Molecular characterization of thecAMP-dependent protein kinase inhibitor.

The molecular characteristics of the heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) were studied using both biochemical and molecular biological methods. First, a synthetic peptide corresponding to the inhibitory domain of PKI was shown to inhibit both the C$\alpha$ and C$\beta$ isoforms of the catalytic subunit of cAMP-dependent protein kinase. Using this peptide, an affinity column was constructed, and C subunit was purified from cells transfected with plasmid expression vectors for the two isoforms. Enzymatic characterization of C$\alpha$ and C$\beta$ demonstrated that both isoforms have similar affinities for ATP, Kemptide, and the PKI peptide. To further address the physiological role of PKI, cDNA clones were isolated and characterized from mouse skeletal muscle and a human neuroblastoma cell line (SH-SY-5Y). The clones from both species contained a 76 amino acid open reading frame which encoded proteins that were 97% identical. Northern blot analysis of various mouse tissues indicated that the 3.8 kilobase mouse mRNA is present at high levels in skeletal muscle and brain and at lower levels in testis, heart, and liver. The human PKI mRNA was found to be 4 kilobases in length in skeletal muscle while shorter transcripts were also present. Southern blot analysis of mouse genomic DNA suggested that other PKI isoforms may exist in the genome. Transient overexpression in COS cells verified that a heat-stable inhibitor of protein kinase was produced by the human PKI cDNA. Protein extracts from the transfected COS cells inhibited both C$\alpha$ and C$\beta$ with equal efficacy. Functional expression of the human PKI was further studied by assaying the ability of PKI expression vectors to inhibit cAMP stimulation of transcription from the human enkephalin promoter. In these studies, elimination of a conserved alternative translation start site was shown to potentiate the inhibition of the PKI expression vector. Its evolutionary conservation and ability to inhibit both C$\alpha$ and C$\beta$ suggest that PKI plays a functional role in the regulation of signal transduction by the cAMP-dependent protein kinase.PhDCellular and Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103653/1/9332145.pdfDescription of 9332145.pdf : Restricted to UM users only