Mitotic arrest-induced phosphorylation of Mcl-1 revisited using two-dimensional gel electrophoresis and phosphoproteomics: nine phosphorylation sites identified
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Rong Chu1,*, Sarah E. Alford1,*, Katherine Hart1, Anisha Kothari1, Samuel G. Mackintosh1, Matthew R. Kovak1, Timothy C. Chambers1
1Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
*These authors have contributed equally to the work
Timothy Chambers, email: firstname.lastname@example.org
Keywords: Mcl-1, phosphorylation sites, mitotic arrest
Abbreviations: MTA, microtubule targeting agent; Cdk, cyclin-dependent kinase; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; MS, mass spectrometry
Received: April 04, 2016 Accepted: September 26, 2016 Published: October 12, 2016
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.
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