Browsing by Author "Mendes, A.I."
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- Antagonistic Regulation of CFTR Cell Surface Expression by Protein Kinases WNK4 and Spleen Tyrosine KinasePublication . Mendes, A.I.; Matos, P.; Amaral, M.D.; Jordan, P.Members 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 that WNK4 promotes the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in mammalian cells. The mechanism by which WNK4 acts on CFTR involves interaction with spleen tyrosine kinase (Syk), which we find to phosphorylate Tyr512 in the first nucleotide-binding domain (NBD) 1 of CFTR. The presence of WNK4 prevents the phosphorylation of NBD1 by Syk in vitro in a kinase-independent manner. In baby hamster kidney cells stably expressing CFTR, catalytically active Syk reduces while WNK4 promotes the cell surface expression of CFTR. 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. Together, our results identify that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and describe an antagonistic role of WNK4 and Syk in this process.
- Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activityPublication . Luz, S.; Kongsuphol, P.; Mendes, A.I.; Romeiras, F.; Sousa, M.; Schreiber, R.; Matos, P.; Jordan, P.; Metha, A.; Amaral, M.D.; Kunzelmann, K.; Farinha, C.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.
- Um novo mecanismo molecular para a regulação do transportador de glicose GLUT1Publication . Mendes, A.I.; Matos, P.; Moniz, S.; Jordan, P.One mechanism by which 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 storage vesicles through inactivation of the Rab GTPase activating protein TBC1D4, also known as AS160. Using protein biochemical techniques we analysed complex formation between TBC1D4 and protein kinase WNK1 in human embryonic kidney (HEK293) cells. We found that WNK1 associates in a protein complex with TBC1D4 and phosphorylates TBC1D4 in vitro. This resulted in increased expression levels of the constitutive glucose transporter GLUT1 at the cell surface. 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 molecular pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases. This pathway may be important in the cellular response to insulin.
- Uma variante do gene WNK4 está associada à osteoporose mas não à hipertensão na população portuguesaPublication . Mendes, A.I.; Mascarenhas, M.R.; Matos, S; Sousa, I; Ferreira, J; Barbosa, A.P.; Bicho, M; Jordan, P.Germline mutations in the WNK4 gene originate Gordon syndrome or pseudohypoaldosteronism type II, a familial form of hypertension with hyperkalemia and hypercalciuria. The WNK4 protein encodes a protein kinase involved in the regulation of various renal ion channels. 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 in a highly conserved arginine residue in exon 17 showed an association to the osteoporosis group. Our data suggest that WNK4 polymorphism rs56116165 is a rare allelic variant of a candidate gene with a biological function in renal calcium homeostasis and thus may contribute to a genetic predisposition to osteoporosis.