The significance of protein-phosphorylating function of DNA-PK in DNA double-strand break repair: XRCC4 as the genuine target

Abstract

DNA-dependent protein kinase (DNA-PK), composed of DNA-PKcs and Ku86/70, is thought to play a critical role in DNA double-strand break (DSB) repair via non-homologous end-joining (NHEJ) pathway. DNA-PK can phosphorylate in vitro a number of proteins and there are lines of evidence indicating the requirement for kinase activity of DNA-PKcs in its DNA repair functios. Nevertheless, it is poorly understood what is/are the in vivo phosphorylation target(s) and why the kinase function of DNA-PK is essential for DNA repair. Several studies have indicated that DNA-PK can phosphorylate XRCC4 in vitro and we have demonstrated that DNA-PK mediates DNA damage-induced phosphorylation of XRCC4 in living cells. Now we identified three serines of XRCC4 as the sites of phosphorylation by DNA-PK in vitro and two of them as the sites of phosphorylation in living cells, requiring DNA-PKcs. Furthermore, the replacement of these serines with alanine substantially reduced the ability of XRCC4 to confer increased radioresistance and proliferation rate to XRCC4-deficient cell line M10, indicating that the phosphorylation by DNA-PK of XRCC4 at these sites might be important for the cooperation of these molecules to sustain cell proliferation in the face of radiation-induced and spontaneous DNA damages. We would also discuss the implication of this phosphorylation event in DSB repair and its possible application in cancer therapy.