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.
Highlights
Mitochondria are critical for energy generation and are recognized as gatekeepers in the control of cell survival
These results support our previous observations using leukemia inhibitory factor that Akt translocates to mitochondria upon activation and leads to increased mitochondrial Hexokinase II (HK-II) binding in cardiomyocytes
It has been demonstrated that an increased level of HK-II at mitochondria is protective and is increased by protective interventions but decreased under stress, it has not been fully determined which molecular signals regulate the level of HK-II at mitochondria
Summary
Cell Culture—Neonatal rat ventricular myocytes (NRVMs) were isolated from 1- to 2-day-old Sprague-Dawley rat pups using a kit (Worthington). Immunocomplexes were washed with ice-cold radioimmune precipitation assay buffer three times, spun down, resuspended in 2ϫ LDS sample buffer (Life Technology), and boiled for 5 min. For the in vitro phosphorylation experiment, immunocomplexes were washed with ice-cold radioimmune precipitation assay buffer and resuspended in kinase reaction buffer containing 20 mM Hepes, 2 mM DTT, and 5 mM MgCl2 with or without 200 M ATP and recombinant Akt (Millipore). Incubations were carried out at 30 °C for 20 min, spun down, resuspended in 2ϫ LDS sample buffer, boiled for 5 min, and subjected to Western blotting. Cells were loaded with 4 M Calcein Blue AM (eBioscience) for 30 min at room temperature, washed twice with DPBS containing 1 mM CaCl2 and 1 mM MgCl2, and fluorescence was detected using an Infinite M200PRO microplate reader (TECAN). Experiments with more than two groups were compared by analysis of variance followed by the Tukey posthoc test. p Ͻ 0.05 was considered statistically significant
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
