Background: Glycogen synthase kinase-3β (GSK-3β) in mitochondria has been shown to facilitate opening of the permeability transition pore, a trigger of cell death in diverse pathological conditions including myocardial ischemia/reperfusion. However, mechanisms of its translocation to mitochondria remain unresolved. Here we examined roles of interacting proteins for GSK3β to translocate to mitochondria and possible presence of a mitochondrial targeting sequence (MTS) in GSK-3β.
Methods and Results: H9c2 cardiomyocytes were transfected with EGFP-tagged GSK-3β (WT) or kinase-deficient GSK-3β (K85R). Forty-eight hours after transfection, the mitochondria were stained with MitoTracker Red. Observation by time-lapse microscopy (Eclipse Ti-E, Nikon) revealed that WT but not K85R translocated from the cytosol to the mitochondria and induced cell death after exposure to H2O2 (10 μM). The prevalence of apoptosis was significantly lower in K85R-transfected cells than in WT-transfected cells (22±4% vs. 31±5%). Two-dimensional gel electrophoresis of anti-GSK-3β-immunoprecipitates obtained from H9c2 cell lysates revealed that exposure to H2O2 increased the density of nine spots on the 2D-gel, one of which was shown to contain voltage-dependent anion channel (VDAC) 2 by TOF/MS analysis. Transfection of H9c2 cells with VDAC2-siRNA significantly attenuated mitochondrial translocation of WT in response to H2O2 challenge compared with transfection of control-siRNA (GSK-3β-positive mitochondria/total mitochondria=36±3% vs. 76±3%). Based on similarities to known mitochondrial targeting sequences (MTSs), we hypothesized that GSK-3β contains an MTS consisting of N-terminal 15 amino acid residues dotted with positively charged amino acid residues, R4, R6 and K15. To analyze the function of the possible MTS, one of these amino acid residues was replaced with alanine by site-directed mutagenesis. Among the mutants, K15A, but not R6A and R4A, showed significant attenuation in mitochondrial translocation after 5 min-exposure to H2O2 compared with WT (45±5%, 51±5%, 75±7% vs. 76±3%).
Conclusion: Kinase activity of GSK-3β and interaction with VDAC2 significantly promote translocation of GSK-3β to mitochondria. An N-terminal domain of GSK-3β, possibly as an MTS, may mediate its mitochondrial translocation in response to oxidative stress. We propose that suppression of mitochondrial translocation of GSK-3β by modifying interaction with VDAC2 and/or N-terminal MTS could be a novel therapeutic strategy to suppress ischemia/reperfusion injury.