Dai, Haiming

Abstract Bcl-2 is phosphorylated on Ser70 after treatment of cells with spindle poisons. On the basis of effects observed in cells overexpressing Bcl-2 S70E or S70A mutants, various studies have concluded that Ser70 phosphorylation either enhances or diminishes Bcl-2 function. In the present study, the ability of phosphorylated Bcl-2, as well as the S70E and S70A mutants, to bind and neutralize proapoptotic Bcl-2 family members under cell-free conditions and in intact cells was examined in an attempt to resolve this controversy. Surface plasmon resonance indicated that phosphorylated Bcl-2, Bcl-2 S70E, and Bcl-2 S70A exhibit enhanced binding to Bim and Bak compared with unmodified Bcl-2. This enhanced binding reflected a readily detectable conformation change in the loop domain of Bcl-2. Furthermore, Bcl-2 S70E and S70A bound more Bak and Bim than wild-type Bcl-2 in pull-downs and afforded greater protection against several chemotherapeutic agents. Importantly, binding of endogenous Bcl-2 to Bim also increased during mitosis, when Bcl-2 is endogenously phosphorylated, and disruption of this mitotic Bcl-2/Bim binding with navitoclax or ABT-199, like Bcl-2 downregulation, enhanced the cytotoxicity of paclitaxel. Collectively, these results provide not only a mechanistic basis for the enhanced antiapoptotic activity of phosphorylated Bcl-2, but also an explanation for the ability of BH3 mimetics to enhance taxane sensitivity. Cancer Res; 73(23); 6998–7008. ©2013 AACR.

The mechanism by which the proapoptotic Bcl-2 family members Bax and Bak release cytochrome c from mitochondria is incompletely understood. In this paper, we show that activator BH3-only proteins bind tightly but transiently to the Bak hydrophobic BH3-binding groove to induce Bak oligomerization, liposome permeabilization, mitochondrial cytochrome c release, and cell death. Analysis by surface plasmon resonance indicated that the initial binding of BH3-only proteins to Bak occurred with similar kinetics with or without detergent or mitochondrial lipids, but these reagents increase the strength of the Bak–BH3-only protein interaction. Point mutations in Bak and reciprocal mutations in the BH3-only proteins not only confirmed the identity of the interacting residues at the Bak–BH3-only protein interface but also demonstrated specificity of complex formation in vitro and in a cellular context. These observations indicate that transient protein–protein interactions involving the Bak BH3-binding groove initiate Bak oligomerization and activation.

Abstract The mechanism of cytotoxicity of farnesyltransferase inhibitors is incompletely understood and seems to vary depending on the cell type. To identify potential determinants of sensitivity or resistance for study in the accompanying clinical trial (Witzig et al, page 4882), we examined the mechanism of cytotoxicity of tipifarnib in human lymphoid cell lines. Based on initial experiments showing that Jurkat variants lacking Fas-associated death domain or procaspase-8 undergo tipifarnib-induced apoptosis, whereas cells lacking caspase-9 or overexpressing Bcl-2 do not, we examined changes in Bcl-2 family members. Tipifarnib caused dose-dependent up-regulation of Bim in lymphoid cell lines (Jurkat, Molt3, H9, DoHH2, and RL) that undergo tipifarnib-induced apoptosis but not in lines (SKW6.4 and Hs445) that resist tipifarnib-induced apoptosis. Further analysis demonstrated that increased Bim levels reflect inhibition of signaling from c-Raf to MEK1/2 and ERK1/2. Additional experiments showed that down-regulation of the Ras guanine nucleotide exchange factor RasGRP1 diminished tipifarnib sensitivity, suggesting that H-Ras or N-Ras is a critical farnesylation target upstream of c-Raf in lymphoid cells. These results not only trace a pathway through c-Raf to Bim that contributes to tipifarnib cytotoxicity in human lymphoid cells but also identify potential determinants of sensitivity to this agent.