Glycogen debranching enzyme association with beta-subunit regulates AMP-activated protein kinase activity.
Glycogen debranching enzyme association with beta-subunit regulates AMP-activated protein kinase activity.
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DOI：
10.1152/ajpendo.00003.2005
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发表时间：
2005-05
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通讯作者：
H. Sakoda;M. Fujishiro;Junko Fujio;N. Shojima;T. Ogihara;A. Kushiyama;Y. Fukushima;M. Anai;Hiraku Ono;M. Kikuchi;N. Horike;A. Viana;Y. Uchijima;H. Kurihara;T. Asano
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作者：
H. Sakoda;M. Fujishiro;Junko Fujio;N. Shojima;T. Ogihara;A. Kushiyama;Y. Fukushima;M. Anai;Hiraku Ono;M. Kikuchi;N. Horike;A. Viana;Y. Uchijima;H. Kurihara;T. Asano
腺苷酸活化蛋白激酶（AMPK）作为细胞内的能量传感器调节糖原和脂质代谢。在这项研究中，我们确定了一个160 kDa的蛋白在小鼠骨骼肌裂解液中使用谷胱甘肽-S-转移酶（GST）-AMPK融合蛋白下拉分析。质谱分析和Mascot搜索显示该蛋白质是糖原脱支酶（GDE）。AMPK与GDE之间的联系不仅在过表达系统中观察到，而且在内源性方面也观察到。接下来，我们显示AMPK的β 1亚基负责与GDE的关联。此外，使用AMPK β 1亚基缺失突变体的实验显示β 1亚基的氨基酸68-123足以与GDE结合。据报道，W100 G和K128 Q都是β 1亚基突变体，不能结合糖原，但都结合GDE，表明AMPK和GDE之间的关联不涉及糖原。相反，AMPK-GDE关联可能是直接的。过表达β 1-亚基的68-123位氨基酸抑制内源性AMPK和GDE之间的关联。虽然GDE活性不受影响，但AMPK的基础磷酸化和激酶活性以及乙酰辅酶A羧化酶的磷酸化显著增加。因此，AMPK-GDE缔合可能是调节AMPK活性以及由此产生的脂肪酸氧化和葡萄糖摄取的新机制。
AMP-activated protein kinase (AMPK) regulates both glycogen and lipid metabolism functioning as an intracellular energy sensor. In this study, we identified a 160-kDa protein in mouse skeletal muscle lysate by using a glutathione-S-transferase (GST)-AMPK fusion protein pull-down assay. Mass spectrometry and a Mascot search revealed this protein to be a glycogen debranching enzyme (GDE). The association between AMPK and GDE was observed not only in the overexpression system but also endogenously. Next, we showed the beta1-subunit of AMPK to be responsible for the association with GDE. Furthermore, experiments using deletion mutants of the beta1-subunit of AMPK revealed amino acids 68-123 of the beta1-subunit to be sufficient for GDE binding. W100G and K128Q, both beta1-subunit mutants, are reportedly incapable of binding to glycogen, but both bound GDE, indicating that the association between AMPK and GDE does not involve glycogen. Rather, the AMPK-GDE association is likely to be direct. Overexpression of amino acids 68-123 of the beta1-subunit inhibited the association between endogenous AMPK and GDE. Although GDE activity was unaffected, basal phosphorylation and kinase activity of AMPK, as well as phosphorylation of acetyl-CoA carboxylase, were significantly increased. Thus it is likely that the AMPK-GDE association is a novel mechanism regulating AMPK activity and the resultant fatty acid oxidation and glucose uptake.