Abstract
Microtubule inhibitors promote phosphorylation and degradation of Mcl-1, and the loss of expression of this key anti-apoptotic Bcl-2 family member appears to be critical for apoptosis induction. While several sites and kinases have been implicated, a phosphorylation/degradation-resistant mutant has not been definitively described. Contrary to previous reports suggesting that Thr92 phosphorylation by Cdk1 regulates Mcl-1 degradation, a Thr92Ala Mcl-1 mutant expressed in HeLa cells was phosphorylated and degraded with the same kinetics as wild-type following vinblastine-treatment. Similarly, when Mcl-1 with alanine replacements of five putative major sites (S64, T70A, T92, S121, T163) was expressed, it was phosphorylated and degraded like the wild-type. These results suggested redundancy or the existence of additional regulatory sites. To analyze Mcl-1 phosphorylation in more detail, cell extracts, made using a novel extraction solution to improve resolution, were subjected to 2D-PAGE/immunoblotting. Extracts from asynchronous control cells showed one major and two minor, more acidic, spots, the latter sensitive to acid phosphatase treatment. Extracts from vinblastine-treated cells showed up to 9 distinct spots all but two of which were eliminated after acid phosphatase treatment. These results reveal an unexpected complexity in Mcl-1 phosphorylation in response to microtubule inhibitors and the existence of many more sites than previously suspected. Identification of the additional sites of phosphorylation, the kinase(s) responsible, and their role in regulation of Mcl-1 degradation are ongoing.
Supported by NIH CA109821.
Citation Format: Rong Chu, Timothy C. Chambers. Unexpected complexity in Mcl-1 phosphorylation in response to microtubule inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1739. doi:10.1158/1538-7445.AM2013-1739
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