The c-fps/fes gene encodes a cytoplasmic tyrosine kinase that is specifically expressed in myeloid cells, in particular in macrophages. The long-term objective of this study is to understand the biological role and mechanism of action of this gene, and the molecular basis of its oncogenic potential. We will investigate the possible role of c-fps/fes in signal transduction by determining if its product undergoes any biochemical changes (i.e. tyrosine phosphorylation and activation of enzymatic activity) during myeloid cell differentiation and macrophage activation, and by examining the biological and biochemical effects of reintroducing biologically active fps/fes genes into these cells. The biochemical parameters of c-fps/fes action will be dissected using temperature sensitive and other mutants of c-fps/fes, which will be introduced into myeloid cells capable of undergoing differentiation and maturation by retroviral mediated gene transfer. These experiments should clarify the biological functions of c-fps/fes and provide the means to identify proliferation-, differentiation-, and maturation-specific substrates. We will also generate antibodies against two previously identified substrates of c-fps/fes, which will be used for their characterization and molecular cloning. We will identify cellular proteins that interact with c-fps/fes kinase using purified c-fps/fes protein expressed in baculovirus, and functional domains expressed in bacteria, as affinity reagents. These proteins will also be used to generate specific monoclonal and polyclonal antibodies, which will be used in conjunction with fps/fes mutants to study functional interactions of c-fps/fes kinase with substrates and other cellular proteins. The c-fps/fes product has unique biological and biochemical properties. The elucidation of its mechanism of action will shed light on the role of tyrosine phosphorylation in myeloid cell differentiation and macrophage activation. As macrophage activation plays a central role in the immune response to infections, and as new experimental therapies rely on the use of myeloid CSFs that utilize tyrosine phosphorylation pathways to achieve differentiation of leukemic cells, the mechanisms uncovered in these studies have broad potential clinical implications.
