Mitotic arrest-induced phosphorylation of Mcl-1 revisited using two-dimensional gel electrophoresis and phosphoproteomics: nine phosphorylation sites identified.
Mitotic arrest-induced phosphorylation of Mcl-1 revisited using two-dimensional gel electrophoresis and phosphoproteomics: nine phosphorylation sites identified.
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DOI：
10.18632/oncotarget.12586
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发表时间：
2016-11-29
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影响因子：
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通讯作者：
Chambers TC
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作者：
Chu R;Alford SE;Hart K;Kothari A;Mackintosh SG;Kovak MR;Chambers TC
微管靶向药物（MTAs）通常促进Mcl - 1的磷酸化和降解，这在有丝分裂死亡中是一个关键的促凋亡信号。虽然有几个磷酸化位点和激酶与有丝分裂阻滞诱导的Mcl - 1磷酸化有关，但一直缺乏全面的生化分析。与先前报道的Cdk1对T92的磷酸化调节Mcl - 1降解相反，在HeLa细胞中表达的T92A Mcl - 1突变体在长春碱处理后，其磷酸化和降解的动力学与野生型Mcl - 1相同。同样，当表达所有五个假定的Cdk位点（S64、T92、S121、S159、T163）都被丙氨酸替代的Mcl - 1时，它在长春碱作用下也会被磷酸化和降解。为了更详细地分析Mcl - 1的磷酸化，进行了二维凝胶电泳（2D - PAGE）。未处理的细胞主要表达未磷酸化的Mcl - 1以及两种少量的磷酸化形式，而来自长春碱处理细胞的Mcl - 1在2D - PAGE过程中迁移为一系列酸性斑点，代表九种或更多的磷酸化形式。对有丝分裂阻滞的HeLa细胞中磷酸化的Mcl - 1进行免疫纯化和质谱分析，揭示了九个不同的位点，包括几个先前未报道的位点。在基础条件下，所有九个位点都被替代的Mcl - 1比野生型Mcl - 1半衰期更长，但在长春碱处理下仍然会发生磷酸化和降解，并且像野生型Mcl - 1一样，无法保护细胞免受MTA处理。这些结果揭示了Mcl - 1在MTAs作用下磷酸化的意想不到的复杂性，并表明先前的研究严重低估了位点的数量，因此需要对当前模型进行重大修订。
Microtubule targeting agents (MTAs) characteristically promote phosphorylation and degradation of Mcl-1, and this represents a critical pro-apoptotic signal in mitotic death. While several phosphorylation sites and kinases have been implicated in mitotic arrest-induced Mcl-1 phosphorylation, a comprehensive biochemical analysis has been lacking. Contrary to previous reports suggesting that T92 phosphorylation by Cdk1 regulates Mcl-1 degradation, a T92A Mcl-1 mutant expressed in HeLa cells was phosphorylated and degraded with the same kinetics as wild-type Mcl-1 following vinblastine treatment. Similarly, when Mcl-1 with alanine replacements of all five putative Cdk sites (S64, T92, S121, S159, T163) was expressed, it was also phosphorylated and degraded in response to vinblastine. To analyze Mcl-1 phosphorylation in more detail, two-dimensional gel electrophoresis (2D-PAGE) was performed. While untreated cells expressed mainly unphosphorylated Mcl-1 with two minor phosphorylated species, Mcl-1 from vinblastine treated cells migrated during 2D-PAGE as a train of acidic spots representing nine or more phosphorylated species. Immunopurification and mass spectrometry of phosphorylated Mcl-1 derived from mitotically arrested HeLa cells revealed nine distinct sites, including several previously unreported. Mcl-1 bearing substitutions of all nine sites had a longer half-life than wild-type Mcl-1 under basal conditions, but still underwent phosphorylation and degradation in response to vinblastine treatment, and, like wild-type Mcl-1, was unable to protect cells from MTA treatment. These results reveal an unexpected complexity in Mcl-1 phosphorylation in response to MTAs and indicate that previous work has severely underestimated the number of sites, and thus encourage major revisions to the current model.