Apoptosis of Mo7e leukemia cells is associated with the cleavage of Bcl-2 into a shortened fragment that is not functional for heterodimerization with Bcl-2 and Bax

Bcl-2 is an integral intracellular membrane protein that can protect cells from apoptosis induced by multiple insults in a variety of cell types. Duri ng apoptosis, Bcl-2 was cleaved into a shortened fragment (Bcl-2/Delta 34) by a caspase-3-like protease in human Mo7e megakaryocytic leukemia cells de prived of exogenous rhGM-CSF. Results from cell fractionation and immunoblo t analyses indicated that both Bcl-2 and Bcl-2/Delta 34 were located exclus ively on the mitochondria of Mo7e cells. Treatment of isolated mitochondria with recombinant caspase-3 induced the same cleavage of Bcl-2 in vitro and caused the release of cytochrome c from the mitochondria into the supernat ant. The antiapoptotic effect of Bcl-2/Delta 34 was investigated using an i n vitro protein translation approach. Both Bcl-2/Delta 34 and Bax proteins generated in wheat germ extract were readily relocated to the mitochondria isolated from control Mo7e cells. Insertion of Bax, but not Bcl2/Delta 34, into mitochondria triggered a rapid release of cytochrome c from the mitoch ondria. Coimmunoprecipitation studies showed that, unlike Bcl-2, the cleave d Bcl-2 fragment was no longer functional for dimerization with either Bcl- 2 or Bax. Taken together, these findings showed that the integrity of Bcl-2 is necessary for its function of heterodimerization with Bax, which appear s to be one of the mechanisms of antiapoptotic effect of Bcl-2. (C) 2000 Ac ademic Press.