BCL-2 DOES NOT REQUIRE RAF KINASE-ACTIVITY FOR ITS DEATH-PROTECTIVE FUNCTION

It has been widely accepted that the oncogene product bcl-2 protects m ammalian cells from programmed cell death (apoptosis). The molecules a nd signalling pathways upon which bcl-2 acts are, however, still ill-d efined. Recently, bcl-2 was shown to interact with c-raf-1 in vitro. F urthermore, an active form of c-raf-1 delayed apoptosis induced by tro phic factor deprivation and enhanced the death-suppressive function of bcl-2 when co-expressed. This has led to the hypothesis that bcl-2 co mmunicates cell-death protection via a raf-dependent signal transducti on pathway. Here we show, by various immunological and biochemical met hods, that bcl-2 does not stably associate with c-raf-1 in cellular ex tracts prepared from fibroblasts before or after treatment with agents that-induce apoptosis. Unexpectedly, bcl-2 function is entirely maint ained, if not improved, when raf-dependent signalling is experimentall y abrogated. In fact, bcl-2 allows the stable overexpression of a kina se-defective dominant-negative raf mutant that usually interferes with cell viability and/or proliferation. Our results indicate that bcl-2 does not require c-raf-1 kinase activity and an associated mitogen-act ivated protein kinase signalling pathway for its survival function. Th is property may be exploited to dissect cellular events that are depen dent or independent of c-raf-1 kinase activity.