DEFECTS IN THE UBIQUITIN PATHWAY INDUCE CASPASE-INDEPENDENT APOPTOSISBLOCKED BY BCL-2

Apoptosis requires the activation of caspases (formerly interleukin 1 beta-converting enzyme-like proteases), in particular those related to the caspase-3/7/6 subfamily. Recent data, however, revealed that, alt hough caspase-specific inhibitors delay apoptosis, they are often inca pable of preventing it. To obtain evidence for caspase-independent ste ps 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 ind uced apoptosis independent of the activation of caspase-3 and -6 and t he cleavage of their respective substrates poly(ADP-ribose) polymerase and Iamin A. In addition, neither the caspase 3/7-specific inhibitor nzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone nor the general cas pase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone were capable of blocking this type of cell death. By contrast, Bcl-2 overe xpression effectively protected cells from apoptosis induced by a defe ct in the E1 enzyme at the nonpermissive temperature. Bcl-2 acted down stream 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 ubiqu itination. These results suggest the existence of short-lived proteins that may serve the role of caspase-independent effecters of apoptosis and attractive targets of the death-protective action of Bcl-2.