IDENTIFICATION OF A 2ND GRB2 BINDING-SITE IN THE V-FMS TYROSINE KINASE

Tyrosine autophosphorylation of the v-Fms oncogene product results in the formation of high-affinity binding sites for cellular proteins con taining Src homology 2 (SH2) domains. These proteins transduce various mitogenic and morphogenic signals. As reported previously, (YKNI)-K-6 96 in the kinase insert domain of v-Fms binds to the growth factor rec eptor bound protein 2 (Grb2), a stimulator of the Ras/Raf1 pathway. He re, we mapped (YTNL)-T-921 within the C-terminal domain of Fms as a no vel autophosphorylation site. We demonstrate that this site constitute s a second Grb2 binding site: a recombinant fusion protein (residues 9 04-944) containing phosphorylated Y921 bound Grb2 from FDCP-1Mac11 cel l extracts significantly more efficiently than a corresponding protein (residues 617-759) containing Y696. A yeast two-hybrid system which a llowed the formation of a functional Fms tyrosine kinase was employed to quantify binding of Grb2. Fms-protein containing either one of the two phosphorylation sites bound Grb2 equally well, binding was increas ed for proteins carrying both sites. In contrast, the simultaneous sub stitution of Y696 and Y921 by phenylalanines abolished Grb2 binding. M ouse NIH3T3 cells expressing the Y921F mutant Fms-protein showed a sub stantially higher content of fibronectin network than wild-type transf ormed cells and had largely lost their serum independent growth phenot ype.