Survival-factor-induced phosphorylation of Bad results in its dissociation from Bcl-x(L) but not Bcl-2.
The pro-apoptotic Bcl-2-family protein Bad heterodimerizes with Bcl-2 and Bcl-x(L) in the outer mitochondrial membranes, nullifying their anti-apoptotic activities and promoting cell death. We report that interleukin-3 (IL-3) stimulation induces Bad phosphorylation and triggers its translocation from mitochondria to cytoplasm in cells expressing Bcl-x(L) but not Bcl-2. Overexpression of Bad sensitized Bcl-x(L)-expressing FL5.12 cells to apoptosis induced by IL-3 deprivation, but had no effect on the viability of cells expressing Bcl-2. IL-3 stimulation induced Bad phosphorylation at Ser-112, impairing its binding to Bcl-x(L) and resulting in its association with 14-3-3 proteins in the cytosol. However, Ser-112 phosphorylation could not trigger Bad dissociation from mitochondria in FL5.12 cells expressing Bcl-2. In 293T cells expressing Bcl-x(L), Bad was phosphorylated at three serines, 112, 136 and 155, and was largely localized in the cytosolic fraction. In contrast, overexpression of Bcl-2 prevented phosphorylation of Bad at Ser-136 and Ser-155, sequestering this protein in the mitochondrial membranes. When the N-terminal regions of Bcl-2 and Bcl-x(L) were swapped with each other, the Bcl-x(L)(N)-Bcl-2 chimaeric protein (containing the N-terminal region of Bcl-x(L)) failed to prevent Bad phosphorylation in cells and was unable to block the cytosolic distribution of this pro-apoptotic protein. Additional experiments with the Bcl-2(N)-Bcl-x(L) chimaeric protein (containing the N-terminal region of Bcl-2) indicated that, although the N-terminal region of Bcl-2 is necessary, it is not sufficient for sequestering Bad in the mitochondrial membranes. These observations suggest that growth-factor-mediated phosphorylation of Bad contributes to the cytoprotective function of Bcl-x(L) but not Bcl-2.
Hirai
I
I
Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Department of Interdisciplinary Oncology, University of South Florida College of Medicine, 12902 Magnolia Drive, Tampa FL 33612, USA.
Wang
H G
HG
D048948:14-3-3 Proteins; D017209:Apoptosis / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D046911:Macromolecular Substances; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein
D048948:14-3-3 Proteins; C494819:BAD protein, human; C494811:BCL2L1 protein, human; D002352:Carrier Proteins; D007377:Interleukin-3; D046911:Macromolecular Substances; D019253:Proto-Oncogene Proteins c-bcl-2; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D012694:Serine; D014446:Tyrosine 3-Monooxygenase