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

Henriques, Andreia F.A.; Matos, Paulo; Carvalho, Ana Sofia; Azkargorta, Mikel; Elortza, Felix; Matthiesen, Rune; Jordan, Peter

http://hdl.handle.net/10400.18/6595

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Authors

Henriques, Andreia F.A.

Abstract(s)

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.

Highlights: Expression levels of protein kinase WNK1 modulate cellular glucose uptake; WNK1 phosphorylates the RAB-GAP proteins TBC1D4 and TBC1D1 in vitro; WNK1-specific phosphorylation sites were identified in TBC1D4 and TBC1D1; Phosphomimetic TBC1D mutants modulate plasma membrane expression of GLUT1 in cells.

Keywords

Arabidopsis Proteins Binding Sites Biological Transport GTPase-Activating Proteins Glucose Glucose Transporter Type 1 HEK293 Cells Humans Immediate-Early Proteins Insulin Phosphorylation Protein-Serine-Threonine Kinases Proto-Oncogene Proteins c-akt WNK Lysine-Deficient Protein Kinase 1 Gene Expression Regulation Vias de Transdução de Sinal e Patologias Associadas