CYCLIC AMP-DEPENDENT PROTEIN KINASE STRUCTURE-FUNCTION

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The proposed studies seek to examine the structural features of the catalytic subunit of cAMP-dependent protein kinase which are necessary for biological activity of the enzyme. In addition, the possible regulation of the production of the enzyme will be examined. The principal approach will involve analysis of catalytic subunit mutants to determine important functional domains of the molecule. The isolation of mutants will be facilitated by in vitro mutagenesis of catalytic subunit cDNA sequences. The function of altered catalytic subunit cDNA sequences will be tested by construction of expression vectors which will be introduced into mammalian cells. Thus, the first specific aim of the proposed studies involves isolation of catalytic subunit cDNA clones containing the complete coding sequence. In preliminary studies a partial cDNA has been isolated and this cDNA will be used as a hybridization probe to obtain a full-length clone. The full-length clone will be inserted in an expression vector which will be introduced into at least two cAMP-responsive cell lines for analysis of the function of the cloned gene. After conditions for expression of the cDNA clone have been determined, random mutations in the cDNA will be introduced by treatment with bisulfite and mutants isolated and characterized. Studies of genomic catalytic subunit sequences will also be performed to determine if there are multiple copies of the gene. Furthermore, analysis of the intron-exon organization of the gene may provide some insight into functional domains of the enzyme. Finally, the possible regulation of protein kinase gene expression will be examined by hybridization analysis of mRNA levels in cAMP treated cells and tissues. The overall goal of these studies is to increase the understanding of the mechanisms which allow cAMP-dependent protein kinase to participate in signal transduction and regulation of biological processes.