Threonine 2609 phosphorylation of the DNA-dependent protein kinase is a critical prerequisite for epidermal growth factor receptor-mediated radiation resistance.
The EGF receptor (EGFR) contributes to tumor radioresistance, in part, through interactions with the catalytic subunit of DNA-dependent protein kinase (DNA-PKc), a key enzyme in the nonhomologous end joining DNA repair pathway. We previously showed 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 seven 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 autophosphorylation site, S2056 had no such effect indicating that DNA-PKcs autophosphorylation is not necessary for EGFR-mediated radioresponse. Our data reveal that in both NSCLCs and human bronchial epithelial cells, 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.
Mesh Terms:
Ataxia Telangiectasia Mutated Proteins, Cell Cycle Proteins, Cell Line, Tumor, DNA-Activated Protein Kinase, DNA-Binding Proteins, Epithelial Cells, ErbB Receptors, Humans, Mutant Proteins, Mutation, Nuclear Proteins, Phosphorylation, Phosphoserine, Phosphothreonine, Protein Binding, Protein Phosphatase 2, Protein Serine-Threonine Kinases, Radiation Tolerance, Radiation, Ionizing, Structure-Activity Relationship, Tumor Suppressor Proteins
Ataxia Telangiectasia Mutated Proteins, Cell Cycle Proteins, Cell Line, Tumor, DNA-Activated Protein Kinase, DNA-Binding Proteins, Epithelial Cells, ErbB Receptors, Humans, Mutant Proteins, Mutation, Nuclear Proteins, Phosphorylation, Phosphoserine, Phosphothreonine, Protein Binding, Protein Phosphatase 2, Protein Serine-Threonine Kinases, Radiation Tolerance, Radiation, Ionizing, Structure-Activity Relationship, Tumor Suppressor Proteins
Mol Cancer Res
Date: Oct. 01, 2012
PubMed ID: 22923485
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