We propose to investigate the determinants of substrate specificity of cyclic AMP-dependent and cyclic GMP-dependent protein kinases. Synthetic peptides will be used in these studies as model substrates and inhibitors of the protein kinases. An undecapeptide that corresponds to the sequence of amino acids at the site of autophosphorylation in cyclic GMP-dependent protein kinase will be used to examine the kinetics and substrate specificity of this enzyme. The contribution of specificic amino acids to the ability of the peptide to be phosphorylated by either cyclic GMP-dependent or cyclic AMP-dependent protein kinase will be determined by replacement of individual residues and evaluation of the kinetic constants for these analogs of the peptide. The autophosphorylation site peptide will also be used to probe the interaction of the regulatory domain of the cyclic GMP-dependent protein kinase with its catalytic site. Additionally, synthetic peptides will be employed in the generation, purification, and characterization of site-specific antibodies to be used as probes of the sites of phosphorylation on protein substrates of the cyclic AMP-dependent protein kinase. L-type pyruvate kinase and peptides corresponding to the sequence of residues at the phosphorylation site in this protein will be used as immunogens to produce monoclonal antibodies. Antibodies directed toward determinants within either the dephospho- or phospho-forms of the phosphorylation site will be purified, and their specificities and affinities will be characterized. These site-specific antibodies will be used to investigate the regulation of pyruvate kinase activity and to immunologically characterize the structures of phosphorylation sites in other known substrates of the protein kinase. These experiments should provide new information about the molecular details that dictate the determinants of specificity of the cyclic nucleotide-dependent protein kinases. An increased understanding of the differences in the specificities between the two protein kinases may allow the development of selective inhibitors of each enzyme. The site-specific immunological reagents developed in these investigations should be useful biochemical and histochemical tools in the study of the physiological roles of the protein kinases and their substrates in hormone action.
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