title: WNK3 abrogates the NEDD4-2-mediated inhibition of the renal Na+-Cl- cotransporter.
abstract: The serine/threonine kinase WNK3 and the ubiquitin-protein ligase NEDD4-2 are key regulators of the thiazide-sensitive Na+-Cl- cotransporter (NCC), WNK3 as an activator and NEDD2-4 as an inhibitor. Nedd4-2 was identified as an interacting partner of WNK3 through a glutathione-S-transferase pull-down assay using the N-terminal domain of WNK3, combined with LC-MS/MS analysis. This was validated by coimmunoprecipitation of WNK3 and NEDD4-2 expressed in HEK293 cells. Our data also revealed that the interaction between Nedd4-2 and WNK3 does not involve the PY-like motif found in WNK3. The level of WNK3 ubiquitylation did not change when NEDD4-2 was expressed in HEK293 cells. Moreover, in contrast to SGK1, WNK3 did not phosphorylate NEDD4-2 on S222 or S328. Coimmunoprecipitation assays showed that WNK3 does not regulate the interaction between NCC and NEDD4-2. Interestingly, in Xenopus laevis oocytes, WNK3 was able to recover the SGK1-resistant NEDD4-2 S222A/S328A-mediated inhibition of NCC and further activate NCC. Furthermore, elimination of the SPAK binding site in the kinase domain of WNK3 (WNK3-F242A, which lacks the capacity to bind the serine/threonine kinase SPAK) prevented the WNK3 NCC-activating effect, but not the Nedd4-2-inhibitory effect. Together, these results suggest that a novel role for WNK3 on NCC expression at the plasma membrane, an effect apparently independent of the SPAK kinase and the aldosterone-SGK1 pathway.
authors.affiliation: Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland; and.
authors.forename: Dagmara
authors.initials: D
authors.lastname: Lagnaz
authors.affiliation: Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico.
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Arroyo
authors.affiliation: Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico.
authors.forename: María
authors.initials: M
authors.lastname: Chávez-Canales
authors.affiliation: Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico.
authors.forename: Norma
authors.initials: N
authors.lastname: Vázquez
authors.affiliation: Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland; and.
authors.forename: Federica
authors.initials: F
authors.lastname: Rizzo
authors.affiliation: Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland; and.
authors.forename: Alessia
authors.initials: A
authors.lastname: Spirlí
authors.affiliation: Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland; and.
authors.forename: Anne
authors.initials: A
authors.lastname: Debonneville
authors.affiliation: Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland; and.
authors.forename: Olivier
authors.initials: O
authors.lastname: Staub
authors.affiliation: Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico gamba@biomedicas.unam.mx.
authors.forename: Gerardo
authors.initials: G
authors.lastname: Gamba
mesh_terms: D000450:Aldosterone / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism* / Q000473:pathology; D023421:Models, Animal; D000075702:Nedd4 Ubiquitin Protein Ligases; D009865:Oocytes / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D049992:Sodium Chloride Symporters / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis; D000099184:Serum-Glucocorticoid Regulated Kinases
chemical_list: D000450:Aldosterone; D056827:Endosomal Sorting Complexes Required for Transport; D017874:Immediate-Early Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; D017346:Protein Serine-Threonine Kinases; D049992:Sodium Chloride Symporters; D044767:Ubiquitin-Protein Ligases; D029867:Xenopus Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618460:nedd4l protein, Xenopus; C519694:STK39 protein, Xenopus; C486409:STK39 protein, human; C000592126:WNK3 protein, Xenopus; C488586:WNK3 protein, human
keywords: WNK; distal convoluted tubule; hypertension; salt transport; ubiquitylation