title: Phosphorylation by PKC and PKA regulate the kinase activity and downstream signaling of WNK4.
abstract: With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain.
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, CT 06510.|Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510.|Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, 14080 Mexico City, Mexico.
authors.forename: Maria
authors.initials: M
authors.lastname: Castañeda-Bueno
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, CT 06510.|Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510.
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Arroyo
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, CT 06510.|Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510.
authors.forename: Junhui
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, CT 06510.|Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Puthumana
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, CT 06510.|Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510.
authors.forename: Orlando
authors.initials: O
authors.lastname: Yarborough
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, CT 06510.|Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Shibata
authors.affiliation: Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, 14080 Mexico City, Mexico.|Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, 14080 Mexico City, Mexico.
authors.forename: Lorena
authors.initials: L
authors.lastname: Rojas-Vega
authors.affiliation: Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, 14080 Mexico City, Mexico.|Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, 14080 Mexico City, Mexico.
authors.forename: Gerardo
authors.initials: G
authors.lastname: Gamba
authors.affiliation: Department of Cellular and Molecular Physiology and Systems Biology Institute, Yale University School of Medicine, New Haven, CT 06510.
authors.forename: Jesse
authors.initials: J
authors.lastname: Rinehart
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, CT 06510.|Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510.|Laboratory of Human Genetics and Genomics, The Rockefeller University, New York, NY 10065.
authors.forename: Richard P
authors.initials: RP
authors.lastname: Lifton
mesh_terms: D000804:Angiotensin II / Q000502:physiology*; D000818:Animals; D001810:Blood Volume; D019556:COS Cells; D002522:Chlorocebus aethiops; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004573:Electrolytes / Q000378:metabolism; D005665:Furosemide / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007686:Kidney Tubules, Distal / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009154:Mutation; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011546:Pseudohypoaldosteronism / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D013148:Spironolactone / Q000494:pharmacology; D014882:Water-Electrolyte Balance / Q000502:physiology
chemical_list: D004573:Electrolytes; D011994:Recombinant Proteins; D000804:Angiotensin II; D010768:Phosphoserine; D013148:Spironolactone; D005665:Furosemide; C499024:Prkwnk4 protein, mouse; D017346:Protein Serine-Threonine Kinases; C433806:WNK4 protein, human; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C
keywords: NCC; distal convoluted tubule; hypertension; renal electrolyte transport; renin-angiotensin-aldosterone system