Browsing by Author "Loureiro, Claudia"
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- Molecular regulation of the plasma membrane retention of disease-related chloride channels CFTR and NKCC2Publication . Loureiro, Claudia; Matos, P; Clarke, Luka; Jordan, PeterThree disease-related chloride transport proteins, CFTR (in cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD)), NKCC2 and KCC3 (in kidney function and hypertension), were found to specific targets for protein kinase Syk which phosphorylates a specific tyrosine residue in each channel. Tyrosine phosphorylation downregulates the amount of CFTR [1] present at the plasma membrane and a better understanding of this process may reveal novel therapeutic options for CF patients. Thus, we determined the cellular adaptor proteins able to recognize the phospho-tyrosine modification and mediate traffic to the plasma membrane.
- A SYK/SHC1 pathway regulates the amount of CFTR in the plasma membranePublication . Loureiro, ClaudiaMutations in the CFTR gene cause the recessive genetic disease Cystic Fibrosis, where the chloride transport across the apical membrane of epithelial cells mediated by the CFTR protein is impaired. CFTR protein trafficking to the plasma membrane (PM) is the result of a complex interplay between the secretory and membrane recycling pathways that control the number of channels present at the membrane. In addition, the ion transport activity of CFTR at the PM is modulated through post-translational protein modifications. Previously we described that spleen tyrosine kinase (SYK) phosphorylates a specific tyrosine residue in the NBD1 domain and this modification can regulate the PM abundance of CFTR. Here we identified the underlying biochemical mechanism using peptide pull-down assays followed by mass spectrometry. We identified in bronchial-epithelial cells that the adaptor protein SHC1 recognizes tyrosine-phosphorylated CFTR through its phosphotyrosine binding (PTB) domain and that the formation of a complex between SHC1 and CFTR is induced at the PM in the presence of activated SYK. The depletion of endogenous SHC1 expression was sufficient to promote an increase in CFTR at the PM of these cells. The results identify a SYK/SHC1 pathway that regulates the PM levels of CFTR channels, contributing to a better understanding of how epithelial chloride secretion is regulated.
- The adaptor protein SHC1 is recruited to tyrosine-phosphorylated plasma membrane chloride channelsPublication . Loureiro, Claudia; Matos, Paulo; Clarke, Luka; Jordan, PeterProtein kinase Syk was recently found to phosphorylate a specific tyrosine residue in three distinct disease-related chloride transport proteins: CFTR (in cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD)), NKCC2 and KCC3 (in kidney function and hypertension).
- Tyrosine phosphorylation modulates cell surface expression of chloride cotransporters NKCC2 and KCC3Publication . Loureiro, Claudia; barros, Patricia; Matos, Paulo; Jordan, PeterIntroduction: Cellular chloride transport has a fundamental role in cell volume regulation and membrane potential, both in normal and tumour cells (1,2). Cellular chloride entry or exit are mediated at the plasma membrane by cotransporter proteins of the solute carrier 12 family. For example, NKCC2 resorbs chloride with sodium and potassium ions at the apical membrane of epithelial cells in the kidney, whereas KCC3 releases chloride with potassium ions at the basolateral membrane. Their ion transport activity is regulated by protein phosphorylation in response to signaling pathways. An additional regulatory mechanism concerns the amount of cotransporter molecules inserted into the plasma membrane. Experimental: Co-transporter constructs were transfected into HEK293 cells and the activity of SYK kinase modulated by incubation with SYK inhibitors or by co-transfection with siRNAs, kinase-dead, or constitutively active SYK mutants. Co-transporter abundance in the plasma membrane was analyzed by biotinylation of cell surface proteins. Results: Here we describe that tyrosine phosphorylation of NKCC2 and KCC3 regulates their plasma membrane expression levels. We identified that spleen tyrosine kinase (SYK) phosphorylates a specific N-terminal tyrosine residue in each cotransporter. Experimental depletion of endogenous SYK or pharmacological inhibition of its kinase activity increased the abundance of NKCC2 at the plasma membrane of human embryonic kidney cells. In contrast, overexpression of a constitutively active SYK mutant decreased NKCC2 membrane abundance. Intriguingly, the same experimental approaches revealed the opposite effect on KCC3 abundance at the plasma membrane, compatible with the known antagonistic roles of NKCC and KCC cotransporters in cell volume regulation. Conclusions: We identified a novel pathway modulating the cell surface expression of NKCC2 and KCC3 and show that this same pathway has opposite functional outcomes for these two cotransporters. The findings have several biomedical implications considering the role of these cotransporters in regulating blood pressure and cell volume.