title: Threonine 2609 phosphorylation of the DNA-dependent protein kinase is a critical prerequisite for epidermal growth factor receptor-mediated radiation resistance.
abstract: 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.
authors.affiliation: Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
authors.forename: Prashanthi
authors.initials: P
authors.lastname: Javvadi
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Makino
authors.forename: Amit K
authors.initials: AK
authors.lastname: Das
authors.forename: Yu-Fen
authors.initials: YF
authors.lastname: Lin
authors.forename: David J
authors.initials: DJ
authors.lastname: Chen
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Chen
authors.forename: Chaitanya S
authors.initials: CS
authors.lastname: Nirodi
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism; D045744:Cell Line, Tumor; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism; D004847:Epithelial Cells / Q000201:enzymology / Q000528:radiation effects; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000528:radiation effects; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism*; D011485:Protein Binding / Q000528:radiation effects; D054648:Protein Phosphatase 2 / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism; D011836:Radiation Tolerance / Q000528:radiation effects; D011839:Radiation, Ionizing; D013329:Structure-Activity Relationship; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050505:Mutant Proteins; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; C512478:EGFR protein, human; D066246:ErbB Receptors; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D054648:Protein Phosphatase 2