title: Translocation of glycogen synthase kinase-3β (GSK-3β), a trigger of permeability transition, is kinase activity-dependent and mediated by interaction with voltage-dependent anion channel 2 (VDAC2).
abstract: Glycogen synthase kinase-3β (GSK-3β) is a major positive regulator of the mitochondrial permeability transition pore (mPTP), a principle trigger of cell death, under the condition of oxidative stress. However, the mechanism by which cytosolic GSK-3β translocates to mitochondria, promoting mPTP opening, remains unclear. Here we addressed this issue by analyses of the effect of site-directed mutations in GSK-3β on mitochondrial translocation and protein/protein interactions upon oxidative stress. H9c2 cardiomyoblasts were transfected with GFP-tagged GSK-3β (WT), a mutant GSK-3β insensitive to inhibitory phosphorylation (S9A), or kinase-deficient GSK-3β (K85R). Time lapse observation revealed that WT and S9A translocated from the cytosol to the mitochondria more promptly than did K85R after exposure to oxidative stress. H2O2 increased the density of nine spots on two-dimensional gel electrophoresis of anti-GSK-3β-immunoprecipitates by more than 3-fold. MALDI-TOF/MS analysis revealed that one of the spots contained voltage-dependent anion channel 2 (VDAC2). Knockdown of VDAC2, but not VDAC1 or VDAC3, by siRNA attenuated both the mitochondrial translocation of GSK-3β and mPTP opening under stress conditions. The mitochondrial translocation of GSK-3β was attenuated also when Lys-15, but not Arg-4 or Arg-6, in the N-terminal domain of GSK-3β was replaced with alanine. The oxidative stress-induced mitochondrial translocation of GSK-3β was associated with an increase in cell death, which was suppressed by lithium chloride (LiCl), a GSK-3β inhibitor. These results demonstrate that GSK-3β translocates from the cytosol to mitochondria in a kinase activity- and VDAC2-dependent manner in which an N-terminal domain of GSK-3β may function as a mitochondrial targeting sequence.
authors.affiliation: From the Departments of Cardiovascular, Renal, and Metabolic Medicine and.
authors.forename: Masaya
authors.initials: M
authors.lastname: Tanno
authors.affiliation: From the Departments of Cardiovascular, Renal, and Metabolic Medicine and Pharmacology, Sapporo Medical University School of Medicine, S1 W16, Chuo-ku, Sapporo 060-8543, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kuno
authors.affiliation: From the Departments of Cardiovascular, Renal, and Metabolic Medicine and.
authors.forename: Satoko
authors.initials: S
authors.lastname: Ishikawa
authors.affiliation: From the Departments of Cardiovascular, Renal, and Metabolic Medicine and.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Miki
authors.affiliation: From the Departments of Cardiovascular, Renal, and Metabolic Medicine and.
authors.forename: Hidemichi
authors.initials: H
authors.lastname: Kouzu
authors.affiliation: From the Departments of Cardiovascular, Renal, and Metabolic Medicine and.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Yano
authors.affiliation: From the Departments of Cardiovascular, Renal, and Metabolic Medicine and.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Murase
authors.affiliation: From the Departments of Cardiovascular, Renal, and Metabolic Medicine and.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Tobisawa
authors.affiliation: From the Departments of Cardiovascular, Renal, and Metabolic Medicine and.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ogasawara
authors.affiliation: Pharmacology, Sapporo Medical University School of Medicine, S1 W16, Chuo-ku, Sapporo 060-8543, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Horio
authors.affiliation: From the Departments of Cardiovascular, Renal, and Metabolic Medicine and miura@sapmed.ac.jp.
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Miura
mesh_terms: D001692:Biological Transport; D016923:Cell Death; D003600:Cytosol / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D008928:Mitochondria / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D009336:Necrosis; D018384:Oxidative Stress; D010539:Permeability; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D050996:Voltage-Dependent Anion Channel 2 / Q000378:metabolism*
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D034741:RNA, Small Interfering; C494765:VDAC2 protein, human; D050996:Voltage-Dependent Anion Channel 2; D006861:Hydrogen Peroxide; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3
keywords: Glycogen Synthase Kinase 3 (GSK-3); Mitochondrial Permeability Transition (MPT); Mitochondrial Transport; Necrosis (Necrotic Death); Protein-Protein Interaction; Voltage-dependent Anion Channel 2