CALCIUM-DEPENDENT STIMULATION OF MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVITY IN A431 CELLS BY LOW-DOSES OF IONIZING-RADIATION

Ionizing radiation at 2 Gy activates the epidermal growth factor recep tor (EGFR) kinase activity in A431 squamous carcinoma cells and as a c onsequence transiently activates a downstream effector, mitogen-activa ted protein kinase (MAPK). A dose-response analysis shows fourfold act ivation 3-5 min after irradiation at 0.5 Gy with no additional activat ion after doses up to 4 Gy. Activation is independent of protein kinas e C as defined by marginal effects of protein kinase C down-regulation and the protein kinase C inhibitor, chelerythrine. In contrast, an in tracellular Ca2+ chelator (BAPTA/AM), a Ca2+ antagonist (TMB-8) and a phospholipase C inhibitor (U73223), which inhibits radiation-induced C a2+ oscillations, all block MARK stimulation. The upstream component, Raf-1, is also activated through a mechanism that is dependent on EGFR and Ca2+. Activation of Raf-1, monitored by tyrosine phosphorylation and co-immunoprecipitation with Ras, was inhibited by BAPTA/AM and TMB -8, indicating that the Ca2+-dependent step occurs at or before the in teraction of Ras and Raf-1. Neither the Ras guanosine triphosphate exc hange protein, SOS, nor Ca2+-activated tyrosine kinases linked to the MAPK pathway, focal adhesion kinase and PYK2, were stimulated by radia tion. In contrast, EGF activated SOS as shown by the enhanced associat ion of SOS with EGFR in co-immunoprecipitation experiments. These resu lts suggest that activation of EGFR-dependent downstream signaling ind uced by radiation differs from that induced by the natural ligands of EGFR. (C) 1998 by Radiation Research Society.