Activation of protein kinase Cε stimulates DNA-repair via epidermal growth factor receptor nuclear accumulation

Purpose: To elucidate the interaction between radioprotector O-phospho-L-tyrosine (P-Tyr) with epidermal growth factor receptor (EGFR). Methods: Molecular effects of P-Tyr at the level of EGFR responses were investigated in vitro with TP53-wildtype bronchial carcinoma cell line A549, which is radio-protected by P-Tyr treatment. Nuclear EGFR accumulation was followed by confocal microscopy and Western blotting. PKC epsilon protein expression was impaired by specific siRNA. Residual DNA-damage was quantified with yH(2)AX foci analysis. Results: P-Tyr mediated radio-protection was associated with nuclear EGFR accumulation. Radiation-induced nuclear EGFR presented increased phosphorylation at residue No. T654. We identified PKC epsilon as responsible for T654-phosphorylation. Knockdown of PKC epsilon by siRNA blocked both radiation- and P-Tyr-triggered nuclear EGFR accumulation. Furthermore, nuclear accumulation of EGFR was associated with increased phosphorylation of DNA-dependent protein kinase (DNA-PK) at residue No. T2609, essential for DNA-repair. Consequently P-Tyr mediated effects upon DNA-PK resulted in a significant reduction of radiation-induced residual gamma H(2)AX-foci. Knockdown of PKC epsilon increased radiation-induced residual damage and abolished the P-Tyr associated radioprotection. In addition, P-Tyr mediated radioprotection was completely absent in colony formation assay. Conclusion: The data presented herein suggest that P-Tyr-treatment mediates activation of PKC epsilon, which triggers nuclear EGFR accumulation. Nuclear EGFR is involved in phosphorylation of DNA-PK at Thr2609, which has a significant impact upon DNA-DSB repair. (c) 2007 Elsevier Ireland Ltd. All rights reserved.