Protein kinase WNK1 contributes to the regulation of GLUT1 expression in the plasma membrane

Henriques, Andreia; Matthiesen, Rune; Matos, Paulo; Jordan, Peter

http://hdl.handle.net/10400.18/6942

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Introduction: One mechanism by which tumour cells regulate the uptake of glucose is overexpression of glucose transporter proteins (GLUT). Besides their expression level, the number of GLUT present at the plasma membrane is regulated by signaling mechanisms (1). Previously we found that protein kinase WNK1 phosphorylates TBC1D4 (2), a GTPase activating protein for RAB-family proteins involved in membrane traffic regulation and regulates the surface expression of the constitutive glucose transporter GLUT1. Phosphorylation of either the TBC1D4 or its paralogue TBC1D1 is a key regulatory step in the kinase cascades leading to changes in glucose uptake (1). Experimental: Putative WNK1 phosphorylation sites in TBC1D1 and 4 were determined by MS following in vitro kinase assays with recombinant proteins. RNA interference, transfection of phosphorylation site mutants, and cell surface protein biotinylation assays were used to analyze the impact of the identified phosphorylation events on GLUT1 plasma membrane abundance. Results: We compared phosphorylation by AKT1, WNK1 and SGK1 and identified two novel WNK1-specific phosphorylation sites at TBC1D1-Ser565 and TBC1D4-Ser704. Transfection of the corresponding phosphomimetic or unphosphorylatable mutants revealed that phosphorylation of either RabGAP by WNK1 at these novel sites participates in the delivery of GLUT1 to the plasma membrane (PM). Consistently, downregulation of WNK1 by RNA interference decreased GLUT1 PM abundance by over 2-fold, which translates to a 60% decrease in Glucose uptake by these cells. Conclusions: Together, our data contribute to a better understanding of the pathways regulating glucose uptake via GLUT1, the upregulation of which is related to cancer progression.