The inhibitor protein (PKI) of the cAMP-dependent protein kinase was first characterized from rabbit skeletal muscle. More recently a form of PKI was isolated and cloned from rat testis which shares relatively limited amino acid sequence with the rabbit skeletal muscle form. We have now isolated a cDNA from rat brain which encodes a protein corresponding to the rabbit skeletal muscle PKI. This establishes the presence of the “skeletal muscle” and “testis” proteins in the same species and therefore that they clearly represent distinct isoforms. We have also demonstrated that the isoform from testis, like the skeletal muscle isoform, is specific for the cAMP-dependent protein kinase and that it is able to inhibit this enzyme when expressed in cultured JEG-3 cells. Both forms contain the five specific amino acid recognition determinants which have been shown to be required for high affinity binding to the protein kinase catalytic site, although there is some noted lack of conservation of codons used for these residues. Overall, the two rat isoforms are only 41% identical at the amino acid level and 46% at the level of coding nucleotides. We propose that the rabbit skeletal muscle and rat testis forms be designated PKIα and PKIβ, respectively. Using Northern blot analysis, we have examined the tissue distribution of the two forms in the rat and their relative expression during development. In the adult rat, mRNA of the PKIα species is highest in muscle (both skeletal and cardiac) and brain (cortex and cerebellum). The PKIβ mRNA is most highly expressed in the adult testis with substantial expression in cerebellum and spleen, but it is undetectable in either form of muscle. The most striking finding of the developmental studies was in the testis, where the PKIα species is present at substantial levels in the neonate but showed a progressive decline with age, while the PKIβ form was undetectable before 20 days but steadily increased with age to become the predominant form of PKI in the testis of the mature male rat. In addition, a second smaller mRNA species for PKIβ appeared in the testis by 30 days and increased by 60 days of age; this mRNA species was not seen in any other tissue.
ASJC Scopus subject areas
- Molecular Biology