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Survival-factor-induced phosphorylation of Bad results in its dissociation from Bcl-x(L) but not Bcl-2.

ABSTRACT: 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.

SUBMITTER: Hirai I

PROVIDER: S-EPMC1222152 | biostudies-other | 2001 Oct

REPOSITORIES: biostudies-other

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Project description:An estimated 40,000 deaths will be attributed to breast cancer in 2016, underscoring the need for improved therapies. Evading cell death is a major hallmark of cancer, driving tumor progression and therapeutic resistance. To evade apoptosis, cancers use antiapoptotic Bcl-2 proteins to bind to and neutralize apoptotic activators, such as Bim. Investigation of antiapoptotic Bcl-2 family members in clinical breast cancer datasets revealed greater expression and more frequent gene amplification of MCL1 as compared with BCL2 or BCL2L1 (Bcl-xL) across three major molecular breast cancer subtypes, Luminal (A and B), HER2-enriched, and Basal-like. While Mcl-1 protein expression was elevated in estrogen receptor α (ERα)-positive and ERα-negative tumors as compared with normal breast, Mcl-1 staining was higher in ERα+ tumors. Targeted Mcl-1 blockade using RNAi increased caspase-mediated cell death in ERα+ breast cancer cells, resulting in sustained growth inhibition. In contrast, combined blockade of Bcl-2 and Bcl-xL only transiently induced apoptosis, as cells rapidly acclimated through Mcl-1 upregulation and enhanced Mcl-1 activity, as measured in situ using Mcl-1/Bim proximity ligation assays. Importantly, MCL1 gene expression levels correlated inversely with sensitivity to pharmacologic Bcl-2/Bcl-xL inhibition in luminal breast cancer cells, whereas no relationship was seen between the gene expression of BCL2 or BCL2L1 and sensitivity to Bcl-2/Bcl-xL inhibition. These results demonstrate that breast cancers rapidly deploy Mcl-1 to promote cell survival, particularly when challenged with blockade of other Bcl-2 family members, warranting the continued development of Mcl-1-selective inhibitors for targeted tumor cell killing.Implications: Mcl-1 levels predict breast cancer response to inhibitors targeting other Bcl-2 family members, and demonstrate the key role played by Mcl-1 in resistance to this drug class. Mol Cancer Res; 15(3); 259-68. ©2016 AACR.

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Survival-factor-induced phosphorylation of Bad results in its dissociation from Bcl-x(L) but not Bcl-2.

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