EVIDENCE FOR 2 ADDITIONAL ISOFORMS OF THE ENDOGENOUS PROTEIN-KINASE INHIBITOR OF CAMP-DEPENDENT PROTEIN-KINASE IN MOUSE

Oligonucleotides derived from the previously published rat testicular protein kinase inhibitor (PKI) sequence (Van Patten, S. M., Ng, D. C., Th'ng, J. P. H., Angelos, K. L., Smith, A. J., and Walsh, D. A. (1991 ) Proc. Natl. Acad. Sci. U. S. A. 88, 5383-5387) were used to isolate a DNA fragment coding for a mouse homologue of the rat testicular PKI. This DNA fragment was then used to screen a mouse brain cDNA library, and two cDNA clones related to the testicular PKI (PKIbeta) were isol ated. Sequencing and comparison showed that the two cDNAs differ only in the presence of a 105-base pair insert, which results in an aminote rminal extension of the predicted PKIbeta2 protein by 20 residues rela tive to the PKIbeta1 protein. By using the appropriate primers, PCR wa s used to amplify specific regions of both of these clones from mouse brain cDNA. When both clones were expressed in vitro, the mRNA for PKI beta2 produced a protein product that was larger and much more effecti vely translated, suggesting a functional role for the inserted sequenc e. Both isoforms were transiently expressed in COS-1 cells to evaluate their ability to inhibit the catalytic subunit of PKA in vivo. Extrac ts from cells expressing the PKIbeta2 isoform showed greater inhibitio n of catalytic subunit kinase activity than extracts expressing the PK Ibeta1 isoform. Northern blot analysis of poly(A)+ RNA from various mo use tissues showed the presence of transcripts of 1.8 kilobases relate d to these cDNAs. Analysis of brain RNA from several species indicated that expression of these PKI mRNAs is evolutionarily conserved. Toget her with previous studies, these results indicate the presence of at l east three PKI proteins in mouse. The skeletal muscle isoform has been designated PKIalpha, while the testicular isoform is PKIbeta. We prop ose to designate the two testicular isoforms described here PKIbeta1 a nd PKIbeta2.