Serine 64 phosphorylation enhances the antiapoptotic function of Mcl-1

Shogo Kobayashi, Sun Hee Lee, Xue W. Meng, Justin L. Mott, Steven F. Bronk, Nathan W. Werneburg, Ruth W. Craig, Scott H. Kaufmann, Gregory J. Gores

Research output: Contribution to journalArticle

88 Scopus citations

Abstract

Mcl-1 is an antiapoptotic Bcl-2 family member that is highly regulated and when dysregulated contributes to cancer. The Mcl-1 protein is phosphorylated at multiple sites in response to different signaling events. Phosphorylations at Thr163 (by ERK) and Ser159 (by glycogen-synthase kinase 3β) have recently been shown to slow and enhance, respectively, Mcl-1 protein turnover. Phosphorylation is also known to be stimulated at other, as-yet uncharacterized sites in the G2/M phase of the cell cycle. Using an S peptide-tagged Mcl-1 T163A mutant, Ser64 was identified as a novel Mcl-1 phosphorylation site by mass spectrometry. Immunoblotting demonstrated that phosphorylation at this site was maximal in cells in G 2/M phase, was enhanced by tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) treatment, was blocked by inhibitors of CDK (but not ERK or glycogen-synthase kinase 3β), and was stimulated in vitro by CDK1, CDK2, and JNK1. The half-life of a nonphosphorylatable S64A Mcl-1 mutant was indistinguishable from that of the wild type polypeptide. In contrast, this mutant failed to protect cells from TRAIL-mediated apoptosis, whereas reconstitution with the phosphomimetic S64E Mcl-1 mutant rendered cells TRAIL resistant. This anti-apoptotic phenotype of the S64E Mcl-1 mutant was also associated with enhanced binding to the proapoptotic proteins Bim, Noxa, and Bak. A pharmacological CDK inhibitor that reduced Ser64 phosphorylation also sensitized cells to TRAIL cytotoxicity. Collectively, these observations not only identify G2/M-associated phosphorylation at Ser64 as a critical determinant of the antiapoptotic activity of Mcl-1 but also elucidate a novel mechanism by which CDK1/2 inhibitors can enhance the effectiveness of the cytotoxic cytokine TRAIL.

Original languageEnglish (US)
Pages (from-to)18407-18417
Number of pages11
JournalJournal of Biological Chemistry
Volume282
Issue number25
DOIs
StatePublished - Jun 22 2007

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Kobayashi, S., Lee, S. H., Meng, X. W., Mott, J. L., Bronk, S. F., Werneburg, N. W., Craig, R. W., Kaufmann, S. H., & Gores, G. J. (2007). Serine 64 phosphorylation enhances the antiapoptotic function of Mcl-1. Journal of Biological Chemistry, 282(25), 18407-18417. https://doi.org/10.1074/jbc.M610010200