The purpose of this proposal is to determine how a myeloid-specific proto- oncogene protein-tyrosine kinase, c-fes can regulate the differentiation of myeloid leukemia cells. The c-fes proto-oncogene encodes a 93 kDa protein- tyrosine kinase that is specifically associated with normal and leukemic myelomonocytic cells. The c-fes gene plays a pivotal role in myeloid differentiation based on studies in our laboratory demonstrating that transfection of immature myeloblast cell line K562 with the genomic c-fes gene results in the stable expression of clonal variants possessing a more mature granulocytic phenotype.
The aims of this proposal are two-fold. The first objective is to examine the manner by which the c-fes gene product, P93c-fes, regulates its protein-tyrosine kinase activity and how this influences differentiation. This will be accomplished in two ways. The first approach will be to express the c-fes cDNA and its various deletions and point mutations in a baculovirus expression system to produce recombinant forms of P93c-fes to investigate in vitro, the role of the amino-terminal domain and autophosphorylation sites in the regulation of its catalytic activity. Secondly, the c-fes cDNA and its variants will be utilized for transfection of K562 cells to study its structure-function relationships in myeloid differentiation. The expression of recombinant P93c-fes will also provide an ample source of protein as an immunogen for the production of polyclonal antibodies for use in the second part of this proposal. The second objective is to assess the role of cytokines which influence myeloid differentiation, such as granulocyte/macrophage colony-stimulating factor (GM-CSF), in the regulation of transcription of c-fes and in the expression of the protein-tyrosine kinase activity of P93c-fes. This will be accomplished by utilizing CSF-dependent or -responsive human myelomonocytic leukemia cell lines to determine the relationship of c-fes expression to the proliferative or differentiative processes. The autophosphorylation of P93c-fes in vivo, and measurement of its kinase activity in vitro will be accomplished by immunoprecipitation with monospecific polyclonal antibodies to P93c-fes, and by affinity chromatography and a non-denaturing polyacrylamide gel assay, respectively. The analysis of mRNA levels will be carried out by RNase protection assay or by competitive polymerase chain reaction assay, and transcription will be measured by nuclear run-on assays. Potential endogenous substrates of P93c-fes in CSF-dependent or -responsive myeloid cell lines will be investigated by immunochemical, electrophoretic and immunoblotting procedures using anti-phosphotyrosine antibodies. The information derived from this study has diagnostic and prognostic utility since the c-fes gene is a specific marker which distinguishes myeloid from other types of leukemias. Understanding the regulation of the c-fes gene has therapeutic implications as well, since the activity of this gene may indicate whether a leukemic cell has the potential for differentiating and thereby reducing its oncogenic potential. Cognizance of this regulatory process should aid in the design of differentiation- specific therapies for myeloid leukemias.
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