New insights into the regulation of insulin-sensitive glucose transporters by protein kinases

Funder

Organizational Unit

Publications

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

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

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.

2019-10-18Conference object

Restricted access

Regulation of glucose uptake in mammalian cells by protein phosphorylation networks

Publication . Henrique, Andreia; Jordan, Peter; Clarke, Luka

Glucose uptake is a key mechanism to maintain cell, tissue and body homeostasis. Among others, glucose transporter proteins (GLUTs) are responsible for glucose transport into the cell. Besides their expression level, the GLUT number present at the plasma membrane (PM) is regulated by signaling mechanisms. Previously, protein kinase WNK1 was found to phosphorylate TBC1D4, a Rab-GAP involved in membrane traffic regulation, and to regulate the surface expression of the constitutive glucose transporter GLUT1. In this work the phosphorylation of either TBC1D4 or its paralogue TBC1D1 was studied as a key step in regulatory cascades modulating glucose uptake. First, we showed that downregulation of WNK1 through RNA interference translates in a 2-fold decrease in PM GLUT1 expression and a 60% decrease in glucose uptake. Then, we compared by mass spectrometry (MS) the in vitro phosphorylation of TBC1D1 and 4 by AKT1, WNK1 and SGK1 and 3. We identified two novel WNK1-specific phosphorylation sites at TBC1D1-Ser565 and TBC1D4-Ser704 and showed that transfection of their phosphomimetic or unphosphorylatable mutants affected cell surface abundance of GLUT1. To define new biological pathways regulated by WNK1, we determined the interactome of WNK1 by MS. Interestingly, the bioinformatic and gene ontology analysis pointed to a previously unrecognized function related to mRNA processing. Our studies identified a novel function of WNK1 in alternative splicing using RAC1B in colorectal HT29 cells as a model. In particular, WNK1 acts as a scaffolding protein through complex formation with GSK3β. This complex protects GSK3β from an inhibitory phosphorylation at Ser9. The active GSK3β allows the translocation of kinase SRPK1 and splicing factor SRSF1 to the nucleus, which is important for RAC1B generation. Considering that RAC1B is known to be essential for cell survival and malignant progression, the results establish a new link between WNK kinases and tumorigenesis. Altogether, this work reinforced a role for WNK1 in cell metabolism and uncovered a new function in regulation of alternative splicing, two events that can contribute to tumor development. The data may provide new targets for pharmacological modulation of RAC1B expression and cellular metabolism, with potential impact for the treatment of cancer and type 2 diabetes.

2019-08-20Doctoral thesis

Restricted access

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

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

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.

2019-12-06Journal article

Restricted access