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 mammalian cells from programmed cell death (apoptosis). The molecules and signalling pathways upon which bcl-2 acts are, however, still ill-defined. Recently, bcl-2 was shown to interact with c-raf-1 in vitro. Furthermore, an active form of c-raf-1 delayed apoptosis induced by trophic factor deprivation and enhanced the death-suppressive function of bcl-2 when co-expressed. This has led to the hypothesis that bcl-2 communicates cell-death protection via a raf-dependent signal transduction pathway. Here we show, by various immunological and biochemical methods, that bcl-2 does not stably associate with c-raf-1 in cellular extracts prepared from fibroblasts before or after treatment with agents that induce apoptosis. Unexpectedly, bcl-2 function is entirely maintained, if not improved, when raf-dependent signalling is experimentally abrogated. In fact, bcl-2 allows the stable overexpression of a kinase-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-activated protein kinase signalling pathway for its survival function. This property may be exploited to dissect cellular events that are dependent or independent of c-raf-1 kinase activity.
Olivier
R
R
Institute of Biochemistry, University of Fribourg, Rue du Musee 5, CH-1700 Fribourg.
Otter
I
I
Monney
L
L
Wartmann
M
M
Borner
C
C
D000818:Animals; D017209:Apoptosis; D020126:Brefeldin A; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology; D003517:Cyclopentanes / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts; D006801:Humans; D007700:Kinetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection
D003517:Cyclopentanes; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D020126:Brefeldin A; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019311:Staurosporine