WNK1 phosphorylation sites in TBC1D1 and TBC1D4 modulate cell surface expression of GLUT1

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WNK1 phosphorylation sites in TBC1D1 and TBC1D4 modulate cell surface expression of GLUT1

Monday, 23.12.2019

Andreia F.A. Henriques (a,b), Paulo Matos (a,b), Ana Sofia Carvalho ()c, Mikel Azkargorta (d), Felix Elortza (d), Rune Matthiesen (c), Peter Jordan (a,b)

a Department of Human Genetics, National Health Institute ‘Dr. Ricardo Jorge’, Lisbon, Portugal

b BioISI - Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisbon, Lisbon, Portugal

c CEDOC-Chronic Diseases Research Centre, Nova Medical School, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisbon, Portugal d Center for Cooperative Research in Biosciences (CIC bioGUNE), Building 800, Science and Technology Park of Bizkaia, 48160, Derio, Spain

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.

Archives of Biochemistry and Biophysics