PP6 Regulatory Subunit R1 Is Bidentate Anchor for Targeting Protein Phosphatase-6 to DNA-dependent Protein Kinase

DNA-dependent protein kinase (DNA-PK) becomes activated in response to DNA double strand breaks, initiating repair by the non-homologous end joining pathway. DNA·PK complexes with the regulatory subunit SAPSR1 (R1) of protein phosphatase-6 (PP6). Knockdown of either R1 or PP6c prevents DNA-PK activation in response to ionizing radiation-induced DNA damage and radiosensitizes glioblastoma cells. Here, we demonstrate that R1 is necessary for and bridges the interaction between DNA-PK and PP6c. Using R1 deletion mutants, DNA-PK binding was mapped to two distinct regions of R1 spanning residues 1–326 and 522–700. Either region expressed alone was sufficient to bind DNA-PK, but only deletion of residues 1–326, not 522–700, eliminated interaction of R1 with DNA-PK. We assign 1–326 as the dominant domain and 522–700 as the supporting region. These results demonstrate that R1 acts as a bidentate anchor to DNA-PK and recruits PP6c. Targeting the dominant interface with small molecule or peptidomimetic inhibitors could specifically prevent activation of DNA-PK and thereby sensitize cells to ionizing radiation and other genotoxic agents. PP6c3 and SAPSR1 bind to DNA-PK and are required for DNA-PK activation following ionizing radiation. R1 associates with DNA-PK independently of PP6c through two distinct regions. Contact between R1 and DNA-PK involves two binding sites: one dominant, the other supporting. DNA-PK-R1 interface may be exploited as a drug target to inhibit DNA-PK activation and radiosensitize cells.