Protein kinases are key enzymes in cellular regulation: cyclic AMP-dependent kinases mediate hormone actin; the isulin and epidermal growth factor receptors also possess kinase activity; Rous-sarcoma virus induced cellular transformation requires a tyrosine protein kinase; and protein kinases are thought to regulate pathways involved in cellular differentiation. Our ultimate goal is to develop methodologies for the specific inhibition of the various protein kinases which will allow unprecedented control of cellular function. Through our own efforts and those of other laboratories the substrate specificity of the cAMP-dependent protein kinase has been defined. We have used this knowledge to develop the first peptide based affinity label for this enzyme and are proposing to develop new affinity labels, photoaffinity labels, and tight binding competitive inhibitors for the kinase. Recently, it was discovered in our laboratory that cAMP-dependent protein kinase will not phosphorylate peptide substrates containing certain N-methylated amide bonds. We plan to determine whether cGMP-dependent protein kinase, which has very similar substrate specificity requirements to the cAMP-dependent enzyme, might differ in these specific peptide substrate requirements. This would mean that inhibitors could be adapted to interact exclusively with one of the two kinases. We intend to continue to investigate the chemistry underlying the mechanism of the catalytic action of cAMP-dependent protein kinase. Those active site residues which are modified by affinity labels or photoaffinity labels will be identified. Additionally, NMR studies in collaboration with Prof. A. S. Mildvan focussed on defining the steric relationship between enzyme bound functional groups and substrates will be performed, as will experiments in collaboration with Prof. J. R. Knowles on the stereochemistry of protein kinase catalyzed phosphoryl group transfer.
