Contribution of Bcl-2 phosphorylation to Bak binding and drug resistance.
Contribution of Bcl-2 phosphorylation to Bak binding and drug resistance.
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DOI：
10.1158/0008-5472.can-13-0940
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发表时间：
2013-12-01
期刊：
影响因子：
11.2
通讯作者：
Kaufmann SH
中科院分区：
文献类型：
--
作者：
Dai H;Ding H;Meng XW;Lee SH;Schneider PA;Kaufmann SH
Bcl-2在用纺锤体毒物处理细胞后在Ser70上磷酸化。基于过表达Bcl-2 S70E或S70A突变体的细胞效应，各种研究得出结论，Ser70磷酸化增强或减弱Bcl-2功能。在本研究中，磷酸化Bcl-2，以及S70E和S70A突变体，结合和中和促凋亡Bcl-2家族成员在无细胞条件下，在完整的细胞中的能力进行了检查，试图解决这一争议。表面等离子体共振表明，与未修饰的Bcl-2相比，磷酸化的Bcl-2、Bcl-2 S70 E和Bcl-2 S70 A表现出增强的与Bim和巴克的结合。这种增强的结合反映了Bcl-2环结构域中容易检测到的构象变化。此外，Bcl-2 S70 E和S70 A在下拉中比野生型Bcl-2结合更多的巴克和Bim，并提供针对几种化疗剂的更大保护。重要的是，在有丝分裂期间，当Bcl-2内源性磷酸化时，内源性Bcl-2与Bim的结合也增加;并且用navitoclax或ABT-199破坏这种有丝分裂Bcl-2/Bim结合，如Bcl-2下调，增强紫杉醇的细胞毒性。总的来说，这些结果不仅为磷酸化Bcl-2的增强的抗凋亡活性提供了机制基础，而且还为BH 3模拟物增强紫杉烷敏感性的能力提供了解释。
Bcl-2 is phosphorylated on Ser70 after treatment of cells with spindle poisons. Based on the cellular effects of 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 to unmodified Bcl-2. This enhanced binding reflected a readily detectable conformation change in the loop domain of Bcl-2. Further, Bcl-2 S70E and S70A bound more Bak and Bim than wildtype Bcl-2 in pulldowns 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 anti-apoptotic activity of phosphorylated Bcl-2, but also an explanation for the ability of BH3 mimetics to enhance taxane sensitivity.