Browsing by Author "Moniz, Sónia"
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- Antagonistic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Cell Surface Expression by Protein Kinases WNK4 and Spleen Tyrosine KinasePublication . Mendes, Ana Isabel; Matos, Paulo; Moniz, Sónia; Luz, Simão; Amaral, Margarida D.; Farinha, Carlos M.; Jordan, PeterMembers of the WNK (with-no-lysine [K]) subfamily of protein kinases regulate various ion channels involved in sodium, potassium, and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe findings demonstrating that the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is also regulated by WNK4 in mammalian cells. This effect of WNK4 is independent of the presence of kinase and involves interaction with and inhibition of spleen tyrosine kinase (Syk), which phosphorylates Tyr512 in the first nucleotide-binding domain 1 (NBD1) of CFTR. Transfection of catalytically active Syk into CFTR-expressing baby hamster kidney cells reduces the cell surface expression of CFTR, whereas that of WNK4 promotes it. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy, and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. In human airway epithelial cells, downregulation of endogenous Syk and WNK4 confirms their roles as physiologic regulators of CFTR surface expression. Together, our results show that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk perform an antagonistic role in this process.
- Emerging roles for WNK kinases in cancerPublication . Moniz, Sónia; Jordan, PeterThe subfamily of WNK protein kinases is composed of four human genes and is characterised by a typical sequence variation within the conserved catalytic domain. Although most research has focussed on the role of WNK1, WNK3 and WNK4 in regulating different ion transporters in both the kidney and extrarenal tissues, there is growing evidence for additional roles of WNK kinases in various signalling cascades related to cancer. Here, we review the connection between WNK kinases and tumorigenesis and describe existing experimental evidence as well as potential new links to major aspects of tumour biology. In particular, we discuss their role in G1/S cell cycle progression, metabolic tumour cell adaptation, evasion of apoptosis and metastasis.
- HGF Stimulation of Rac1 Signaling Enhances Pharmacological Correction of the Most Prevalent Cystic Fibrosis Mutant F508del-CFTRPublication . Moniz, Sónia; Sousa, Marisa; Moraes, Bruno José; Mendes, Ana Isabel; Palma, Marta; Barreto, Celeste; Fragata, José I.; Amaral, Margarida D; Matos, PauloCystic fibrosis (CF), a major life-limiting genetic disease leading to severe respiratory symptoms, is caused by mutations in CF transmembrane conductance regulator (CFTR), a chloride (Cl(-)) channel expressed at the apical membrane of epithelial cells. Absence of functional CFTR from the surface of respiratory cells reduces mucociliary clearance, promoting airways obstruction, chronic infection, and ultimately lung failure. The most frequent mutation, F508del, causes the channel to misfold, triggering its premature degradation and preventing it from reaching the cell surface. Recently, novel small-molecule correctors rescuing plasma membrane localization of F508del-CFTR underwent clinical trials but with limited success. Plausibly, this may be due to the mutant intrinsic plasma membrane (PM) instability. Herein, we show that restoration of F508del-CFTR PM localization by correctors can be dramatically improved through a novel pathway involving stimulation of signaling by the endogenous small GTPase Rac1 via hepatocyte growth factor (HGF). We first show that CFTR anchors to apical actin cytoskeleton (via Ezrin) upon activation of Rac1 signaling through PIP5K and Arp2/3. We then found that such anchoring retains pharmacologically rescued F508del-CFTR at the cell surface, boosting functional restoration by correctors up to 30% of wild-type channel levels in human airway epithelial cells. Our findings reveal that surface anchoring and retention is a major target pathway for CF pharmacotherapy, namely, to achieve maximal restoration of F508del-CFTR in patients in combination with correctors. Moreover, this approach may also translate to other disorders caused by trafficking-deficient surface proteins.
- Loss of WNK2 expression by promoter gene methylation occurs in adult gliomas and triggers Rac1-mediated tumour cell invasivenessPublication . Moniz, Sónia; Martinho, Olga; Pinto, Filipe; Sousa, Bárbara; Loureiro, Cláudia; Oliveira, Maria José; Moita, Luís Ferreira; Honavar, Mrinalini; Pinheiro, Célia; Pires, Manuel; Lopes, José Manuel; Jones, Chris; Costello, Joseph F; Paredes, Joana; Reris, Rui Manuel; Jordan, PeterThe gene encoding protein kinase WNK2 was recently identified to be silenced by promoter hypermethylation in gliomas and meningiomas, suggesting a tumour-suppressor role in these brain tumours. Following experimental depletion in cell lines, WNK2 was further found to control GTP-loading of Rac1, a signalling guanosine triphosphatase involved in cell migration and motility. Here we show that WNK2 promoter methylation also occurs in 17.5% (29 out of 166) of adult gliomas, whereas it is infrequent in its paediatric forms (1.6%; 1 out of 66). Re-expression of WNK2 in glioblastoma cells presenting WNK2 gene silencing reduced cell proliferation in vitro, tumour growth in vivo and also cell migration and invasion, an effect correlated with reduced activation of Rac1. In contrast, when endogenous WNK2 was depleted from glioblastoma cells with unmethylated WNK2 promoter, changes in cell morphology, an increase in invasion and activation of Rac1 were observed. Together, these results validate the WNK2 gene as a recurrent target for epigenetic silencing in glia-derived brain tumours and provide first mechanistic evidence for a tumour-suppressing role of WNK2 that is related to Rac1 signalling and tumour cell invasion and proliferation.
- Protein kinase WNK1 promotes cell surface expression of glucose transporter GLUT1 by regulating a Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4)-Rab8A complexPublication . Mendes, Ana Isabel; Matos, Paulo; Moniz, Sónia; Jordan, PeterOne mechanism by which mammalian cells regulate the uptake of glucose is the number of glucose transporter proteins (GLUT) present at the plasma membrane. In insulin-responsive cells types, GLUT4 is released from intracellular stores through inactivation of the Rab GTPase activating protein Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4) (also known as AS160). Here we describe that TBC1D4 forms a protein complex with protein kinase WNK1 in human embryonic kidney (HEK293) cells. We show that WNK1 phosphorylates TBC1D4 in vitro and that the expression levels of WNK1 in these cells regulate surface expression of the constitutive glucose transporter GLUT1. WNK1 was found to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. These effects were dependent on the catalytic activity because expression of a kinase-dead WNK1 mutant had no effect on binding of 14-3-3 and Rab8A, or on surface GLUT1 levels. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases.
- Stimulation of Rac1 signalling enhances apical retention of chemically corrected F508del-CFTR in human airway cells.Publication . Matos, Paulo; Moniz, Sónia; Moraes, Bruno; Mendes, Ana Isabel; Amaral, Margarida D.; Jordan, Peter