Identification of a second Grb2 binding site in the v-Fms tyrosine kinase

Abstract

Tyrosine autophosphorylation of the v-Fms oncogene product results in the formation of high-a nity binding sites for cellular proteins containing Src homology 2 (SH2) domains. These proteins transduce various mitogenic and morphogenic signals. As reported previously, Y 696 KNI in the kinase insert domain of v-Fms binds to the growth factor receptor bound protein 2 (Grb2), a stimulator of the Ras/Raf1 pathway. Here, we mapped Y 921 TNL within the C-terminal domain of Fms as a novel autophosphorylation site. We demonstrate that this site constitutes a second Grb2 binding site: a recombinant fusion protein (residues 904 ± 944) containing phosphorylated Y921 bound Grb2 from FDCP1Mac11 cell extracts signi®cantly more e ciently than a corresponding protein (residues 617 ± 759) containing Y696. A yeast two-hybrid system which allowed 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 increased for proteins carrying both sites. In contrast, the simultaneous substitution of Y696 and Y921 by phenylalanines abolished Grb2 binding. Mouse NIH3T3 cells expressing the Y921F mutant Fms-protein showed a substantially higher content of ®bronectin network than wild-type transformed cells and had largely lost their serum independent growth phenotype.

FMIP, a novel Fms-interacting protein, affects granulocyte/macrophage differentiation

“…The reduced binding capacity of this latter mutant is likely, however, to be caused by a diminished tyrosine kinase activity in yeast cells which was observed exclusively with this mutant. In keeping with this notion, PI-3' kinase and Grb2 also showed about 50% reduced binding to this mutant (Mancini et al, 1997). Since it is unclear which of the remaining ten tyrosine residues of the cytoplasmic domain of Fms become also phosphorylated, additional extensive experiments will be required to solve the issue whether the interaction between FMIP and Fms depends on one or more phosphotyrosine residues.…”

“…The bait consisted of the cytoplasmic domain of mouse c-Fms fused to the C terminus of the DNA-binding and dimerization domain of LexA. Upon expression of the fusion protein in yeast cells, dimerization leads to interchain tyrosine phosphorylation of the two Fms receptor segments (Mancini et al, 1997), thus allowing us to screen a mouse embryo cDNA library with a Fms molecule representing the activated receptor phenotype. A total of 157 cDNA clones of various length were obtained, together encoding seven di erent proteins.…”

“…These mutants included Y543F devoid of the p55 binding site (Joos et al, 1996), Y558/568F in which the reported Src attachment sites were mutated (Courtneidge et al, 1993), Y705F lacking the binding site for Stat1 (Novak et al, 1996), Y720F with a mutation of the PI-3'-kinase binding site (Reedijk et al, 1992, Y807F lacking the putative binding site of p120RasGAP (Trouliaris et al, 1995), and Y696/921F in which the two Grb2 binding sites are destroyed (Mancini et al, 1997). However, with the exception of the Y807F mutant, none of the aforementioned mutations in¯uenced binding of FMIP to a signi®cant extent (Figure 4b).…”

“…The construction of LexA fusion genes encoding the cytoplasmic domains of c-Fms, TrkA, c-Met, and c-Kit downstream of LexA, the expression in E. coli pBTM116 and S. cerevisiae strain L40 were described previously (Weidner et al, 1996;Mancini et al, 1997). Fms-mutants containing tyrosine/phenylalanine replacements have been described (Mancini et al, 1997).…”

“…Fms-mutants containing tyrosine/phenylalanine replacements have been described (Mancini et al, 1997). Yeast clones were selected in medium lacking uracil and tryptophan.…”