INTERACTION OF THE FLT-1 TYROSINE KINASE RECEPTOR WITH THE P85 SUBUNIT OF PHOSPHATIDYLINOSITOL 3-KINASE - MAPPING OF A NOVEL SITE INVOLVED IN BINDING

We have examined the interactions of the p85 regulatory subunit of pho sphatidylinositol 3-kinase with the endothelium-specific Flt-1 recepto r tyrosine kinase using the yeast two-hybrid system. We find that both the amino- and carboxyl-terminal SH2 domains of p85 bind to Flt-1. We have performed site-directed mutagenesis on the carboxyl-terminal tai l of the Flt-1 receptor in order to identify the site(s) that is respo nsible for the p85 interactions. A single tyrosine to phenylalanine ch ange at position 1213 inhibits the binding of both p85 SH2 domains. Ph osphopeptide mapping of the wild type and mutant protein expressed in insect cells verifies that this amino acid is a target for autophospho rylation. The amino acids following this tyrosine are VNA and thus def ine a novel binding site for p85.