Extracellular Zinc Ions Induces Mitogen-Activated Protein Kinase Activity and Protein Tyrosine Phosphorylation in Bombesin-Sensitive Swiss 3T3 Fibroblasts

Abstract

The growth factor-like effect of zincin vitroandin vivo,which has long been recognized was investigated with respect to its mechanisms of action. Addition of zinc chloride to bombesin-sensitive Swiss 3T3 mouse fibroblasts induced a fourfold stimulation in the cytosolic myelin basic protein kinase activity. The response was dose- and time-dependent, with an ED50of around 100 μMand a peak at 5 min. The kinase activity coeluted with p42 MAP kinase using chromatography on Mono-Q ion exchanger. Intracellular loading of cells with the heavy metal chelator BTC-5N did not attenuate the response to zinc. The action of zinc was not suppressed by long-term pretreatment with 4β-phorbol dibutyrate (48 h). Addition of 0.3 mMvanadate alone did not increase the kinase activity, but prolonged the action of zinc when added simultaneously. Addition of zinc (0.3 mM) or epidermal growth factor for 1 min resulted in a marked increase in tyrosine phosphorylation of proteins with apparent molecular weights of ≈100, 105–120, 215, and 240 kDa in whole cell extracts. Immunoprecipitation against the p85 subunit of phosphatidylinositol 3-kinase resulted in the appearance of two phosphotyrosine-containing proteins, 100 and 115 kDa, in extracts from cells treated with zinc or epidermal growth factor, indicating that the tyrosine phosphorylation was recognized by the corresponding SH2-domains. The present study demonstrates that extracellular zinc has the potential to partially mimic the action of growth factors on intracellular MAP kinase activation and protein tyrosine phosphorylation.