The aims of this proposal are to characterize the role of proto- oncogene tyrosine protein kinase activities in myeloid differentiation. p93c-fes tyrosine protein kinase has been purified from DMSO-differentiated HL-60 leukemia cells. Therefore one goal of this proposal is to assess the relationship between the expression of p93c-fes and myeloid differentiation in various myelomonocytic cell lines which are sensitive and resistant to differentiating agents or colony stimulating factors. This will be accomplished by protein blotting with antibodies to specific antigenic determinants of p93c-fes, by determining the effects of tyrosine protein kinase inhibitors and by using antisense oligodeoxynucleotides to the initiation codon of p93c-fes mRNA. A second goal of this proposal is to determine the effects of overexpression of the c-fes gene in myeloid cell lines deficient in such expression. This will be accomplished by transfecting the genomic DNA for c-fes into HL-60, KG-1a and K562 cells. The regulation of expression of the mRNA and c-fes protein will be analyzed by hybridization analysis using a cDNA restriction fragment as well as by protein blotting using polyclonal antibodies to various regions of the recombinant p93c-fes. A third goal of these studies will be to determine the cellular protein substrates of p93c-fes by using antibodies against phosphotyrosine to detect phosphoproteins radiolabeled with 32P in vivo or in vitro in over- expressing cells.
