CRITICAL TYROSINE RESIDUES REGULATE THE ENZYMATIC AND BIOLOGICAL-ACTIVITY OF RAF-1 KINASE

The serine/threonine kinase activity of the Raf-1 proto-oncogene produ ct is stimulated by the activation of many tyrosine kinases, including growth factor receptors and pp60v-src. Recent studies of growth facto r signal transduction pathways demonstrate that Raf-1 functions downst ream of activated tyrosine kinases and p21ras and upstream of mitogen- activated protein kinase. However, coexpression of both activated tyro sine kinases and p21ras is required for maximal activation of Raf-1 in the baculovirus-Sf9 expression system. In this study, we investigated the role of tyrosine kinases and tyrosine phosphorylation in the regu lation of Raf-I activity. Using the baculovirus-Sf9 expression system, we identified Tyr-340 and Tyr-341 as the major tyrosine phosphorylati on sites of Raf-1 when coexpressed with activated tyrosine kinases. In troduction of a negatively charged residue that may mimic the effect o f phosphorylation at these sites activated the catalytic activity of R af-1 and generated proteins that could transform BALB/3T3 cells and in duce the meiotic maturation of Xenopus oocytes. In contrast, substitut ion of noncharged residues that were unable to be phosphorylated produ ced a protein that could not be enzymatically activated by tyrosine ki nases and that could block the meiotic maturation of oocytes induced b y components of the receptor tyrosine kinase pathway. These findings d emonstrate that mutation of the tyrosine phosphorylation sites can dra matically alter the function of Raf-1. In addition, this is the first report that a transforming Raf-1 protein can be generated by a single amino acid substitution.