Threonine 2609 phosphorylation of the DNA-dependent Protein Kinase is a critical prerequisite for epidermal growth factor receptor mediated radiation resistance

The epidermal growth factor receptor (EGFR) contributes to tumor radioresistance, in part, through interactions with the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs), a key enzyme in the non homologous end joining DNA repair pathway. We previously demonstrated that EGFR-DNA-PKcs interactions are significantly compromised in the context of activating mutations in EGFR in non small cell lung carcinoma (NSCLC) and human bronchial epithelial cells. Here, we investigate the reciprocal relationship between phosphorylation status of DNA-PKcs and EGFR-mediated radiation response. The data reveal that both the kinase activity of DNA-PKcs and radiation-induced phosphorylation of DNA-PKcs by the Ataxia Telangiectasia Mutated (ATM) kinase are critical prerequisites for EGFR-mediated radioresponse. Alanine substitutions at 7 key serine/threonine residues in DNA-PKcs or inhibition of DNA-PKcs by NU7441 completely abrogated EGFR-mediated radioresponse and blocked EGFR binding. ATM-deficiency or ATM inhibition with KU55933 produced a similar effect. Importantly, alanine substitution at an ATM-dependent DNA-PKcs phosphorylation site, T2609, was sufficient to block binding or radioresponse of EGFR. However, mutation of a DNA-PKcs auto-phosphorylation site, S2056 had no such effect indicating that DNA-PKcs auto-phosphorylation is not necessary for EGFR-mediated radioresponse. Our data reveal that in both NSCLCs and HBECs, activating mutations in EGFR specifically abolished the DNA-PKcs phosphorylation at T2609, but not S2056. Our study underscores the critical importance of a reciprocal relationship between DNA-PKcs phosphorylation and EGFR mediated radiation response and elucidates mechanisms underlying mutant EGFR associated radiosensitivity in NSCLCs.