Abstract 18386: ROS-Induced Mitochondrial Translocation of Phosphatases Cancels Cell-Protective Signals Activated by Phosphorylation of Mitochondrial Protective Kinases
Background: Protein kinases localizing in mitochondria are thought to mediate pro-survival signaling. However, their regulatory mechanisms remain unclear. Here, we examined intra-mitochondrial localizations of pro-survival kinases and the role of reactive oxygen species (ROS) in their regulation.
Methods and Results: Mitochondria were isolated from HEK293 cells and incubated in trypsin at 1-1,000 μg/ml for stepwise digestion; trypsin eliminated TOM20, an outer membrane (OM) protein, and cytochrome oxidase IV, an inner membrane (IM) protein, at 1 and 1,000 μg/ml, respectively. We estimated the amounts of proteins in the OM and IM by Western blot analyses. At baseline, 62% and 22% of mitochondrial GSK-3β were localized in the OM and IM, respectively, the remaining 16% presumably being in the matrix. Sub-mitochondrial localization of ERK was similar to that of GSK-3β (53% in the OM and 25% in the IM), whereas Akt was mainly localized in the IM (41%), with 31% residing in the OM. Treatment with IGF-1 (50 nmol/L, 45 min) significantly increased mitochondrial GSK-3β, Akt and ERK by 35%, 83% and 86%, respectively, without marked change in their localization. The levels of protective Ser9-phospho-GSK-3β in both the OM and IM were significantly elevated by IGF-1 (47% and 60%, respectively). The levels of phospho-Akt in the OM and IM were also increased by IGF1 (174% and 76%, respectively, p<0.05 for the OM), but phosphorylation of ERK was modest (5% and 50%, respectively, p<0.05 for the IM). After careful cell fractionation, PHLPP-1 and DUSP5, phosphatases targeting Akt and ERK, respectively, were detected in the mitochondria. Both PHLPP-1 and DUSP5 were significantly increased in the OM (52% and 86%, respectively) after treatment with antimycin A (100 μmol/L, 30 min), a mitochondrial ROS inducer. Addition of antimycin A to IGF-1 eliminated phosphorylation of Akt, ERK and GSK-3β both in the OM and IM.
Conclusion: Since GSK-3β is a substrate of both Akt and ERK, the results suggest that protective Ser9-phosphorylation of GSK-3β in mitochondria is mediated by Akt and ERK in the OM and/or IM and that mitochondrial ROS cancel the protective signaling by mitochondrial recruitment of PHLPP-1 and DUSP5, which dephosphorylate Akt and ERK, respectively, in mitochondria.
Author Disclosures: W. Ohwada: None. M. Tanno: None. T. Yano: None. A. Kuno: None. T. Miki: None. S. Ishikawa: None. Y. Tatekoshi: None. K. Abe: None. K. Ohno: None. M. Mizuno: None. K. Nakata: None. T. Miura: None.
- © 2016 by American Heart Association, Inc.