Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death.
Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death.
复制标题
DOI：
10.1038/cddis.2013.360
复制
发表时间：
2013-10-03
影响因子：
9
通讯作者：
中科院分区：
文献类型：
--
作者：
微管抑制剂（MIAs）通常会诱导主要的抗凋亡Bcl - 2家族成员Mcl - 1、Bcl - 2和Bcl - xL磷酸化，尽管这会导致Mcl - 1降解，但Bcl - 2/Bcl - xL磷酸化在有丝分裂死亡中的作用一直存在争议。这部分是由于癌细胞系之间对MIA的敏感性存在差异、细胞命运对药物浓度的依赖性以及所发生的细胞死亡方式的不确定性，从而使得对已发表报告进行比较变得困难。为了规避与MIAs相关的问题，我们使用后期促进复合物激活因子Cdc20的小干扰RNA（siRNA）敲低作为一个明确的分子系统，来研究单个抗凋亡Bcl - 2蛋白及其磷酸化形式在有丝分裂死亡中的具体作用。我们发现，在HeLa细胞中敲低Cdc20会诱导有丝分裂停滞以及随后的有丝分裂死亡。在稳定过表达无标签野生型Bcl - 2、Bcl - xL或Mcl - 1的HeLa细胞中敲低Cdc20，会促进过表达的蛋白与其内源性对应物同时磷酸化。Bcl - 2或Bcl - xL的过表达会阻断由Cdc20敲低诱导的有丝分裂死亡；磷酸化缺陷型突变体比野生型蛋白更具保护性，而磷酸化模拟型Bcl - xL无法阻断有丝分裂死亡。过表达的Mcl - 1无法防止Cdc20 siRNA介导的死亡，因为过表达的蛋白与内源性Mcl - 1类似，容易发生降解。这些结果提供了令人信服的证据，表明抗凋亡Bcl - 2蛋白的磷酸化在有丝分裂死亡的调控中具有关键作用。这些发现对我们理解MIAs的分子作用机制做出了重要贡献，这对其在临床上的合理使用至关重要。
Microtubule inhibiting agents (MIAs) characteristically induce phosphorylation of the major anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-xL, and although this leads to Mcl-1 degradation, the role of Bcl-2/Bcl-xL phosphorylation in mitotic death has remained controversial. This is in part due to variation in MIA sensitivity among cancer cell lines, the dependency of cell fate on drug concentration and uncertainty about the modes of cell death occurring, thus making comparisons of published reports difficult. To circumvent problems associated with MIAs, we used siRNA knockdown of the anaphase-promoting complex activator, Cdc20, as a defined molecular system to investigate the role, specifically in mitotic death, of individual anti-apoptotic Bcl-2 proteins and their phosphorylated forms. We show that Cdc20 knockdown in HeLa cells induces mitotic arrest and subsequent mitotic death. Knockdown of Cdc20 in HeLa cells stably overexpressing untagged wild-type Bcl-2, Bcl-xL or Mcl-1 promoted phosphorylation of the overexpressed proteins in parallel with their endogenous counterparts. Overexpression of Bcl-2 or Bcl-xL blocked mitotic death induced by Cdc20 knockdown; phospho-defective mutants were more protective than wild-type proteins, and phospho-mimic Bcl-xL was unable to block mitotic death. Overexpressed Mcl-1 failed to protect from Cdc20 siRNA-mediated death, as the overexpressed protein was susceptible to degradation similar to endogenous Mcl-1. These results provide compelling evidence that phosphorylation of anti-apoptotic Bcl-2 proteins has a critical role in regulation of mitotic death. These findings make an important contribution toward our understanding of the molecular mechanisms of action of MIAs, which is critical for their rational use clinically.
登录
查看更多内容
影响因子：
2.9
作者：
Skube, Susan B.;Chaverri, Jose M.;Goodson, Holly V.
通讯作者：
Goodson, Holly V.
影响因子：
5.8
作者：
Chu, Rong;Terrano, David T.;Chambers, Timothy C.
通讯作者：
Chambers, Timothy C.
影响因子：
8
作者：
Hernandez-Vargas, H.;Palacios, J.;Moreno-Bueno, G.
通讯作者：
Moreno-Bueno, G.
影响因子：
2.7
作者：
Shitashige, M;Toi, M;Shibasaki, F
通讯作者：
Shibasaki, F
影响因子：
5.8
作者：
Russo, Maria;Spagnuolo, Carmela;Russo, Gian Luigi
通讯作者：
Russo, Gian Luigi