Serine 64 phosphorylation enhances the antiapoptotic function of Mcl-1.
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 3beta) 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-alpha-related apoptosis-inducing ligand (TRAIL) treatment, was blocked by inhibitors of CDK (but not ERK or glycogen-synthase kinase 3beta), 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.
Kobayashi
Shogo
S
Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.
Lee
Sun-Hee
SH
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.
Meng
Xue W
XW
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; Division of Oncology Research, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.
Mott
Justin L
JL
Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.
Bronk
Steven F
SF
Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.
Werneburg
Nathan W
NW
Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.
Craig
Ruth W
RW
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755.
Kaufmann
Scott H
SH
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; Division of Oncology Research, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.
Gores
Gregory J
GJ
Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905. Electronic address: gores.gregory@mayo.edu.
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D002455:Cell Division; D045744:Cell Line, Tumor; D016195:G2 Phase; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000502:physiology*; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*; D012694:Serine / Q000737:chemistry*
D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; D012694:Serine; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3