title: Akt phosphorylates HK-II at Thr-473 and increases mitochondrial HK-II association to protect cardiomyocytes.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, California 92093, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Roberts
authors.forename: Valerie P
authors.initials: VP
authors.lastname: Tan-Sah
authors.forename: Jeffery M
authors.initials: JM
authors.lastname: Smith
authors.forename: Shigeki
authors.initials: S
authors.lastname: Miyamoto
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D019610:Cytoprotection / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D019298:Glucose-6-Phosphate / Q000494:pharmacology; D006593:Hexokinase / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D051379:Mice; D008929:Mitochondria, Heart / Q000187:drug effects / Q000201:enzymology*; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D050505:Mutant Proteins; D010769:Phosphothreonine; D019298:Glucose-6-Phosphate; D007334:Insulin-Like Growth Factor I; D006861:Hydrogen Peroxide; D006593:Hexokinase; D051057:Proto-Oncogene Proteins c-akt
keywords: Akt PKB; Cardiomyocytes; Cell Death; Glucose 6-Phosphate; Hexokinase; Insulin-like Growth Factor (IGF); Mitochondria; Phosphorylation