Activation of PKCalpha downstream from caspases during apoptosis induced by 7-hydroxystaurosporine or the topoisomerase inhibitors, camptothecin and etoposide, in human myeloid leukemia HL60 cells.
We previously demonstrated that the anticancer agent and protein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) induces apoptosis independently of p53 and protein synthesis in HL60 cells. We now report the associated changes of PKC isoforms. PKCalpha, betaI, betaII, delta, and zeta activities were measured after immunoprecipitation of cytosols from UCN-01-treated HL60 cells. UCN-01 had no effect on PKCzeta and inhibited kinase activity of PKCbetaI, betaII, and delta. PKCalpha activity was initially inhibited at 1 h, and subsequently increased as cells underwent apoptosis 3 h after the beginning of UCN-01 treatment. Camptothecin (CPT) and etoposide (VP-16) also markedly enhanced PKCalpha activity during apoptosis in HL60 cells. However, CPT did not affect PKCbetaI, betaII and zeta, and activated PKCdelta. PKCalpha activation was not due to increased protein levels or proteolytic cleavage but was associated with PKCalpha autophosphorylation in vitro and increased phosphorylation in vivo. We also found that not only PKC delta but also PKC betaI was proteolytically activated in HL60 cells during apoptosis. The PKCalpha activation and hyperphosphorylation were abrogated by N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk) under conditions that abrogated apoptosis. z-VAD-fmk also prevented PKCdelta and betaI proteolytic activation. Together these findings suggest that caspases regulate PKC activity during apoptosis in HL60 cells. At least two modes of activation were observed: hyperphosphorylation for PKCalpha and proteolytic activation for PKC delta and betaI.
Shao
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RG
Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Cao
C X
CX
Pommier
Y
Y
D000470:Alkaloids / Q000494:pharmacology*; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002166:Camptothecin / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D019311:Staurosporine / Q000031:analogs & derivatives; D015854:Up-Regulation
D000470:Alkaloids; D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; D000972:Antineoplastic Agents, Phytogenic; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D005047:Etoposide; C054852:7-hydroxystaurosporine; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D003546:Cysteine Endopeptidases; D019311:Staurosporine; D002166:Camptothecin