Abstract 103: Mechanisms Underlying Mitochondrial Translocation Of GSK-3β, A Crucial Inducer Of Mitochondrial Permeability Transition: GSK-3β Activity, Interaction With VDAC2 And A Mitochondrial Targeting Sequence.

Background: Although glycogen synthase kinase-3β (GSK-3β) is mainly localized in the cytosol in cardiomyocytes, mitochondrial GSK-3β has been shown to trigger permeability transition and cell death. In this study, we examined molecular mechanisms of the mitochondrial translocation of GSK-3β. Methods and Results: H9c2 cells were transfected with EGFP-tagged GSK-3β (WT) or kinase-deficient GSK-3β (K85R) and stained with MitoTracker Red. Observation by time-lapse microscopy (Eclipse Ti-E, Nikon) revealed that WT, but not K85R, translocated from the cytosol to mitochondria and induced cell death after exposure to H2O2. Two-dimensional gel electrophoresis of anti-GSK-3β-immunoprecipitates obtained from H9c2 cell lysates indicated that exposure to H2O2 down-regulated 4 spots and up-regulated 9 spots. LC/MS analysis revealed that one of the up-regulated spots contained voltage-dependent anion channel (VDAC) 2. Transfection of H9c2 cells with VDAC2-siRNA attenuated mitochondrial translocation of WT in response to H2O2 challenge (% of GSK-3β-positive mitochondria to total mitochondria =36±3% vs. 76±3%, p<0.05). Furthermore, observation with a super-resolution microscope (N-SIM, Nikon) revealed that knockdown of VDAC2 reduced mitochondrial swelling and fragmentation caused by repetitive laser exposure compared with control cells (78±3% vs. 53±12%, p<0.05). 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 test this hypothesis, one of these amino acid residues was replaced with alanine. R6A and K15A, but not R4A, showed significant attenuation in mitochondrial translocation after exposure to H2O2 compared with WT (51±5%, 45±5%, 75±7% vs. 76±3%, respectively). Conclusion: Kinase activity of GSK-3β and interaction with VDAC2 significantly promote translocation of GSK-3β to mitochondria, contributing to cell death. An N-terminal domain of GSK-3β, possibly as an MTS, mediates its mitochondrial translocation in response to oxidative stress.