Kawaguchi T, Takenoshita M, Kabashima T, Uyeda K
Department of Biochemistry, Dallas Veterans Affairs Medical Center and University of Texas Southwestern Medical Center at Dallas, 4500 South Lancaster Road, Dallas, TX 75223, USA.
Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):13710-5. doi: 10.1073/pnas.231370798. Epub 2001 Nov 6.
Recently we purified and identified a previously uncharacterized transcription factor from rat liver binding to the carbohydrate responsive element of the L-type pyruvate kinase (L-PK) gene. This factor was named carbohydrate responsive element binding protein (ChREBP). ChREBP, essential for L-PK gene transcription, is activated by high glucose and inhibited by cAMP. Here, we demonstrated that (i) nuclear localization signal and basic helix-loop-helix/leucine-zipper domains of ChREBP were essential for the transcription, and (ii) these domains were the targets of regulation by cAMP and glucose. Among three cAMP-dependent protein kinase phosphorylation sites, Ser(196) and Thr(666) were the target sites. Phosphorylation of the former resulted in inactivation of nuclear import, and that of the latter resulted in loss of the DNA-binding activity and L-PK transcription. On the other hand, glucose activated the nuclear import by dephosphorylation of Ser(196) in the cytoplasm and also stimulated the DNA-binding activity by dephosphorylation of Thr(666) in the nucleus. These results thus reveal mechanisms for regulation of ChREBP and the L-PK transcription by excess carbohydrate and cAMP.
最近，我们从大鼠肝脏中纯化并鉴定出一种以前未被表征的转录因子，它能与L型丙酮酸激酶（L-PK）基因的碳水化合物反应元件结合。该因子被命名为碳水化合物反应元件结合蛋白（ChREBP）。ChREBP对L-PK基因转录至关重要，它被高糖激活并被cAMP抑制。在此，我们证明：（i）ChREBP的核定位信号和碱性螺旋-环-螺旋/亮氨酸拉链结构域对转录至关重要，以及（ii）这些结构域是cAMP和葡萄糖调控的靶点。在三个依赖cAMP的蛋白激酶磷酸化位点中，Ser(196)和Thr(666)是靶点。前者的磷酸化导致核输入失活，后者的磷酸化导致DNA结合活性丧失和L-PK转录受阻。另一方面，葡萄糖通过使细胞质中的Ser(196)去磷酸化激活核输入，并通过使细胞核中的Thr(666)去磷酸化刺激DNA结合活性。因此，这些结果揭示了过量碳水化合物和cAMP对ChREBP及L-PK转录的调控机制。
