WNK1 Phosphorylation Sites in TBC1D1 and TBC1D4 Modulate Cell-Surface Abundance of GLUT1
TL;DR
It is described that downregulation of WNK1 through RNA interference in HEK293 cells led to a two-fold decrease in cell-surface GLUT1 abundance, concomitant with a 40% decrease in glucose uptake.
Abstract
Highlights This manuscript is the corrected version of a previous publication that was retracted due to errors detected in the assembly of Figure 3, as documented in the respective publisher’s note at https://doi.org/10.1016/j.abb.2019.108223. Glucose uptake is a key mechanism for cellular growth and survival and involves the regulated insertion of glucose transporter proteins (GLUTs) from storage vesicles into the plasma membrane (PM). Receptor-stimulated signaling pathways control this process, including the insulin receptor that activates protein kinase AKT to phosphorylate TBC1D4, a RAB-GTPase–activating protein involved in membrane traffic regulation. What are the main findings? Protein kinase WNK1 modulates cell-surface abundance of the constitutive glucose transporter GLUT1 in human cells and also phosphorylates TBC1D4. Mass spectrometry identified serine 704 in TBC1D4, but also serine 565 in its paralogue TBC1D1 as candidate WNK1 phosphorylation sites in vitro, and mutational analysis supports a functional role for these residues in regulating cell-surface GLUT1 abundance. What are the implications of the main findings? The results reinforce a regulatory role for WNK1 in glucose metabolism besides AKT. Data reveal a better understanding of how GLUT1 trafficking and glucose uptake are regulated, which may have potential relevance for understanding metabolic dysregulation, as observed in many cancer cells or insulin-responsive cell types. Abstract This manuscript is the corrected version of a previously published paper. 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 is 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 abundance of the constitutive glucose transporter GLUT1. Here we describe that downregulation of WNK1 through RNA interference in HEK293 cells led to a two-fold decrease in cell-surface GLUT1 abundance, concomitant with a 40% decrease in glucose uptake. By mass spectrometry, we identified serine (S) 704 in TBC1D4 and also S565 in its paralogue TBC1D1 as candidate WNK1 phosphorylation sites. 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 GLUT1 trafficking and glucose uptake and may have potential impact for the understanding of metabolic dysregulation, as observed in many cancer cells or insulin-responsive cell types.
