Hematopoietic cell growth, di erentiation, and commitment to a restricted lineage are guided by a set of cytokines acting exclusively on cells expressing the corresponding cytokine receptor. The macrophage colony stimulating factor (M-CSF, also termed CSF-1) and its cognate receptor, the tyrosine kinase c-Fms, are essential for monocyte and macrophage development. The underlying molecular mechanism, however, is poorly understood. Here we identi®ed a novel Fms-interacting protein (FMIP, MW 78 kDa) which binds transiently via its Nterminal 144 residues to the cytoplasmic domain of activated Fms-molecules. Binding of FMIP was paralleled by rapid tyrosine phosphorylation within the binding domain which drastically reduced its ability to associate with Fms. Binding was speci®c as evidenced by co-immunoprecipitation and association with recombinant GST-Fms fusion proteins. No binding was observed with the tyrosine phosphorylated cytoplasmic domains of c-Kit, TrkA, c-Met, and the insulin receptor. The role of FMIP in hematopoietic di erentiation was studied in the bipotential myeloid progenitor cell line, FDC-P1Mac11. Overexpression of FMIP prevented M-CSF induced macrophage di erentiation. Instead, cells di erentiated into granulocytes. Our data suggest that the level of FMIP expression could form a threshold that decides about di erentiation either into macrophages or into granulocytes.

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.

Apoptotic cell death was shown to be accompanied or preceded by an elevated expression of the c-fos protooncogene and DNA binding activity of transcription factor AP-1. We used Fos-de®cient mice to study the role of c-Fos during programmed cell death in the prostate. In normal mice apoptosis is induced in the prostate within 2 ± 4 days after castration. Histological features of reduced secretory activity and morphological signs of programmed cell death become obvious. No apparent decrease in secretory activity and no epithelial cell death were observed in Fos-de®cient animals after castration. Fragmentation of nuclear DNA was measured by in situ terminal transferase reaction. DNA fragmentation was observed in the prostate epithelium of control mice after castration whereas no similar fragmentation was found in Fos-de®cient animals. After castration an AP-1 complex accumulated in the prostate of Fos de®cient mice which mainly consists of FosB, Fra-2 and JunD whereas in control animals the AP-1 complex in addition contained c-Fos. Our data strongly suggest that c-Fos is required for programmed cell death of prostate epithelial cells.