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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

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Tyrosine phosphorylation modulates cell surface expression of chloride cotransporters NKCC2 and KCC3

Publication . Loureiro, Claudia; barros, Patricia; Matos, Paulo; Jordan, Peter

Introduction: Cellular chloride transport has a fundamental role in cell volume regulation and membrane potential, both in normal and tumour cells (1,2). Cellular chloride entry or exit are mediated at the plasma membrane by cotransporter proteins of the solute carrier 12 family. For example, NKCC2 resorbs chloride with sodium and potassium ions at the apical membrane of epithelial cells in the kidney, whereas KCC3 releases chloride with potassium ions at the basolateral membrane. Their ion transport activity is regulated by protein phosphorylation in response to signaling pathways. An additional regulatory mechanism concerns the amount of cotransporter molecules inserted into the plasma membrane. Experimental: Co-transporter constructs were transfected into HEK293 cells and the activity of SYK kinase modulated by incubation with SYK inhibitors or by co-transfection with siRNAs, kinase-dead, or constitutively active SYK mutants. Co-transporter abundance in the plasma membrane was analyzed by biotinylation of cell surface proteins. Results: Here we describe that tyrosine phosphorylation of NKCC2 and KCC3 regulates their plasma membrane expression levels. We identified that spleen tyrosine kinase (SYK) phosphorylates a specific N-terminal tyrosine residue in each cotransporter. Experimental depletion of endogenous SYK or pharmacological inhibition of its kinase activity increased the abundance of NKCC2 at the plasma membrane of human embryonic kidney cells. In contrast, overexpression of a constitutively active SYK mutant decreased NKCC2 membrane abundance. Intriguingly, the same experimental approaches revealed the opposite effect on KCC3 abundance at the plasma membrane, compatible with the known antagonistic roles of NKCC and KCC cotransporters in cell volume regulation. Conclusions: We identified a novel pathway modulating the cell surface expression of NKCC2 and KCC3 and show that this same pathway has opposite functional outcomes for these two cotransporters. The findings have several biomedical implications considering the role of these cotransporters in regulating blood pressure and cell volume.

2019-10-18Conference object

Open access

A SYK/SHC1 pathway regulates the amount of CFTR in the plasma membrane

Publication . Loureiro, Claudia

Mutations in the CFTR gene cause the recessive genetic disease Cystic Fibrosis, where the chloride transport across the apical membrane of epithelial cells mediated by the CFTR protein is impaired. CFTR protein trafficking to the plasma membrane (PM) is the result of a complex interplay between the secretory and membrane recycling pathways that control the number of channels present at the membrane. In addition, the ion transport activity of CFTR at the PM is modulated through post-translational protein modifications. Previously we described that spleen tyrosine kinase (SYK) phosphorylates a specific tyrosine residue in the NBD1 domain and this modification can regulate the PM abundance of CFTR. Here we identified the underlying biochemical mechanism using peptide pull-down assays followed by mass spectrometry. We identified in bronchial-epithelial cells that the adaptor protein SHC1 recognizes tyrosine-phosphorylated CFTR through its phosphotyrosine binding (PTB) domain and that the formation of a complex between SHC1 and CFTR is induced at the PM in the presence of activated SYK. The depletion of endogenous SHC1 expression was sufficient to promote an increase in CFTR at the PM of these cells. The results identify a SYK/SHC1 pathway that regulates the PM levels of CFTR channels, contributing to a better understanding of how epithelial chloride secretion is regulated.

2019-10-18Conference object

Open 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

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Regulation of epithelial chloride transport by phospho-tyrosine-initiated protein networks

Publication . Loureiro, Cláudia; Jordan, Peter; Clarke, Luka

Ion transport is crucial for cell volume regulation by compensating variations in extracellular tonicity, playing an important role in maintaining the structural integrity and intracellular milieu in cells. These functions require a dynamic, spatio-temporally coordinated regulation of ion transport, which occurs in cells by two mechanisms: first, the amount of channel or cotransporter inserted into the plasma membrane (PM) from a pool of endosomal storage vesicles, and second, the ion transport activity regulated by post-translational modifications such as phosphorylation of channel or transporter proteins. Previous results from the host laboratory showed that phosphorylation by spleen tyrosine kinase (SYK) of Tyr512 in the NBD1 domain regulates PM abundance of CFTR, the chloride channel involved in cystic fibrosis. The main objective of this PhD project was to identify phospho-tyrosine-binding proteins involved in the regulation of chloride transport proteins and the underlying molecular mechanism. First, it was found that besides CFTR two further renal ion cotransporters, NKCC2 and KCC3, are phosphorylated by SYK in vitro on an N-terminal tyrosine residue and that experimental manipulation of either SYK expression levels or its catalytic activity affect the cell surface abundance of these cotransporters. Interestingly, the very same phosphorylation pathway leads to a decrease in NKCC2 but to an increase in KCC3 PM levels. Second, the underlying biochemical mechanism was identified using peptide pulldown assays followed by mass spectrometry and revealed that the adaptor protein SHC1 binds to phospho-tyrosine in NKCC2, KCC3 and CFTR through its PTB domain. Upon depletion of endogenous SHC1 expression, KCC3 decreased at the PM, whereas NKCC2 and CFTR levels increased. In the case of phosphorylated NKCC2, SNX27 and NCK1 were identified as additional binding partners. Lastly, SHC1 was shown to form a complex with CFTR following activation of protein kinase SYK, but does not affect the PM level of the most frequent mutant F508del-CFTR. The results described in this work identified a novel SYK/SHC1 pathway that regulates the cotransporters NKCC2 and KCC3 and the chloride channel CFTR and have potential biomedical implications for the identification of new therapeutic targets in diseases like hypertension or cystic fibrosis, or those involving regulation of cell volume.

2019-05-03Doctoral thesis

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