Survival-factor-induced phosphorylation of Bad results in its dissociation from Bcl-xL but not Bcl-2

Abstract

The pro-apoptotic Bcl-2-family protein Bad heterodimerizes with Bcl-2 and Bcl-xL 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-xL but not Bcl-2. Overexpression of Bad sensitized Bcl-xL-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-xL 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-xL, 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-xL were swapped with each other, the Bcl-xL(N)–Bcl-2 chimaeric protein (containing the N-terminal region of Bcl-xL) 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-xL 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-xL but not Bcl-2.