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J. Biol. Chem., Vol. 282, Issue 25, 18407-18417, June 22, 2007

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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^¶, and Gregory J. Gores¹

From the Division of Gastroenterology and Hepatology, the Department of Molecular Pharmacology and Experimental Therapeutics, and the ^¶Division of Oncology Research, Mayo Clinic College of Medicine, Rochester, Minnesota 55905 and the ^||Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755

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 3) 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 G₂/M phase, was enhanced by tumor necrosis factor--related apoptosis-inducing ligand (TRAIL) treatment, was blocked by inhibitors of CDK (but not ERK or glycogen-synthase kinase 3), and was stimulated in vitro by CDK 1, 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.

Received for publication, October 25, 2006 , and in revised form, April 25, 2007.

^* This work was supported by National Institutes of Health Grants DK 59427 (to G. J. G.), CA 69008 (to S. H. K.), and CA 57359 (to R. W. C.) and funds from the Mayo and Palumbo Foundations (to G. J. G.) and the Japanese Society for Science Promotion (to S. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, 200 First St. SW, Rochester MN, 55905. Tel.: 507-284-0686; Fax: 507-284-0762; E-mail: gores.gregory{at}mayo.edu.

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