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 James Gores

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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

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Phosphorylation
Serine
Apoptosis
Tumor Necrosis Factor-alpha
Ligands
Glycogen Synthase Kinase 3
Cell Division
Noxae
Gastrin-Secreting Cells
Peptides
Proteins
G2 Phase
Cytotoxicity
Immunoblotting
Mass spectrometry
Half-Life
Mass Spectrometry
Cell Cycle
Cells
Pharmacology

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kobayashi, S., Lee, S. H., Meng, X. W., Mott, J. L., Bronk, S. F., Werneburg, N. W., ... 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

Serine 64 phosphorylation enhances the antiapoptotic function of Mcl-1. / Kobayashi, Shogo; Lee, Sun Hee; Meng, Xue W.; Mott, Justin L.; Bronk, Steven F.; Werneburg, Nathan W.; Craig, Ruth W.; Kaufmann, Scott H; Gores, Gregory James.

In: Journal of Biological Chemistry, Vol. 282, No. 25, 22.06.2007, p. 18407-18417.

Research output: Contribution to journalArticle

Kobayashi, S, Lee, SH, Meng, XW, Mott, JL, Bronk, SF, Werneburg, NW, Craig, RW, Kaufmann, SH & Gores, GJ 2007, 'Serine 64 phosphorylation enhances the antiapoptotic function of Mcl-1', Journal of Biological Chemistry, vol. 282, no. 25, pp. 18407-18417. https://doi.org/10.1074/jbc.M610010200
Kobayashi S, Lee SH, Meng XW, Mott JL, Bronk SF, Werneburg NW et al. Serine 64 phosphorylation enhances the antiapoptotic function of Mcl-1. Journal of Biological Chemistry. 2007 Jun 22;282(25):18407-18417. https://doi.org/10.1074/jbc.M610010200
Kobayashi, Shogo ; Lee, Sun Hee ; Meng, Xue W. ; Mott, Justin L. ; Bronk, Steven F. ; Werneburg, Nathan W. ; Craig, Ruth W. ; Kaufmann, Scott H ; Gores, Gregory James. / Serine 64 phosphorylation enhances the antiapoptotic function of Mcl-1. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 25. pp. 18407-18417.
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