Preservation of caspase-3 subunits from degradation contributes to apoptosis evoked by lactacystin: any single lysine or lysine pair of the small subunit is sufficient for ubiquitination.
Procaspase-3 (p32) is processed by upstream caspases to p12 and p20 subunits, which heterodimerize. Concomitant with formation of the active heterotetramer, p20 is autoprocessed to p17. Treatment of HL-60 cells with lactacystin, a selective inhibitor of the proteasome, exponentially increased caspase-3-like hydrolytic activity and induced apoptosis but had little or no effect on the activity of upstream caspase-8, caspase-9, or granzyme B. Lactacystin treatment decreased the p32 zymogen and evoked the accumulation of the p17 and p12 subunits. Treatment of transfected human retinoblast 911 cells with a proteasome inhibitor evoked the accumulation of epitope-tagged p12, p17, and p20 but had no effect on p32 zymogen. This result suggests that caspase-3 subunits, in contrast to the zymogen, are unstable because of degradation by the ubiquitin-proteasome system. Ubiquitin conjugates of p12 and p17 accumulated in cells that were cotransfected with p12 and a caspase inactive mutant of p17. Substitution of arginine for all eight lysines of p12 almost abolished its ubiquitination. Any single lysine or lysine pair was sufficient for p12 ubiquitination. Lactacystin treatment of HL-60 cells induced proteolytic processing of the X-linked inhibitor of apoptosis (XIAP) and decreased full-length XIAP, which is known to have ubiquitin-protein ligase activity for active caspase-3. These findings indicate that caspase-3 subunits can be degraded by the ubiquitin-proteasome system and suggest that lactacystin induces apoptosis in part by disabling the ubiquitin-protein ligase function of XIAP and by stabilizing active caspase-3 subunits.
Chen
Lei
L
Department of Pharmacology and Toxicology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294-0019, USA.
Smith
Lucinda
L
Wang
Zhi
Z
Smith
Jeffrey B
JB
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D017209:Apoptosis; D001120:Arginine / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002461:Cell Line, Transformed; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D008239:Lysine / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D051636:X-Linked Inhibitor of Apoptosis Protein
D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; D009097:Multienzyme Complexes; D021122:Protein Subunits; D011506:Proteins; D025801:Ubiquitin; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C067713:lactacystin; D001120:Arginine; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008239:Lysine; D000111:Acetylcysteine