The long-term goal of this research program is to elucidate the structural mechanisms which regulate protein kinases. these enzymes catalyze the transfer of phosphate onto the surface of target proteins. Phosphorylation of these target proteins mediates many important cellular events such as glycogenolysis, mitosis and possibly gene transcription. Since protein phosphorylation mediates many key cellular events, it is very important to have the phosphotransferase activity of the protein kinases highly regulated. The proposed research will identify the structures which regulate the cAMP-dependent protein kinase. Structural analysis of several protein kinases has identified separate catalytic and regulatory domains. The function of the regulatory domain is to inhibit phosphotransferase activity in the catalytic domain. These domains can be on the same polypeptide or separate subunits of a holoenzyme complex. It is postulated that inhibitors contain regions of amino acid sequence resembling substrates called """"""""pseudosubstrates"""""""", which blind to the catalytic domain preventing phosphoryl transfer.
The aim of this proposal are to identify the inhibitory regions within the type II regulatory subunit, RII, of the cAMP-dependent protein kinase and isolate cDNA clones for the heat-stable inhibitor of the kinase (PKI). Since cDNA for RII is available and RII protein can be expressed in bacteria, this experimental approach will involve protein engineering. The RII cDNA will be manipulated by various mutagenesis techniques and the resulting mutant proteins will be expressed in E. coli. Each mutant RII will be purified and kinetic measurements of its inhibitory properties will be performed.
The specific aims of this proposal are: to define the minimum structure required for RII inhibition of the C subunit: identify specific amino acids at the inhibitory site of RII; examine the conformation of the inhibitory site by Nuclear Magnetic Resonance; and locate amino acids essential for holoenzyme dissociation. Once cDNA clones for PK1 have been isolated, they will be used as probes to investigate the inhibitor function. The PKI mRNA size, amount and tissue distribution will be examined using the cDNA as a probe. Expression of PKI in E. coli will yield protein which will be used to raise anti-PKI antisera. These antibodies will be used to investigate the protein under more physiological conditions.
