The primary goals are to elucidate the physiological role of the Inhibitor Protein of the cAMP-dependent protein kinase, to understand its mode of interaction at the catalytic site of the protein kinase, and to gain a fuller understanding of the role and regulation of the cAMP-dependent protein kinase in cellular control. These objectives will be approached as follows: (i) A 20 amino acid peptide (IP20) has recently been obtained by us, both by proteolysis of the Inhibitor Protein and by direct synthesis. This peptide has about 30% of the activity of the native molecule (Ki about 0.5 nM) and clearly contains most of the key domains for interaction with the catalytic site. We propose to study the structural features of IP20 that provide for this high affinity interaction by synthesizing a variety of peptides based upon the sequence of IP20. With these, the interactions with the catalytic subunit will be examined kinetically, and by NMR, fluorescence spectroscopy and circular dichroism. We plan to compare the interaction of the Inhibitor Protein and the inhibitory peptides with the catalytic site, with those of substrates and regulatory subunit. (ii) FLuoresceinated derivatives of catalytic subunit, regulatory subunit, Inhibitor Protein and IP20 have or will be synthesized. These will be used as cytochemical probes to examine the subcellular localization of protein kinase subunits and the Inhibitor Protein, the dissociation reaction of the protein kinase in cells, and the function of the Inhibitor Protein and its interaction with catalytic subunit. These studies will be conducted in H-35 Hepatoma cells, Chinese Hamster Ovary cells mutant in component proteins of the protein kinase, C1C12 clonal myocytes and gonadotrophs. (iii) An oligonucleotide, whose sequence is based upon IP20, is to be introduced into yeast cells such that its synthesis can be regulated. By controlling its level of expression, this should provide a new avenue to probe for cAMP function in growth control and other cellular functions in these cells.
