Caspase 3 specifically cleaves p21WAF1/CIP1 in the earlier stage of apoptosis in SK-HEP-1 human hepatoma cells.
We report here that p21WAF1/CIP1, an inhibitor of cyclin kinases, underwent proteolytic processing into a smaller fragment, p14, in the early stage of apoptosis in SK-HEP-1 cells. Apoptosis was induced by either staurosporine or ginsenoside Rh2, a ginseng saponin with a dammarane skeleton. Proteolytic processing was the result of caspase-3 activity, which accompanied the early changes in cell morphology and DNA fragmentation. p21WAF1/CIP1 translated in vitro was cleaved into a p14 fragment when incubated with cell extracts obtained from either ginsenoside Rh2-treated or staurosporine-treated cells. Cleavage was equally inhibited in both cases by adding Ac-DEVD-CHO, a specific caspase-3 inhibitor, but not by Ac-YVAD-CHO, a specific caspase-1 inhibitor. Similarly, p21WAF1/CIP1 was efficiently cleaved by recombinant caspase-3, overexpressed in Escherichia coli. Moreover, the endogenous p21WAF1/CIP1 of untreated cell extracts was also cleaved by recombinant caspase 3, as measured by immunoblotting. Mutation analysis allowed identification of two caspase-3 cleavage sites, DHVD112/L and SMTD149/F, which are located within or near the interaction domains for cyclins, Cdks, and proliferating cell nuclear antigen (PCNA). Taken together, these results show that ginsenoside Rh2 and staurosporine increase caspase-3 activity, which in turn directly cleaves p21WAF1/CIP1 during the early stages of apoptosis. We propose that proteolytic cleavage of p21WAF1/CIP1 is a functionally relevant event that allows release of the cyclin/Cdk complex from the p21WAF1/CIP1 inhibitor, resulting in the elevated levels of cyclin/Cdk kinase activity seen in the earlier stage of apoptosis.
Park
J A
JA
College of Pharmacy, Seoul National University, Korea.
Kim
K W
KW
Kim
S I
SI
Lee
S K
SK
D000595:Amino Acid Sequence; D017209:Apoptosis; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D036145:Ginsenosides; D006801:Humans; D006868:Hydrolysis; D008113:Liver Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D008969:Molecular Sequence Data; D012503:Saponins / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D019311:Staurosporine / Q000494:pharmacology; D014407:Tumor Cells, Cultured
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D017931:DNA Primers; D004791:Enzyme Inhibitors; D036145:Ginsenosides; D012503:Saponins; C055305:ginsenoside Rh2; D018844:Cyclin-Dependent Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine