The 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

Abstract

Protein kinase C (PKC) has been implicated in signaling induced by diverse sets of stimuli regulating growth, differentiation, and apoptosis. The present study focused on the fate of PKC isotype proteins during Fas-mediated apoptosis of human leukemic cell lines. Among the PKC isotypes expressed in different cell types, such as Jurkat, HPB-ALL, U937, and HL60, all the nPKC isotypes including nPKC6, nPKCE, and nPKCB, but not cPKCa and fl1 and aPKCC (n, c, and a represent novel, conventional and atypical, respectively), showed limited proteolytic cleavage during Fas-mediated apoptosis. The limited proteolysis of nPKC isotypes means the disappearance of the intact protein band concomitant with the appearance of two fragments, most likely containing the kinase and regulatory domains, in contrast to the so-called down-regulation known for both cPKC and nPKC isotypes following exposure to stimuli such as 12-0-tetradecanoyl-phorbol 13-acetate (TPA). The time course of Fas-mediated apoptosis in Jurkat cells parallels that of the activation of a 32-kDa cysteine protease (CPP32)-like protease and also closely parallels the proteolytic cleavage of nPKC isotypes. 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 catalytic fragment of nPKCG to COSl cells resulted in the apoptotic morphology of cells and nuclei.The effect of TPA on apoptosis depends on the cell type. TPA significantly suppressed Fas-mediated apoptosis 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 into apoptotic and non-apoptotic differentiating cells revealed that the proteolytic fragmentation of nPKC isotypes occurs only in apoptotic cells and, in adherent differentiating cells, nPKC isotypes as well as cPKCa were downregulated without the generation of nPKC fragments. These results are consistent with the idea that nPKC isotypes meet two different fates, down-regulation and proteolytic cleavage generating kinase and regulatory fragments, and that the proteolytic cleavage of nPKC isotypes is a step in the signaling pathway involved in Fas-mediated and TPA-induced apoptosis.

Fas mediated apoptosis of human Jurkat T-cells: intracellular events and potentiation by redox-active α-lipoic acid

“…40 Also, transfection of recombinant protein coding for the catalytic fragment of PKCd results in the apoptotic morphology of cells and nuclei supporting that PKCd activity is indeed involved in mediating Fas mediated apoptosis. 26 In summary, this study provides first evidence showing that inducible caspase 3 activity may be pharmacologically up-regulated by intracellular reducing agents such as DHLA. Both mitochondria and PKCd are targets of caspase 3 activity.…”

Calcium and apoptosis: facts and hypotheses

“…A catalytically active fragment of PKC is generated by proteolysis in cells undergoing apoptosis in response to ionizing radiation, DNA-damaging drugs, and anti-Fas antibody (Emoto et al, 1995;Mizuno et al, 1997). As to the site of action, PKC was shown to translocate during apoptosis to mitochondria (Majumder et al, 2000), as well as to the Golgi complex (Emoto et al, 1995;Konishi et al, 1999), where it is activated through a tyrosine-phosphorylation step.…”

Protein kinase Cε makes the life and death decision

“…Several PKC isozymes, including PKCδ, -ε, -θ and -ζ are substrates for caspases [23][24][25][26][27]. Cleavage of PKCs by caspases at the hinge region causes cofactor-independent activation [28].…”