2 SIGNALING MOLECULES SHARE A PHOSPHOTYROSINE-CONTAINING BINDING-SITEIN THE PLATELET-DERIVED GROWTH-FACTOR RECEPTOR

Autophosphorylation sites of growth factor receptors with tyrosine kin ase activity function as specific binding sites for Src homology 2 (SH 2) domains of signaling molecules. This interaction appears to be a cr ucial step in a mechanism by which receptor tyrosine kinases relay sig nals to downstream signaling pathways. Nck is a widely expressed prote in consisting exclusively of SH2 and SH3 domains, the overexpression o f which causes cell transformation. It has been shown that various gro wth factors stimulate the phosphorylation of Nck and its association w ith autophosphorylated growth factor receptors. A panel of platelet-de rived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutatio n at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant fa iled to mediate PDGF-stimulated phosphorylation of Nck in intact cells . A phosphorylated Tyr-751 is also required for binding of phosphatidy linositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experi ments with different phosphopeptides derived from the PDGF receptor su ggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is ab le to link the PDGF receptor to two distinct SH2 domain-containing sig naling molecules.