Glucose uptake by mammalian cells is a key mechanism to maintain cell and tissue homeostasis and relies mostly on plasma membrane-localized glucose transporter proteins (GLUTs). Two main cellular mechanisms regulate GLUT proteins in the cell: first, expression of GLUT genes is under dynamic transcriptional control and is used by cancer cells to increase glucose availability. Second, GLUT proteins are regulated by membrane traffic from storage vesicles to the plasma membrane (PM). This latter process is triggered by signaling mechanisms and well-studied in the case of insulin-responsive cells, which activate protein kinase AKT to phosphorylate TBC1D4, a RAB-GTPase activating protein involved in membrane traffic regulation. Previously, we identified protein kinase WNK1 as another kinase able to phosphorylate TBC1D4 and regulate the surface expression of the constitutive glucose transporter GLUT1. Here we describe that downregulation of WNK1 through RNA interference in HEK293 cells led to a 2-fold decrease in PM GLUT1 expression, concomitant with a 60% decrease in glucose uptake. By mass spectrometry, we identified serine (S) 704 in TBC1D4 as a WNK1-regulated phosphorylation site, and also S565 in the paralogue TBC1D1. Transfection of the respective phosphomimetic or unphosphorylatable TBC1D mutants into cells revealed that both affected the cell surface abundance of GLUT1. The results reinforce a regulatory role for WNK1 in cell metabolism and have potential impact for the understanding of cancer cell metabolism and therapeutic options in type 2 diabetes.

中文翻译：

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TBC1D1和TBC1D4中的WNK1磷酸化位点调节GLUT1的细胞表面表达。

哺乳动物细胞摄取葡萄糖是维持细胞和组织动态平衡的关键机制，并且主要依赖于质膜定位的葡萄糖转运蛋白（GLUT）。两种主要的细胞机制可调节细胞中的GLUT蛋白：首先，GLUT基因的表达处于动态转录控制之下，并被癌细胞用于增加葡萄糖的利用率。其次，GLUT蛋白受从存储囊泡到质膜（PM）的膜运输的调节。后一个过程是由信号传导机制触发的，并且在胰岛素反应性细胞的情况下进行了充分研究，该细胞激活蛋白激酶AKT来磷酸化TBC1D4，TBC1D4是一种参与膜交通调节的RAB-GTPase激活蛋白。之前，我们将蛋白激酶WNK1鉴定为能够磷酸化TBC1D4并调节组成型葡萄糖转运蛋白GLUT1的表面表达的另一种激酶。在这里，我们描述了通过HEK293细胞中RNA干扰对WNK1的下调导致PM GLUT1表达降低2倍，同时葡萄糖摄取降低60％。通过质谱，我们在TBC1D4中鉴定出丝氨酸（S）704为WNK1调控的磷酸化位点，在旁系同源物TBC1D1中也鉴定为S565。将各自的拟磷酸酶或不可磷酸化的TBC1D突变体转染到细胞中，发现它们都影响了GLUT1的细胞表面丰度。结果加强了WNK1在细胞代谢中的调节作用，并可能对理解2型糖尿病的癌细胞代谢和治疗选择产生潜在影响。