BIM, a pro-apoptotic “BH3-only” member of the BCL-2 family, is required for apoptosis induced by chemotherapeutic drugs (i.e. Taxol, dexamethasone, and Gleevec). For instance, BIM down-regulation by gene-targeting or small interfering RNA (siRNA) shows strong resistance to treatment with these drugs. This indicates that BIM could be a key regulator in anticancer therapy against human malignancies. In this study, we focus on the post-translational modification of BIM and its effect with other BCL-2 family members. We used HeLa cells to study the precise mechanisms of BIM-mediated apoptosis by Taxol treatment. Down regulation of BIM by siRNA improves cell viability, confirming its importance. Taxol treatment results in phosphorylation of BIM prior to the onset of apoptosis. Using pharmacological inhibitors, inhibition of Jun N-terminal kinase (JNK) prevents BIM phosphorylation and reduces apoptosis, but the inhibition of extracellular signal-regulated kinase, p38-mitogen activated protein kinase or phosphoinositide 3-kinase has no effect. Transient expression of non-phosphorylatable mutations (Ser100, Thr112 and Ser114) eradicates the phosphorylation-specific BIM band-shift on SDS-PAGE. Most importantly, non-phosphorylatable mutations significantly curtail taxol-induced apoptosis. After Taxol treatment, the BIM/ anti-apoptotic protein MCL-1 interaction is dramatically decreased and the BIM/ multi-domain pro-apoptotic effector Bax interaction is increased, although the localization and overall protein expression levels of these proteins are constant. In contrast, a non-phosphorylatable BIM mutant treated by Taxol does not show significant change of the interaction with MCL-1 and Bax. These results demonstrate that phosphorylation of BIM is required for down-regulating the interaction with MCL-1 and up-regulating the interaction with Bax, and this regulation enhances the pro-apoptotic activity of BIM. The mechanisms of Taxol-induced BIM phosphorylation and the interaction with other Bcl-2 family members provide new insights into BIM regulation and function.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]