Translocation of GSK-3{beta}, a Trigger of Permeability Transition, Is Kinase Activity-dependent and Mediated by Interaction with VDAC2 [Molecular Bases of Disease]

September 3rd, 2014 by Tanno, M., Kuno, A., Ishikawa, S., Miki, T., Kouzu, H., Yano, T., Murase, H., Tobisawa, T., Ogasawara, M., Horio, Y., Miura, T.

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 2-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 lys15, but not arg4 or arg6, in the N-terminal domain of GSK-3β was replaced with alanine. The oxidative stress-induced mitochondrial translocation of GSK-3β was associated with 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.
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