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 receptor (EGFR) kinase activity in A431 squamous carcinoma cells and as a consequence transiently activates a downstream effector, mitogen-activated protein kinase (MAPK). A dose-response analysis shows fourfold activation 3-5 min after irradiation at 0.5 Gy with no additional activation after doses up to 4 Gy. Activation is independent of protein kinase C as defined by marginal effects of protein kinase C down-regulation and the protein kinase C inhibitor, chelerythrine. In contrast, an intracellular Ca²⁺ chelator (BAPTA/AM), a Ca²⁺ antagonist (TMB-8) and a phospholipase C inhibitor (U73223), which inhibits radiation-induced Ca²⁺ oscillations, all block MAPK 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 interaction of Ras and Raf-1. Neither the Ras guanosine triphosphate exchange protein, SOS, nor Ca²⁺-activated tyrosine kinases linked to the MAPK pathway, focal adhesion kinase and PYK2, were stimulated by radiation. In contrast, EGF activated SOS as shown by the enhanced association of SOS with EGFR in co-immunoprecipitation experiments. These results suggest that activation of EGFR-dependent downstream signaling induced by radiation differs from that induced by the natural ligands of EGFR.