Akt Phosphorylates HK-II at Thr-473 and Increases Mitochondrial HK-II Association to Protect Cardiomyocytes

• Published in: The Journal of biological chemistry 2013-08, Vol.288 (33), p.23798-23806
• Main Authors: Roberts, David J., Tan-Sah, Valerie P., Smith, Jeffery M., Miyamoto, Shigeki
• Format: Article
• Language: English
• Subjects: Akt PKB; Animals; Animals, Newborn; Cardiomyocytes; Cell Death; Cytoprotection - drug effects; Enzyme Activation - drug effects; Glucose 6-Phosphate; Glucose-6-Phosphate - pharmacology; Hexokinase; Hexokinase - metabolism; Hydrogen Peroxide - pharmacology; Insulin-like Growth Factor (IGF); Insulin-Like Growth Factor I - pharmacology; Mice; Mitochondria; Mitochondria, Heart - drug effects; Mitochondria, Heart - enzymology; Models, Biological; Mutant Proteins - metabolism; Myocytes, Cardiac - cytology; Myocytes, Cardiac - drug effects; Myocytes, Cardiac - enzymology; Phosphorylation; Phosphorylation - drug effects; Phosphothreonine - metabolism; Protein Binding - drug effects; Proto-Oncogene Proteins c-akt - metabolism; Rats; Rats, Sprague-Dawley; Signal Transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.482026

Hexokinase II (HK-II) is an enzyme that catalyzes the first step in glycolysis and localizes not only in the cytosol but also at mitochondria. Akt, activated by insulin-like growth factor 1 (IGF-1) treatment in neonatal rat ventricular myocytes, translocates to mitochondria and increases mitochondrial HK-II binding. Expression of an HK-II-dissociating peptide diminished IGF-1-induced increases in mitochondrial HK-II as well as protection against hydrogen peroxide treatment, suggesting an important role of mitochondrial HK-II in IGF-1/Akt-mediated protection. We hypothesized, on the basis of an Akt phosphorylation consensus sequence present in HK-II, that Thr-473 is the target of Akt kinase activity. Indeed, recombinant kinase-active Akt robustly phosphorylates WT HK-II, but not Thr-473 mutants. Phosphomimetic (T473D)HK-II, but not non-phosphorylatable (T473A)HK-II, constitutively increased mitochondrial binding compared with WT HK-II and concomitantly confers greater protection against hydrogen peroxide. Glucose 6-phosphate (G-6P), a product of the catalytic activity of HK-II, is well known to dissociate HK-II from mitochondria. Addition of G-6P to isolated mitochondria dose-dependently dissociates WT HK-II, and this response is inhibited significantly in mitochondria isolated from cardiomyocytes expressing T473D HK-II. Pretreatment with IGF-1 also inhibits G-6P-induced overexpressed or endogenous HK-II dissociation, and this response was blocked by Akt inhibition. These results show that Akt phosphorylation of HK-II at Thr-473 is responsible for the Akt-mediated increase in HK-II binding to mitochondria. This increase is, at least in part, due to the decreased sensitivity to G-6P-induced dissociation. Thus, phosphorylation-mediated regulation of mitochondrial HK-II would be a critical component of the protective effect of Akt. Backgound: Hexokinase II binds to mitochondria and promotes cell survival. Results: Akt phosphorylates HK-II but not the threonine 473 mutant. The phosphomimetic T473D mutant decreases its dissociation from mitochondria induced by G-6P and increases cell viability against stress. Conclusion: Akt phosphorylates HK-II at Thr-473, resulting in increased mitochondrial HK-II and cell protection. Significance: The Akt-HK-II signaling nexus is important in cell survival.