Research Papers:

Mitotic arrest-induced phosphorylation of Mcl-1 revisited using two-dimensional gel electrophoresis and phosphoproteomics: nine phosphorylation sites identified

Rong Chu, Sarah E. Alford, Katherine Hart, Anisha Kothari, Samuel G. Mackintosh, Matthew R. Kovak and Timothy C. Chambers _

PDF  |  HTML  |  Supplementary Files  |  How to cite  |  Order a Reprint

Oncotarget. 2016; 7:78958-78970. https://doi.org/10.18632/oncotarget.12586

Metrics: PDF 720 views  |   HTML 1129 views  |   ?  


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

Correspondence to:

Timothy Chambers, email: chamberstimothyc@uams.edu

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.

Creative Commons License All site content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 License.
PII: 12586