The colony-stimulating factor 1 receptor (CSF-1R), encoded by the c-fms proto-oncogene, exhibits intrinsic, ligand-dependent, protein tyrosine kinase (PTK) activity. Mutations at Leu-301 within the extracellular domain of human CSF-1R unmask its oncogenicity, and induce constitutive receptor PTK activity. In principle, activating mutations in human FMS might contribute to the etiology of myeloid proliferative disorders, including leukemias and myelodysplastic syndromes, but no bona fide activating mutations at codon 301 have thus far been identified.
Specific Aim #1 is to use an unbiased approach to search for additional activating mutations in human FMS gene. Chemically mutagenized FMS """"""""target cassettes"""""""" will be recloned en masse into a parental vector which will be transfected into NIH/3T3 cells. Foci of transformed cells will be isolated, and the relevant portions of their FMS genes will be directly sequenced after amplification of the target cassette by polymerase chain reaction. We expect to catalog alternative activating mutations that may potentially contribute to the neoplastic transformation of cells of the mononuclear phagocyte series. The locations of multiple activating mutations should circumscribe structural motifs within CSF-1R that determine its ligand-independent activation. Signal transduction by CSF-1R involves the association of the autophosphorylated receptor with """"""""downstream"""""""" effectors of the mitogenic response. Selective mutagenesis of mapped sites of receptor autophosphorylation abrogate its ability to couple to these proteins, generating mutants that are partially defective in signal transduction. Mutation of Tyr-809 uncouples the ability of CSF-1R to induce c-myc expression and greatly reduces CSF-1 stimulated mitogenicity. In contrast, the ligand-stimulated CSF-1R[Phe-809] mutant exhibits wild- type levels of PTK activity, binds to a phosphatidylinositol 3-kinase, and induces the activity of c-fos, c-jun and junB with unperturbed kinetics. Introduction of an exogenous c-myc gene into NIH/3T3 cells bearing CSF-1R[Phe-809] restores CSF-1 induced mitogenicity in serum- free medium.
Under Specific Aim #2, we propose to clone the putative cellular effector protein that interacts with CSF-1R in the region flanking PTyr-809, elucidate its function, and study its regulation of CSF-1-responsive genes. The latter experiments focus on mechanisms controlling normal growth which are likely to be perturbed in cancer cells.
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