CHARACTERIZATION OF NEWLY-IDENTIFIED PROTEIN KINASES

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The long term objective of this proposal is to work toward the systematic identification and characterization of protein-serine kinases expressed in a growing HeLa cell population. A thorough survey of this imporant class of regulatory enzymes in a proliferating human cell population should provide new insight into our understanding of cellular control mechanisms in general and specifically should help to define the chain of events involved in the ordered duplication and segregation of the genetic material which underlies cell division. This informalion, in turn, will suggest pathways for transduction of signals induced by binding of growth factors. A more complete understanding of the enzymatic and metabolic events involved in the regulation of mammalian cell reproduction will lead to more effective therapies for the treatment of human cancers. cDNA clones encoding members of the protein-serine kinase family will be identified by screening a HeLa library with oligonucleotide probes directed at highly conserved regions within the catalytic domain. Familial relationships will be confirmed by DNA sequence analysis. Structural and functional information about the putative protein-serine kinases encoded by the clones will be acquired through an analysis of deduced amino acid sequences as well as through heterologous eukaryotic expression systems. Antisera will be produced against the encoded proteins for use in 1) immunoprecipitation studies to allow enzymatic and further structural characterization, 2) indirect immunofluorescence studies to determine intracellular localization, and 3) immunoblotting studies to measure cell and tissue specificity of expression. Preliminary studies have clearly demonstrated the utility and value of the oligonucleotide homology screening approach. A cDNA clone has been identified which encodes a protein with considerable homology to protein-serine kinases encoded by cell cycle regulating genes in yeast. A major thrust of the proposed studies will be to further explore the structural and functional relationships between these proteins.