Browsing by Author "Jordan, P."
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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.
- Effect of Aflatoxin B1 in both Caco-2 and Caco-2/HT29-MTX modelsPublication . Serrenho, I.; Vidal, N.; Rolo, D.; Louro, H.; Pereira, J.; Matos, P.; Jordan, P.; Alvito, P.About effect of Aflatoxin B1 in both Caco-2 and Caco-2/HT29-MTX models within the scope of the intestinal absorption assessment and enabling formulations.
- Microcystin-LR activates the ERK1/2 kinases and stimulates the proliferation of the monkey kidney-derived cell line Vero-E6Publication . Dias, E.; Matos, P.; Pereira, P.; Batoréu, M.C.C.; Silva, M.J.; Jordan, P.Microcystin-LR (MCLR) is a peptide produced by freshwater cyanobacteria that induces severe hepatotoxicity in humans and animals. MCLR is also a potent tumour promoter and it has been proposed that this activity is mediated by the inhibition of protein phosphatases PP1/PP2A, possibly through the activation of proto-oncogenes c-jun, c-fos and c-myc. However, the mechanisms underlying MCLR-induced tumour promotion are still largely unknown, particularly in non-liver cells. In previous studies we have demonstrated that micromolar concentrations of MCLR induce cytotoxic effects in the kidney Vero-E6 cell line. The purpose of the present work was to evaluate whether the exposure to subcytotoxic concentrations of MCLR was sufficient to induce the proliferation of Vero-E6 cells. Through BrdU incorporation assay we show that at nanomolar concentrations MCLR stimulates cell cycle progression in Vero-E6 kidney cell line. Moreover, the analysis of mitogen-activated protein kinases p38, JNK and ERK1/2 activity revealed that the proliferative effect of MCLR is associated with the activation of the pro-proliferative ERK1/2 pathway. These results emphasise the importance to confirm in vivo the impact of MCLR on tumour promotion at kidney level.
- 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.
- Protein kinase WNK2 as a tumour suppressor gene in malignant gliomasPublication . Moniz, S.; Martinho, O.; Reis, R.M.; Jordan, P.Malignant glioblastomas are the most common and lethal adult brain tumours, with patients dying within two years from diagnosis. Little is known about the molecular mechanisms underlying the formation and/or development of these tumours, which present a very invasive phenotype within the brain and are genetically heterogeneous and highly resistant to both chemo- and radio-therapies. Recently, the promoter region of the protein kinase WNK2 gene was found to be hypermethylated in 29 of 31 infiltrative gliomas and about 80% of meningiomas. We have previously described that the experimental depletion of WNK2 expression decreases RhoA activity whilst leading to increased Rac1 activity. Because RhoA/Rac1 activities are important for cell migration and glioblastomas are very invasive tumours, we tested the effects of WNK2 on wound-healing assays in glioma cell lines SW1088 and A172. SW1088 cells express endogenous WNK2 and we observed that wound closure was increased upon experimental depletion of endogenous WNK2. In contrast, A172 cells display complete promoter region methylation and WNK2 re-expression was found to decrease migration. Consistently, we observed an increase in Rac1 activity in SW1088 cells upon WNK2 down-regulation, but lower levels of active Rac1 in A172 cells stably expressing WNK2 cDNA when compared with an equivalent cell line stably transfected with the same empty vector. Our studies indicate that loss of WNK2 expression promotes Rac1 activation and may contribute to the highly invasive phenotype that glioblastomas present. We also observed that, in a panel of glioblastoma cell lines, WNK2 promoter methylation correlates with a marked deregulation in Akt, MEK1/2 and ERK1/2 activities, suggesting WNK2 may also be important for tumour cell survival and cell cycle progression.
- Protein kinase WNK2 regulates cell migration in malignant gliomasPublication . Moniz, S.; Martinho, O.; Reis, R.M.; Jordan, P.Malignant glioblastomas are the most common and lethal adult brain tumours, with patients dying within two years from diagnosis. Little is known about the molecular mechanisms underlying the formation and/or development of these tumours, which present a very invasive phenotype within the brain and are genetically heterogeneous and highly resistant to both chemo- and radio-therapies. Recently, the promoter region of the protein kinase WNK2 gene was found to be hypermethylated in 29 of 31 infiltrative gliomas and about 80% of meningiomas. We have previously described that the experimental depletion of WNK2 expression decreases RhoA activity whilst leading to increased Rac1 activity. Because RhoA/Rac1 activities are important for cell migration and glioblastomas are very invasive tumours, we tested the effects of WNK2 on wound-healing assays in glioma cell lines SW1088 and A172. SW1088 cells express endogenous WNK2 and we observed that wound closure was increased upon experimental depletion of endogenous WNK2. In contrast, A172 cells display complete promoter region methylation and WNK2 re-expression was found to decrease migration. Consistently, we observed an increase in Rac1 activity in SW1088 cells upon WNK2 down-regulation, but lower levels of active Rac1 in A172 cells stably expressing WNK2 cDNA when compared with an equivalent cell line stably transfected with the same empty vector. Our studies indicate that loss of WNK2 expression promotes Rac1 activation and may contribute to the highly invasive phenotype that glioblastomas present. We also observed that, in a panel of glioblastoma cell lines, WNK2 promoter methylation correlates with a marked deregulation in Akt, MEK1/2 and ERK1/2 activities, suggesting WNK2 may also be important for tumour cell survival and cell cycle progression.
- Unverricht-Lundborg disease: development of splicing therapeutic approaches for a patient with an homozygous mutation in the cystatin B genePublication . Matos, L.; Duarte, A.J.; Ribeiro, D.; Jordan, P.; Prata, M.J.; Chaves, J.; Desviat, L.R.; Pérez, B.; Amaral, O.; Alves, S.Unverricht-Lundborg disease (ULD) is the most common form of progressive myoclonic epilepsy worldwide. It is an autosomal recessive neurodegenerative disorder caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. An unstable expansion, missense, nonsense, frameshift and mutations that may lead to alternative splicing have been described as causal of ULD. Recently, our group described an ULD patient who is homozygous for a new synonymous mutation (c.66G>A; p.Q22Q) located at the last nucleotide of exon 1. The transcriptional profile analysis allowed the identification of two CSTB splice variants, one of normal size with the G>A change and other with partial inclusion of intron 1 due to activation of a cryptic splice-site inside the intronic sequence. To correct the splice defect, here we developed antisense oligonucleotide and U1snRNA mediated therapeutic strategies. U1 is required for splice donor site (SDS) recognition of pre-mRNAs and initiates the splicing process. The mutation c.66G>A interferes with the recognition of the SDS by U1. In a first approach, to reduce missplicing we generated four U1 construct isoforms with increasing complementarity to the SDS. Transfection of patient-derived fibroblasts with different concentrations of the adapted U1 vectors did not allowed the correction of the aberrant transcript. In a second strategy, we have designed a specific lock nucleic-acid (LNA) oligonucleotide to block the activated cryptic splice-site in intron 1. Normal splicing pattern of a single transcript with the synonymous change G>A was successfully rescued after LNA transfection in patient cells. The therapeutic effect showed to be dose-dependent. These results suggest that antisense therapy might be a potential alternative or adjunct treatment strategy for patients holding splicing changes in CSTB gene. As far as we know this is the first report of a patient tailored therapy in cells of an ULD patient.
- 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.