title: Regulation of the expression of the Na/Cl cotransporter by WNK4 and WNK1: evidence that accelerated dynamin-dependent endocytosis is not involved.
abstract: The novel serine/threonine kinases (with no lysine kinases or WNKs), WNK1 and WNK4, are encoded by the disease genes for Gordon syndrome (PRKWNK1 and PRKWNK4), a rare monogenic syndrome of hypertension and hyperkalemia. These proteins alter the expression of the thiazide-sensitive Na/Cl cotransporter (NCCT) in Xenopus laevis oocytes, although the details are controversial. We describe here our own experience and confirm that kinase-dead WNK4 (318D>A) is unable to affect Na+ fluxes through the thiazide-sensitive Na/Cl transporter (NCCT) or its membrane expression as an ECFP-NCCT fusion protein. However, the kinase domain is not sufficient for a functional WNK4 since deletion of the acidic motif (a motif unique to WNK family members) completely abolishes functional activity. Indeed, the NH2 terminal of WNK4 (1-620) containing the kinase domain and acidic motif retains full activity, but does not interact directly with NCCT in pull-down assays. Coexpression of WNK1 antagonizes the action of WNK4, and kinase-dead WNK1 (368D>A) or WNK1 carrying a WNK4 disease mutation (565Q>E) behaves in the same way as wild-type WNK1. This suggests kinase activity and charge conservation within the acidic motif are not essential for the WNK1-WNK4 interaction. We also report that WNK4 probably reduces surface expression largely through an effect on forward trafficking. Hence, the effect of WNK4 on NCCT expression is mimicked by dynamin, but the dominant-negative K44A dynamin mutant does not block the action of WNK4 itself. These results further highlight important differences in the mechanism by which WNK kinases affect expression of NCCT vs. other membrane proteins such as ROMK.
authors.affiliation: Department of Medicine, University of Cambridge, UK.
authors.forename: Amir P
authors.initials: AP
authors.lastname: Golbang
authors.forename: Georgina
authors.initials: G
authors.lastname: Cope
authors.forename: Abbas
authors.initials: A
authors.lastname: Hamad
authors.forename: Meena
authors.initials: M
authors.lastname: Murthy
authors.forename: Che-Hsiung
authors.initials: CH
authors.lastname: Liu
authors.forename: Alan W
authors.initials: AW
authors.lastname: Cuthbert
authors.forename: Kevin M
authors.initials: KM
authors.lastname: O'shaughnessy
mesh_terms: D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D034281:Dynamins / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D017353:Gene Deletion; D006056:Golgi Apparatus / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D015778:Minor Histocompatibility Antigens; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D049992:Sodium Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D012979:Sodium Radioisotopes; D000076204:WNK Lysine-Deficient Protein Kinase 1; D014982:Xenopus laevis
chemical_list: D008565:Membrane Proteins; D015778:Minor Histocompatibility Antigens; D049992:Sodium Chloride Symporters; D012979:Sodium Radioisotopes; C499024:Prkwnk4 protein, mouse; D017346:Protein Serine-Threonine Kinases; D000076204:WNK Lysine-Deficient Protein Kinase 1; C498744:Wnk1 protein, mouse; D034281:Dynamins