Characterization of PKIγ, a novel isoform of the protein kinase inhibitor of cAMP-dependent protein kinase

Abstract

Attempts to understand the physiological roles of the protein kinase inhibitor (PKI) proteins have been hampered by a lack of knowledge concerning the molecular heterogeneity of the PKI family. The PKIγ cDNA sequence determined here predicted an open reading frame of 75 amino acids, showing 35% identity to PKIα and 30% identity to PKI/3β1. Residues important for the high affinity of PKIα and PKIβ1 as well as nuclear export of the catalytic (C) subunit of cAMP-dependent protein kinase were found to be conserved in PKIγ. Northern blot analysis showed that a 1.3-kilobase PKIγ message is widely expressed, with highest levels in heart, skeletal muscle, and testis. RNase protection analysis revealed that in most tissues examined pKIγ is expressed at levels equal to or higher than the other known PKI isoforms and that in several mouse-derived cell lines, PKIγ is the predominant PKI message. Partial purification of PKI activities from mouse heart by DEAE ion exchange chromatography resolved two major inhibitory peaks, and isoform-specific polyclonal antibodies raised against recombinant PKIα and PKIγ identified these inhibitory activities to be PKIα and PKIγ. A comparison of inhibitory potencies of PKIα and PKIγ expressed in Escherichia coli revealed that PKIγ was a potent competitive inhibitor of Cα phosphotransferase activity in vitro (K(i) = 0.44 nM) but is 6-fold less potent than PKIα (K(i) = 0.073 nM). Like PKIα, PKIγ was capable of blocking the nuclear accumulation of Flag-tagged C subunit in transiently transfected mammalian cells. Finally, the murine PKIγ gene was found to overlap the murine adenosine deaminase gene on mouse chromosome 2. These results demonstrate that PKIγ is a novel, functional PKI isoform that accounts for the previously observed discrepancy between PKI activity and PKI mRNA levels in several mammalian tissues.