Binding Proteins, Etoposide, Humans, Nuclear Proteins, Phosphatidylinositol 3-Kinases, Phosphorylation, Phosphothreonine, Protein Serine-Threonine Kinases, Protein Subunits, Radiation, Ionizing, Replication Protein A, Threonine, Tumor Suppressor Proteins
Nucleic Acids Res
Date: Feb. 12, 2004
PubMed ID: 14872059
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