Browsing by Author "Mendes, Ana Isabel"
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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.
- 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.
- 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.
- Signal transduction pathways involving the hypertension-related WNK1 and WNK4 protein kinasesPublication . Mendes, Ana IsabelThe genes WNK1 and WNK4 belong to the subfamily of WNK protein kinases and their mutation causes pseudohypoaldosteronism type II, a rare familial form of hypertension with hyperkalemia and hypercalciuria. The molecular mechanisms underlying this condition involve the regulation of renal electrolyte homeostasis and the modulation of diverse ion channels and transporters via WNK kinases. Additionally, WNKs have also been reported to participate in signal transduction pathways related to cell survival and proliferation. The objective of the present thesis was to identify novel WNK1 and WNK4 interacting proteins and the underlying signal transduction pathways. First, it was found that WNK1 forms a protein complex with the Rab-GAP TBC1D4 and phosphorylates it in vitro. It was shown that the expression levels of WNK1 regulate surface expression of the constitutive glucose transporter GLUT1 in HEK293 cells. WNK1 is shown to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. Moreover, these effects were kinase activity-dependent. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases. Second, WNK4 was found to promote the cell surface expression of the CFTR chloride channel in mammalian cells. The mechanism by which WNK4 acts on CFTR involves interaction with the tyrosine kinase Syk, which we found to phosphorylate tyrosine 512 (Tyr512) in the first nucleotide-binding domain of CFTR. The presence of WNK4 prevents this in vitro phosphorylation in a kinase-independent manner. In BHK21 cells stably expressing CFTR, Syk reduces, while WNK4 promotes, the cell surface expression of CFTR. Mutation of Tyr512 revealed that its phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk play an antagonistic role in this process. Finally, ten WNK4 variants were detected in a cohort of Portuguese patients and control individuals, which subsequently were tested for association to hypertension and/or osteoporosis. Despite none of the variants yield any significant association to hypertension, a rare missense alteration (rs56116165) in a highly conserved arginine residue showed a nominal association to osteoporosis. This finding advocates that this polymorphism is a rare allelic variant, in a candidate gene with a biological function in renal calcium homeostasis, that may contribute to a genetic predisposition to osteoporosis.
- 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
- The contribution of CK2 and spleen tyrosine kinase (SYK) to CFTR trafficking and PKA-induced activityPublication . Luz, Simão; Kongsuphol, Patthara; Mendes, Ana Isabel; Romeiras, Francisco; Sousa, Marisa; Schreiber, Rainer; Matos, Paulo; Jordan, Peter; Mehta, Anil; Amaral, Margarida D.; Kunzelmann, Karl; Farinha, Carlos M.Previously, the pleiotropic “master kinase” casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.
- A WNK4 gene variant relates to osteoporosis and not to hypertension in the Portuguese populationPublication . Mendes, Ana Isabel; Mascarenhas, Mário Rui; Matos, Sónia; Sousa, Inês; Ferreira, Joana; Barbosa, Ana Paula; Bicho, Manuel; Jordan, PeterGermline mutations in the WNK4 gene originate Gordon syndrome or pseudohypoaldosteronism type II, a familial form of hypertension with hyperkalemia and hypercalciuria. In order to elucidate the contribution of WNK4 genetic variants to hypertension and/or osteoporosis, we analyzed 271 control individuals and a cohort of 448 hypertensive and 372 osteoporosis patients from the Portuguese population. Ten genetic variants were detected in 4.3% of the population under study, none of which revealed any significant association to the hypertension phenotype. In contrast, a rare missense alteration within exon 17 in a highly conserved arginine residue showed a possible tendency for association to the osteoporosis group. Our data suggest that WNK4 polymorphism rs56116165 is a rare allelic variant in a candidate gene with a biological function in renal calcium homeostasis that may contribute to a genetic predisposition to osteoporosis.