Defects in the ubiquitin pathway induce caspase-independent apoptosis blocked by Bcl-2.
Apoptosis requires the activation of caspases (formerly interleukin 1beta-converting enzyme-like proteases), in particular those related to the caspase-3/7/6 subfamily. Recent data, however, revealed that, although caspase-specific inhibitors delay apoptosis, they are often incapable of preventing it. To obtain evidence for caspase-independent steps of apoptosis, we artificially created a high amount of short-lived or aberrant proteins by blocking the ubiquitin degradation pathway. A temperature-sensitive defect in the ubiquitin-activating enzyme E1 induced apoptosis independent of the activation of caspase-3 and -6 and the cleavage of their respective substrates poly(ADP-ribose) polymerase and lamin A. In addition, neither the caspase 3/7-specific inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone nor the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone were capable of blocking this type of cell death. By contrast, Bcl-2 overexpression effectively protected cells from apoptosis induced by a defect in the E1 enzyme at the nonpermissive temperature. Bcl-2 acted downstream of the accumulation of short-lived or aberrant proteins because it did not prevent the overexpression of the short-lived proteins p53, p27(kip1), and cyclins D1 and B1 under conditions of decreased ubiquitination. These results suggest the existence of short-lived proteins that may serve the role of caspase-independent effectors of apoptosis and attractive targets of the death-protective action of Bcl-2.
Monney
L
L
Institute of Biochemistry, University of Fribourg, 1700 Fribourg, Switzerland.
Otter
I
I
Olivier
R
R
Ozer
H L
HL
Haas
A L
AL
Omura
S
S
Borner
C
C
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000473:pathology; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D019938:Cyclin D1 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D008025:Ligases / Q000172:deficiency* / Q000235:genetics; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases
D000590:Amino Acid Chloromethyl Ketones; C534700:Ccnb1 protein, mouse; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D019926:Cyclin B; D056744:Cyclin B1; D015853:Cysteine Proteinase Inhibitors; D008869:Microtubule-Associated Proteins; D009097:Multienzyme Complexes; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003513:Cycloheximide; D044767:Ubiquitin-Protein Ligases; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes