SELECTIVITY OF PHOSPHOLIPASE-C PHOSPHORYLATION BY THE EPIDERMAL GROWTH-FACTOR RECEPTOR, THE INSULIN-RECEPTOR, AND THEIR CYTOPLASMIC DOMAINS

Phosphatidylinositol-specific phospholipase C isozyme γ (PLC-γ, Mr 145,000) is an excellent substrate for the epidermal growth factor (EGF) receptor both in vivo and in vitro. PLC-β-1, another PLC isozyme, is a poor substrate for the EGF receptor. We examined the relative phosphorylation of PLC-γ and PLC-β-1 by the 170-kDa native EGF receptor molecule, the 66-kDa cytoplasmic kinase domain of the EGF receptor (Arg647Ala1186), the α2β2 native insulin receptor, and the 48-kDa cytoplasmic kinase domain of the insulin receptor β subunit (Gly947-Ser1343). Similar to the intact EGF receptor, the cytoplasmic kinase domain of the EGF receptor preferentially phosphorylated PLC-γ. High-performance liquid Chromatographic comparison of tryptic phosphopeptides from PLC-γ phosphorylated by both forms of the EGF receptor kinase indicated similar patterns of multiple tyrosine phosphorylations. These results imply that substrate selectivity, at least in terms of PLC isozymes, is independent of the extracellular ligand-binding and membrane anchor domains of the EGF receptor. In comparison, neither the intact insulin receptor nor the β-chain kinase domain was able to phosphorylate PLC-γ to a significant extent. Also, insulin failed to stimulate the phosphorylation of PLC-γ in NIH 3T3/HIR cells, which overexpress the human insulin receptor. Thus PLC-γ is not a phosphorylation substrate for the insulin receptor in vitro or in the intact cell.