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
K. Mizuno et al., 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, European journal of biochemistry, 250(1), 1997, pp. 7-18
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.