Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c

Caspases are cysteine proteases that mediate apoptosis by proteolysis of sp ecific substrates, Although many caspase substrates have been identified, f or most substrates the physiologic caspase(s) required for cleavage is unkn own. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by c aspases during apoptosis and by recombinant caspase-3 in vitro. In the pres ent study, we show that endogenous caspase-3 is a physiologic caspase for B cl-2, Apoptotic extracts from 293 cells cleave Bcl-2 but not Bar, even thou gh Bar is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizi ng radiation. In contrast to Bcl-2, cleavage of Bar was only partially bloc ked by caspase inhibitors. Inhibitor profiles indicate that Bar may be clea ved by more than one type of noncaspase protease, Immunodepletion of caspas e-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas im munodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MC F-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following s taurosporine-induced cell death. However, transient transfection of caspase -3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleav age of Bcl-2 requires activation of caspase-3, When the pro-apoptotic caspa se cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells , it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to pr omote further caspase activation as part of a positive feedback loop for ex ecuting the cell.