PMID:9218452

Citation	Collins, SP and Uhler, MD (1997) Characterization of PKIgamma, a novel isoform of the protein kinase inhibitor of cAMP-dependent protein kinase. J. Biol. Chem. 272:18169-78
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 PKIgamma cDNA sequence determined here predicted an open reading frame of 75 amino acids, showing 35% identity to PKIalpha and 30% identity to PKIbeta1. Residues important for the high affinity of PKIalpha and PKIbeta1 as well as nuclear export of the catalytic (C) subunit of cAMP-dependent protein kinase were found to be conserved in PKIgamma. Northern blot analysis showed that a 1.3-kilobase PKIgamma message is widely expressed, with highest levels in heart, skeletal muscle, and testis. RNase protection analysis revealed that in most tissues examined PKIgamma is expressed at levels equal to or higher than the other known PKI isoforms and that in several mouse-derived cell lines, PKIgamma 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 PKIalpha and PKIgamma identified these inhibitory activities to be PKIalpha and PKIgamma. A comparison of inhibitory potencies of PKIalpha and PKIgamma expressed in Escherichia coli revealed that PKIgamma was a potent competitive inhibitor of Calpha phosphotransferase activity in vitro (Ki = 0.44 nM) but is 6-fold less potent than PKIalpha (Ki = 0.073 nM). Like PKIalpha, PKIgamma was capable of blocking the nuclear accumulation of Flag-tagged C subunit in transiently transfected mammalian cells. Finally, the murine PKIgamma gene was found to overlap the murine adenosine deaminase gene on mouse chromosome 2. These results demonstrate that PKIgamma is a novel, functional PKI isoform that accounts for the previously observed discrepancy between PKI activity and PKI mRNA levels in several mammalian tissues.
Links	PubMed
Keywords	Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins/biosynthesis; Carrier Proteins/chemistry; Carrier Proteins/pharmacology; Cell Line; Chromatography, DEAE-Cellulose; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors; DNA Primers; Enzyme Inhibitors/chemistry; Humans; Intracellular Signaling Peptides and Proteins; Kinetics; Mice; Molecular Sequence Data; Myocardium/metabolism; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Recombinant Proteins/pharmacology; Sequence Alignment; Sequence Homology, Amino Acid; Transfection
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