MOLECULAR-CLONING AND CHARACTERIZATION OF PKC(IOTA), AN ATYPICAL ISOFORM OF PROTEIN-KINASE-C DERIVED FROM INSULIN-SECRETING CELLS

The protein kinase C (PKC) family of serine-threonine kinases comprises at least eight members. These are differentially expressed, show varying affinities for activators such as Ca2+ and lipid species, and are therefore thought to play wide-ranging roles in the regulation of such cellular processes as differentiation, growth, and secretion. The aim of this study was to identify new PKC isoforms in the insulin-secreting cell line RINm5F that might be activated by the alterations in lipid metabolism that accompany nutrient-stimulated insulin release. Fragments of cDNA, derived from RINm5F cell mRNA, were amplified by the polymerase chain reaction using degenerate oligonucleotide primers corresponding to highly conserved regions in the catalytic domains of all known PKCs. A novel sequence generated by this approach was subsequently used to screen cDNA libraries. The entire 587-amino acid coding region of a new PKC isoform, PKCiota, was deduced from two overlapping clones isolated from a human kidney cDNA library. The amino acid sequence of PKCiota showed greatest homology to PKCzeta, with 72% identity overall rising to 84% in the catalytic domain. In contrast, the homology of PKCiota to the other isoforms was less pronounced, with <53% identity even in the highly conserved catalytic region. Further similarities between PKCzeta and PKCiota included a highly conserved pseudosubstrate sequence, the absence of an apparent Ca2+-binding region, and the presence of only one cysteine-rich, zinc finger-like domain. Northern blot analysis, using the full-length PKCiota clone as a probe, revealed a single 4.6-kilobase transcript present predominantly in lung and brain, but also expressed at lower levels in many tissues including pancreatic islets. In CHO-K1 cells stably expressing the PKCiota cDNA under the human beta-actin promoter, the protein was detected as a 65-kDa band by Western blotting using an antibody to the COOH terminus of PKCzeta (conserved in PKCiota). Extracts of transfected CHO-K1 cells also displayed a significantly increased kinase activity using myelin basic protein as a substrate. The results suggest that PKCiota should be included in the atypical subgroup of PKCs whose definitive member is PKCzeta. As such, PKCiota is unlikely to be activated by the diacylglycerol that is derived from phosphoinositide hydrolysis, but might be a target for novel lipid activators that are elevated during nutrient-stimulated insulin secretion.