The human c-fes locus encodes a 93 kDa cytoplasmic protein-tyrosine kinase (PTK) that is activated during the terminal differentiation of myeloid leukemia cell lines in vitro. Transfection of an immature myeloid leukemia cell line (K562) with the c-fes gene induced differentiation, suggesting that p93(c-fes) plays an active role in myeloid growth control. Little is known about the regulation of c-fes PTK activity, or the identity of the substrates phosphorylated by p93(c- fes) that mediate differentiation. This proposal will investigate these questions by focusing on a unique structural feature of p93(c-fes) known as the src homology 2 (SH2) domain. This non-catalytic domain is located N-terminal to the kinase domain in p93(c-fes) and other PTKs, including the viral c-fes homolog, v-fps. The SH2 domain of v-fps has been shown to regulate PTK activity by binding to autophosphorylated tyrosine residues in the kinase domain, and to mediate interactions with transformation-related substrates and regulatory proteins. To test the hypothesis that the c-fes SH2 domain regulates the adjacent kinase domain, a series of deletions will be made in the SH2 domain of the c-fes cDNA. The resulting family of mutants will be expressed in E. coli, and assayed for PTK activity using an immune-complex kinase assay. Changes in activity will be correlated with altered SH2-kinase domain association using a protease-resistance assay. The role of the c-fes SH2 domain in biological function will be assessed by transfecting K562 cells with the SH2 deletion mutants, and assaying the cells for functional markers of differentiation. Patterns of phosphotyrosine-containing proteins will be compared between K562 cells transfected with the wild-type and SH2 mutants. Proteins phosphorylated on tyrosine that are present in cells transfected with the wild-type but not in differentiation-defective SH2 mutants will be good candidates for c-fes substrates. To directly test the hypothesis that the c-fes SH2 domain can recognize c-fes substrates and regulatory proteins, recombinant c-fes SH2 domain protein will be synthesized in bacteria, biotinylated, and used to probe western blots of proteins from K562 cells. Additionally, the recombinant SH2 protein will be attached to beaded agarose and used as an affinity matrix to purify SH2-binding proteins. Finally, c-fes autophosphorylation sites will be identified using 2-D tryptic phosphopeptide mapping and site- directed mutagenesis. Mutagenesis of these sites is expected to affect p93(c-fes) PTK activity, structure and biological function due to disrupted interaction with the SH2 domain. Successful completion of these studies will provide novel information regarding the role of the SH2 domain in c-fes PTK regulation and complex formation with substrates and/or regulatory proteins. Further understanding of this growth- regulatory pathway may provide a molecular basis for the design of novel anti-leukemic agents that induce terminal differentiation by stimulating p93(c-fes) PTK activity.
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