DNA Damage and Cellular Stress Responses Effect of the Inositol Polyphosphate InsP6 on DNA-PK–Dependent Phosphorylation

Inositol hexakisphosphate (InsP6) is a member of the inositol polyphosphate group that participates in numerous intracellular signaling pathways. Cheung and colleagues previously reported that InsP6 stimulated double-strand break repair by nonhomologous end joining (NHEJ) in cell-free extracts and that InsP6 binding by the Ku70/80 subunit of the DNA-dependent protein kinase (DNA-PK) was required for stimulation of NHEJ in vitro. This report describes InsP6-dependent phosphorylation of two NHEJ factors, XRCC4 and XLF, in partially purified human cell extracts. XRCC4 and XLF are known substrates for DNA-PK, which does not require InsP6 for protein kinase activity. Consistent with a role for DNA-PK in these reactions, InsP6-dependent phosphorylation of XRCC4 and XLF was DNA dependent and not observed in the presence of DNA-PK inhibitors. Depletion of the Ku70/80 DNA-, InsP6-binding subunit of DNA-PK resulted in loss of InsP6-dependent phosphorylation and showed a requirement for Ku70/80 in these reactions. Complementation of Ku70/80-depleted reactions with recombinant wild-type Ku70/80 restored InsP6-dependent phosphorylation of XRCC4 and XLF. In contrast, addition of a Ku70/80 mutant with reduced InsP6 binding failed to restore InsP6-dependent phosphorylation. While additional protein kinases may participate in InsP6-dependent phosphorylation of XRCC4 and XLF, data presented here describe a clear requirement for DNA-PK in these phosphorylation events. Furthermore, these data suggest that binding of the inositol polyphosphate InsP6 by Ku70/80 may modulate the substrate specificity of the phosphoinositide-3-kinase–related protein kinase DNA-PK. Mol Cancer Res; 9(10); 1366–76. 2011 AACR.