PROTEOLYTIC CLEAVAGE OF PROTEIN-KINASE-C ISOTYPES, WHICH GENERATES KINASE AND REGULATORY FRAGMENTS, CORRELATES WITH FAS-MEDIATED AND 12-O-TETRADECANOYL-PHORBOL-13-ACETATE-INDUCED APOPTOSIS

Protein kinase C (PKC) has been implicated in signaling induced by div erse sets of stimuli regulating growth differentiation, and apoptosis. The present study focused on the fate of PKC isotype proteins during Pas-mediated apoptosis of human leukemic cell lines. Among the PKC iso types expressed in different cell types, such as Jurkat, HPB-ALL, U937 , and HL60, all the nPKC isotypes including nPKC delta, nPKC epsilon, and nPKC theta, but not cPKC alpha and beta II and aPKC zeta (n, c, an d a represent novel, conventional and atypical, respectively), showed limited proteolytic cleavage during Fas-mediated apoptosis. The limite d proteolysis of nPKC isotypes means the disappearance of the in:act p rotein band concomitant with the appearance of two fragments, most lik ely containing the kinase and regulatory domains, in contrast to the s o-called down-regulation known for both cPKC and nPKC isotypes followi ng exposure to stimuli such as 12-0-tetradecanoyl-phorbol 13-acetate ( TPA). The time course of Fas-mediated apoptosis in Jurkat cells parall els that of the activation of a 32-kDa cysteine protease (CPP32)-like protease and also closely parallels the proteolytic cleavage of nPKC i sotypes. A peptide inhibitor of the CPP32-like protease, Ac-DEVD-CHO, blocked the proteolytic cleavage of nPKC isotypes as well as apoptosis mediated by Fas. Transfection of recombinant protein coding for the c atalytic fragment of nPKC delta to COS1 cells resulted in the apoptoti c morphology of cells and nuclei. The effect of TPA on apoptosis depen ds on the cell type. TPA significantly suppressed Fas-mediated apoptos is in Jurkat, whereas TPA alone caused apoptosis in HPB-ALL, U937, and HL60, only slight apoptosis in Jurkat. The proteolytic fragmentation of nPKC isotypes again closely correlated with the degree of apoptosis even in apoptosis induced by TPA. Separation of TPA-treated cells int o apoptotic and non-apoptotic differentiating cells revealed that the proteolytic fragmentation of nPKC isotypes occurs only in apoptotic ce lls and, in adherent differentiating cells, nPKC isotypes as well as c PKC alpha were down-regulated without the generation of nPKC fragments . These results are consistent with the idea that nPKC isotypes meet t wo different fates, down-regulation and proteolytic cleavage generatin g kinase and regulatory fragments, and that the proteolytic cleavage o f nPKC isotypes is a step in the signaling pathway involved in Fas-med iated and TPA-induced apoptosis.