PHOSPHORYLATION OF SERINE-985 NEGATIVELY REGULATES THE HEPATOCYTE GROWTH-FACTOR RECEPTOR KINASE

The receptor for hepatocyte growth factor/scatter factor (HGF/SF) is a n alphabeta tyrosine kinase of 190 kDa which mediates growth and motil ity in several cell types. We have previously shown that tyrosine auto phosphorylation enhances the receptor kinase activity, while serine ph osphorylation by protein kinase C or other Ca2+-dependent kinase(s) is inhibitory. We now identify Ser985 as the major phosphorylation site for the protein kinases responsible for such inhibition. Both phorbol esters or Ca2+ ionophore treatment induces phosphorylation of the same tryptic phosphopeptide corresponding to the sequence Leu983-Arg987 lo cated in the juxtamembrane domain of the receptor beta chain. Purified protein kinase C phosphorylates in vitro a synthetic peptide (V14S) i ncluding Ser985. Trypsin digestion of the phosphorylated V14S generate s a single phosphopeptide comigrating in reverse-phase high performanc e liquid chromatography with the tryptic peptide phosphorylated in viv o. Phorbol ester treatment of cultured cells inhibits the ligand-induc ed tyrosine autophosphorylation of the receptor. In vitro, Ser985 phos phorylation inhibits the receptor tyrosine kinase activity on exogenou s substrates. Substitution of Ser985 by site-directed mutagenesis resu lts in increased tyrosine phosphorylation of the receptor and abolishe s down-modulation by protein kinase C. These data show that phosphoryl ation of Ser985 is a key mechanism for the negative regulation of HGF/ SF receptor.