Browsing by Issue Date, starting with "2013-12-06"
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- A new WNK4/SYK signaling pathway regulating membrane levels of chloride cotransporters in kidney cellsPublication . Loureiro, Cláudia; Jordan, PeterArterial hypertension affects one third of the Western population and constitutes a remarkable risk factor for cardiovascular disease and stroke. Pseudohypoaldosteronism type II or Gordon's syndrome is a rare familial form of hypertension characterized by increased renal salt reabsorption accompanied by hyperkalemia, and caused by mutations in the WNK1 and WNK4 genes. These encode protein kinases regulating renal electrolyte homeostasis through modulation of several membrane transporters and ion channels but the molecular pathways involved are only partially understood. In a recent study on the regulation of the chloride channel CFTR (Cystic Fibrosis Transmembrane Regulator Condutance), a novel WNK4 signaling pathway was described to regulate the amount of CFTR at the cell surface. The underlying mechanism involves tyrosine kinase Syk (Spleen tyrosine kinase), that phosphorylates CFTR protein and promotes its removal from the plasma membrane whereas this activity is inhibited in the presence of WNK4 [1]. In order to study whether the WNK4/Syk interplay may also operate in the regulation of other chloride co-transporters, their protein sequences were inspected for the presence of a Syk consensus motif. Among 20 different transport proteins, the Syk motif was identified only in the sequence of chloride co-transporters NKCC2 and KCC3, which are important for electrolyte homeostasis in the kidney and blood pressure regulation. In order to investigate whether Syk can phosphorylate these channels in vitro, recombinant NKCC2 and KCC3 fragments were produced and tested as substrates in in vitro phosphorylation assays. Strong phosphorylation of renal channels NKCC2 and KCC3 by Syk was observed. The results of this study are a first step to understand the regulation of these ion channels via the WNK4/Syk pathway and possible identify a new mode regulating electrolyte homeostasis in the kidney, potentially related to hypertension.
- Impact of microcystins on the growth and antioxidant system of aquatic bacteriaPublication . Miguéns, Diana; Valério, Elisabete; Reis, Ana[EN] Microcystins (MC) are the type of hepatotoxins more abundantly produced by cyanobacteria. Studies have shown that these toxins affect many multicellular organisms that inhabit aquatic ecosystems, however their impact on bacteria that cohabit with freshwater cyanobacteria is still unclear. In this work the impact of three variants of the MC (-LR,-RR,-YR) was evaluated on growth and antioxidant system of heterotrophic bacteria isolated from three Portuguese reservoirs where blooms of cyanobacteria are often observed, some having microcystin-producing strains, and also in bacteria isolated from a reservoir where these phenomena do not occur. To this end, morphological and molecular characterization of the bacterial isolates was proceeded and these bacteria were exposed to three different concentrations of each variant of the MC, and the effect on the bacterial growth curves was evaluated. The enzymatic activity of catalase (CAT) and SuperOxide Dismutase (SOD1 and SOD2) was determined spectrophotometrically at 240 nm and 550 nm, respectively, in cells exposed to the microcystin variants. It was found that MC can reduce the growth of most bacteria tested (62.5%), and some bacterial cultures grown with no effect (37.5%), while others reacted differently depending on the variant and concentration used on the same isolate. However, in two isolated bacteria a slight stimulation of growth was observed, although with no statistical significance. The results of the determination of CAT and SOD activities showed that the bacterial isolates were susceptible to 10 nM of each variant MC. In all strains tested there was an increase in CAT activity and, in relation to the SOD1 and SOD2 activities it was observed that, most bacteria had an increase of the each SOD activity when exposed to MC. However, not all isolates showed effects on SOD1 or SOD2 activities in the three variants of the MC used. This study showed that MCs can induce a reduction on the growth of most bacteria isolated from freshwater. In respect to the antioxidant system enzymes, all results point out that microcystins can induce oxidative stress in the bacteria tested and that CAT and SOD activities were activated as a defense mechanism to scavenge reactive oxygen species (ROS) increment.
- Phosphorylation of SRSF1 by SRPK1 regulates alternative splicing of tumor-related Rac1bPublication . Gonçalves, Vânia; Matos, Paulo; Neves-Costa, Ana; Moyer, M.P.; Ferreira-Moita, Luís; Gama-Carvalho, Margarida; Jordan, PeterThe pre-messenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications such as protein phosphorylation, which are determined by signal transduction pathways. Here we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells we found that depletion of AKT2, AKT3, GSK3β and SRPK1 significantly decreased endogenous Rac1b levels. Whereas knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3β or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insights into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival.
- A novel Rac1/PAK1/BCL-6/STAT5 pathway modulates the expression of cell-cycle-associated genesPublication . Barros, Patrícia; Lam, Eric; Jordan, Peter; Matos, PauloGene expression depends on binding of transcriptional regulators to gene promoters, a process controlled by signalling pathways. The transcriptional repressor BCL-6 downregulates genes involved in cell cycle progression and becomes inactivated following phosphorylation by the Rac1 GTPase activated protein kinase PAK1. Interestingly, the DNA motifs recognized by BCL-6 and STAT5 are similar. Because STAT5 stimulation in epithelial cells can also be triggered by Rac1 signalling, we asked whether both factors have opposing roles in transcriptional regulation and whether Rac1 signalling may coordinate a transcription factor switch. We used chromatin immunoprecipitation to show that active Rac1 promotes release of the repressor BCL-6 while increasing binding of STAT5A to a BCL-6-regulated reporter gene. We further show in colorectal cell lines that the endogenous activation status of the Rac1/PAK1 pathway correlated with the phosphorylation status of BCL-6 and STAT5A. Three cellular genes (cyclin D2, p15INK4B, SUMO1) were identified to be inversely regulated by BCL-6 and STAT5A and responded to Rac1 signalling with increased expression and corresponding changes in promoter occupancy. Together, our data show that Rac1 signalling controls a group of target genes that are repressed by BCL-6 and activated by STAT5A, providing novel insights into the modulation of gene transcription by GTPase signalling.
- Sistema de organização e classificação documental - o exemplo da UOVPublication . Brazão, Roberto