Bcl-2, Raf-1 and mitochondrial regulation of apoptosis

Raf-1 kinase was shown to bind via its catalytic domain (Cat) to Bcl-2 in a BH4 domain-dependent manner. Using a green fluorescent protein (GFP)- Raf-1 (Cat) fusion protein, Bcl-2 but not Bcl-2(ΔBH4) was found to target Raf-1 to mitochondria in cells. Targeting Raf-1 (Cat) to mitochondrial membranes by fusing with the transmembrane domain of an outer mitochondrial membrane protein protected cells from apoptosis and resulted in phosphorylation of BAD protein, whereas plasma-membrane targeted Raf-1 failed to phosphorylate BAD and did not protect against cell death. Moreover, a Bcl- 2 binding protein, BAG-1, was shown to not only bind Raf-1 but also increase the activity of this kinase through a protein-protein interaction. The findings suggest that Bcl-2 targets Raf-1 to mitochondria, allowing this kinase to contribute to cellular survival by phosphorylating BAD or possibly other protein substrates in the vicinity of Bcl-2.