Importin-α Protein Binding to a Nuclear Localization Signal of Carbohydrate Response Element-Binding Protein (ChREBP)
Importin-α Protein Binding to a Nuclear Localization Signal of Carbohydrate Response Element-Binding Protein (ChREBP)
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DOI：
10.1074/jbc.m111.237016
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发表时间：
2011-08-12
影响因子：
4.8
通讯作者：
Uyeda, Kosaku
中科院分区：
文献类型：
--
作者：
Ge, Qiang;Nakagawa, Tsutomu;Uyeda, Kosaku
碳水化合物反应元件结合蛋白（carbon response element-binding protein, ChREBP）是一种葡萄糖反应转录因子，在葡萄糖介导的肝糖酵解和脂肪生成相关基因诱导中起关键作用。循环血糖水平在很大程度上通过翻译后机制影响肝细胞中的ChREBP活性，包括磷酸化依赖性亚细胞定位。先前，我们发现ChREBP通过磷酸化依赖结合14-3-3蛋白二聚体保留在细胞质中，并确定α 2螺旋（残基125-135）磷酸化- ser（140）结构域是主要的14-3-3结合位点(Sakiyama， H., Wynn， R. M., Lee， W. R., Fukasawa， M., Mizuguchi， H., Gardner， K. H., Repa， J. J., and Uyeda， K. (2008) J. Biol。化学，283,24899-24908)。为了响应高葡萄糖进入细胞核，ChREBP必须结合importin- α；这种异源二聚体然后与进口蛋白形成复合物与核孔复合物相互作用。在这项工作中，我们重新表征了大鼠ChREBP的importin- α结合核定位信号（NLS），将其确定为包含最小残基158-190的扩展的经典二部NLS。用丙氨酸取代ChREBP的Lys(159)/Lys（190）残基导致importin- α结合丧失，葡萄糖刺激的转录活性和核定位丧失。14-3-3蛋白的二级结合位点为α 3螺旋（残基170 ~ 190）磷酸丝氨酸（196）结构域。发现Importin-alpha和14-3-3与该次要位点具有竞争性结合。这些结果提示了一个重要的机制，通过进口蛋白- α和14-3-3控制ChREBP进出细胞核的运动，以响应肝脏葡萄糖水平的变化，从而进一步表明ChREBP的延伸NLS是一个关键的葡萄糖感知和葡萄糖反应位点。
Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. Circulating blood glucose levels affect ChREBP activity in hepatocytes largely by post-translational mechanisms that include phosphorylation-dependent subcellular localization. Previously, we showed that ChREBP is retained in the cytosol by phosphorylation-dependent binding to 14-3-3 protein dimers and identified the alpha 2 helix (residues 125-135) phospho-Ser(140) domain as the primary 14-3-3 binding site (Sakiyama, H., Wynn, R. M., Lee, W. R., Fukasawa, M., Mizuguchi, H., Gardner, K. H., Repa, J. J., and Uyeda, K. (2008) J. Biol. Chem. 283, 24899-24908). To enter the nucleus in response to high glucose, ChREBP must bind importin-alpha; this heterodimer then forms a complex with importin-beta to interact with the nuclear pore complex. In this work, we recharacterized the importin-alpha binding nuclear localization signal (NLS) of rat ChREBP, identifying it as an extended classical bipartite NLS encompassing minimally residues 158-190. Replacing Lys(159)/Lys(190) residues of ChREBP with alanine resulted in loss of importin-alpha binding, glucose-stimulated transcriptional activity and nuclear localization. Asecondary 14-3-3 protein binding site also was identified, the alpha 3 helix (residues 170-190) phospho-Ser(196) domain. Importin-alpha and 14-3-3 were found to bind competitively to this secondary site. These results suggest an important mechanism by which importin-alpha and 14-3-3 control movement of ChREBP in and out of the nucleus in response to changes in glucose levels in liver and thus further suggest that the extended NLS of ChREBP is a critical glucose-sensing, glucose-responsive site.