Untitled

Funder

Organizational Unit

Publications

Regulatory crosstalk by protein kinases on CFTR trafficking and activity

Publication . Farinha, Carlos M.; Swiatecka-Urban, Agnieszka; Brautigan, David L.; Jordan, Peter

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

2016-01Journal article

Open access

Regulation of epithelial chloride transport by phospho-tyrosine-initiated protein networks

Publication . Loureiro, Cláudia; Jordan, Peter; Clarke, Luka

Ion transport is crucial for cell volume regulation by compensating variations in extracellular tonicity, playing an important role in maintaining the structural integrity and intracellular milieu in cells. These functions require a dynamic, spatio-temporally coordinated regulation of ion transport, which occurs in cells by two mechanisms: first, the amount of channel or cotransporter inserted into the plasma membrane (PM) from a pool of endosomal storage vesicles, and second, the ion transport activity regulated by post-translational modifications such as phosphorylation of channel or transporter proteins. Previous results from the host laboratory showed that phosphorylation by spleen tyrosine kinase (SYK) of Tyr512 in the NBD1 domain regulates PM abundance of CFTR, the chloride channel involved in cystic fibrosis. The main objective of this PhD project was to identify phospho-tyrosine-binding proteins involved in the regulation of chloride transport proteins and the underlying molecular mechanism. First, it was found that besides CFTR two further renal ion cotransporters, NKCC2 and KCC3, are phosphorylated by SYK in vitro on an N-terminal tyrosine residue and that experimental manipulation of either SYK expression levels or its catalytic activity affect the cell surface abundance of these cotransporters. Interestingly, the very same phosphorylation pathway leads to a decrease in NKCC2 but to an increase in KCC3 PM levels. Second, the underlying biochemical mechanism was identified using peptide pulldown assays followed by mass spectrometry and revealed that the adaptor protein SHC1 binds to phospho-tyrosine in NKCC2, KCC3 and CFTR through its PTB domain. Upon depletion of endogenous SHC1 expression, KCC3 decreased at the PM, whereas NKCC2 and CFTR levels increased. In the case of phosphorylated NKCC2, SNX27 and NCK1 were identified as additional binding partners. Lastly, SHC1 was shown to form a complex with CFTR following activation of protein kinase SYK, but does not affect the PM level of the most frequent mutant F508del-CFTR. The results described in this work identified a novel SYK/SHC1 pathway that regulates the cotransporters NKCC2 and KCC3 and the chloride channel CFTR and have potential biomedical implications for the identification of new therapeutic targets in diseases like hypertension or cystic fibrosis, or those involving regulation of cell volume.

2019-05-03Doctoral thesis

Restricted access

Metabolic tumor cell adaptation: tyrosine phosphorylation modulates cell surface expression of NKCC2 and KCC3

Publication . Loureiro, Cláudia; Barros, Patrícia; Matos, Paulo; Jordan, Peter

Introduction: Tumor cells require cellular chloride and potassium transport to adapt to a changing microenvironment, both for cell volume regulation and membrane potential maintenance. Cellular chloride and potassium 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. Material and Methods: Cotransporter constructs were transfected into HEK293 cells and the activity of spleen tyrosine kinase (SYK) modulated by incubation with SYK inhibitors or by co-transfection with siRNAs, kinase-dead, or constitutively active SYK mutants. Cotransporter abundance in the plasma membrane was analyzed by biotinylation of cell surface proteins. Results and Discussions: Here we describe that tyrosine phosphorylation of NKCC2 and KCC3 regulates their plasma membrane expression levels. We identified that 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. Conclusion: 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 add knowledge on how tumor cells may respond to microenvironmental changes that affect their cell volume or metabolic crosstalk.

2020-03-03Conference object

Open access