Akt phosphorylates HK-II at Thr-473 and increases mitochondrial HK-II association to protect cardiomyocytes.
Akt phosphorylates HK-II at Thr-473 and increases mitochondrial HK-II association to protect cardiomyocytes.
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DOI：
10.1074/jbc.m113.482026
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发表时间：
2013-08-16
期刊：
影响因子：
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通讯作者：
Miyamoto S
中科院分区：
文献类型：
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作者：
Roberts DJ;Tan-Sah VP;Smith JM;Miyamoto S
背景：己糖激酶II与线粒体结合，促进细胞存活。结果：AKT可使HK-II磷酸化，但不能使苏氨酸473突变体磷酸化。拟磷突变体T473D减少了G-6P诱导的线粒体解离，提高了细胞在应激状态下的存活率。结论：AKT在Thr-473位使HK-II磷酸化，导致线粒体HK-II增强，细胞保护作用增强。意义：Akt-HK-II信号通路在细胞存活中起重要作用。己糖激酶II(HK-II)是一种催化糖酵解第一步的酶，它不仅定位于胞浆中，还定位于线粒体中。胰岛素样生长因子-1(IGF-1)激活的AKT移位到线粒体，增加线粒体HK-II的结合。HK-II解离肽的表达降低了IGF-1诱导的线粒体HK-II的增加以及对过氧化氢处理的保护作用，提示线粒体HK-II在IGF-1/Akt介导的保护中起重要作用。根据HK-II中存在的Akt磷酸化共识序列，我们推测Thr-473是Akt激酶活性的靶标。事实上，重组激酶活性Akt强效地使WTHK-II磷酸化，但不能使Thr-473突变体磷酸化。与WT HK-II相比，拟磷酸化的(T473D)HK-II，但不是非磷酸化的(T473A)HK-II，结构性地增加了线粒体的结合，并伴随着对过氧化氢的更大保护。葡萄糖6-磷酸(G-6P)是HK-II催化活性的产物，能将HK-II从线粒体中解离出来。在分离的线粒体中加入G-6P呈剂量依赖性地解离WT HK-II，并且这种反应在表达T473D HK-II的心肌细胞的线粒体中被显著抑制。IGF-1可抑制G-6P诱导的细胞过度表达或内源性HK-II解离，这种作用可被Akt抑制所阻断。这些结果表明，Akt对Thr-473位HK-II的磷酸化是Akt介导的HK-II与线粒体结合增加的原因。这种增加至少部分是由于对G-6P诱导的解离的敏感性降低。因此，线粒体HK-II的磷酸化调节可能是Akt保护作用的重要组成部分。
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. 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.