title: Mitotic arrest-induced phosphorylation of Mcl-1 revisited using two-dimensional gel electrophoresis and phosphoproteomics: nine phosphorylation sites identified.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Chu
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Alford
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Hart
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Anisha
authors.initials: A
authors.lastname: Kothari
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Samuel G
authors.initials: SG
authors.lastname: Mackintosh
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Kovak
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Chambers
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D016203:CDC2 Protein Kinase / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008938:Mitosis / Q000187:drug effects; D009154:Mutation; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D050257:Tubulin Modulators / Q000494:pharmacology; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D014747:Vinblastine / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; C579095:MCL1 protein, human; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D050257:Tubulin Modulators; D014747:Vinblastine; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human
keywords: Mcl-1; mitotic arrest; phosphorylation sites