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 comprise s at least eight members. These are differentially expressed, show var ying affinities for activators such as Ca2+ and lipid species, and are therefore thought to play wide-ranging roles in the regulation of suc h cellular processes as differentiation, growth, and secretion. The ai m of this study was to identify new PKC isoforms in the insulin-secret ing cell line RINm5F that might be activated by the alterations in lip id metabolism that accompany nutrient-stimulated insulin release. Frag ments of cDNA, derived from RINm5F cell mRNA, were amplified by the po lymerase chain reaction using degenerate oligonucleotide primers corre sponding to highly conserved regions in the catalytic domains of all k nown PKCs. A novel sequence generated by this approach was subsequentl y used to screen cDNA libraries. The entire 587-amino acid coding regi on of a new PKC isoform, PKCiota, was deduced from two overlapping clo nes isolated from a human kidney cDNA library. The amino acid sequence of PKCiota showed greatest homology to PKCzeta, with 72% identity ove rall rising to 84% in the catalytic domain. In contrast, the homology of PKCiota to the other isoforms was less pronounced, with <53% identi ty even in the highly conserved catalytic region. Further similarities between PKCzeta and PKCiota included a highly conserved pseudosubstra te sequence, the absence of an apparent Ca2+-binding region, and the p resence of only one cysteine-rich, zinc finger-like domain. Northern b lot analysis, using the full-length PKCiota clone as a probe, revealed a single 4.6-kilobase transcript present predominantly in lung and br ain, but also expressed at lower levels in many tissues including panc reatic islets. In CHO-K1 cells stably expressing the PKCiota cDNA unde r 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 PKC zeta (conserved in PKCiota). Extracts of transfected CHO-K1 cells also displayed a significantly increased kinase activity using myelin basi c protein as a substrate. The results suggest that PKCiota should be i ncluded in the atypical subgroup of PKCs whose definitive member is PK Czeta. As such, PKCiota is unlikely to be activated by the diacylglyce rol that is derived from phosphoinositide hydrolysis, but might be a t arget for novel lipid activators that are elevated during nutrient-sti mulated insulin secretion.