{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,19]],"date-time":"2026-10-19T17:45:10Z","timestamp":1792431910493,"version":"4.5.0"},"reference-count":32,"publisher":"Portland Press Ltd.","issue":"1","content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2005,10,1]]},"abstract":"<jats:p>Mutations in the human genes encoding WNK1 [with no K (lysine) protein kinase-1] and the related protein kinase WNK4 are the cause of Gordon's hypertension syndrome. Little is known about the molecular mechanism by which WNK isoforms regulate cellular processes. We immunoprecipitated WNK1 from extracts of rat testis and found that it was specifically associated with a protein kinase of the STE20 family termed \u2018STE20\/SPS1-related proline\/alanine-rich kinase\u2019 (SPAK). We demonstrated that WNK1 and WNK4 both interacted with SPAK as well as a closely related kinase, termed \u2018oxidative stress response kinase-1\u2019 (OSR1). Wildtype (wt) but not catalytically inactive WNK1 and WNK4 phosphorylated SPAK and OSR1 to a much greater extent than with other substrates utilized previously, such as myelin basic protein and claudin-4. Phosphorylation by WNK1 or WNK4 markedly increased SPAK and OSR1 activity. Phosphopeptide mapping studies demonstrated that WNK1 phosphorylated kinase-inactive SPAK and OSR1 at an equivalent residue located within the T-loop of the catalytic domain (Thr233 in SPAK, Thr185 in OSR1) and a serine residue located within a C-terminal non-catalytic region (Ser373 in SPAK, Ser325 in OSR1). Mutation of Thr185 to alanine prevented the activation of OSR1 by WNK1, whereas mutation of Thr185 to glutamic acid (to mimic phosphorylation) increased the basal activity of OSR1 over 20-fold and prevented further activation by WNK1. Mutation of Ser325 in OSR1 to alanine or glutamic acid did not affect the basal activity of OSR1 or its ability to be activated by WNK1. These findings suggest that WNK isoforms operate as protein kinases that activate SPAK and OSR1 by phosphorylating the T-loops of these enzymes, resulting in their activation. Our analysis also describes the first facile assay that can be employed to quantitatively assess WNK1 and WNK4 activity.<\/jats:p>","DOI":"10.1042\/bj20051180","type":"journal-article","created":{"date-parts":[[2005,9,26]],"date-time":"2005-09-26T10:46:34Z","timestamp":1127731594000},"page":"17-24","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":488,"title":["The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases"],"prefix":"10.1042","volume":"391","author":[{"given":"Alberto\u00a0C.","family":"Vitari","sequence":"first","affiliation":[{"name":"MRC Protein Phosphorylation Unit, School of Life Sciences, MSI\/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Deak","sequence":"additional","affiliation":[{"name":"MRC Protein Phosphorylation Unit, School of Life Sciences, MSI\/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nick\u00a0A.","family":"Morrice","sequence":"additional","affiliation":[{"name":"MRC Protein Phosphorylation Unit, School of Life Sciences, MSI\/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dario\u00a0R.","family":"Alessi","sequence":"additional","affiliation":[{"name":"MRC Protein Phosphorylation Unit, School of Life Sciences, MSI\/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"288","published-online":{"date-parts":[[2005,9,26]]},"reference":[{"key":"2021112217133360200_B1","doi-asserted-by":"crossref","first-page":"16795","DOI":"10.1074\/jbc.275.22.16795","article-title":"WNK1, a novel mammalian serine\/threonine protein kinase lacking the catalytic lysine in subdomain II","volume":"275","author":"Xu","year":"2000","journal-title":"J. Biol. Chem."},{"key":"2021112217133360200_B2","doi-asserted-by":"crossref","first-page":"5562","DOI":"10.1038\/sj.onc.1204726","article-title":"WNK kinases, a novel protein kinase subfamily in multi-cellular organisms","volume":"20","author":"Verissimo","year":"2001","journal-title":"Oncogene"},{"key":"2021112217133360200_B3","doi-asserted-by":"crossref","first-page":"1107","DOI":"10.1126\/science.1062844","article-title":"Human hypertension caused by mutations in WNK kinases","volume":"293","author":"Wilson","year":"2001","journal-title":"Science"},{"key":"2021112217133360200_B4","doi-asserted-by":"crossref","first-page":"14109","DOI":"10.1073\/pnas.2336103100","article-title":"Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention","volume":"100","author":"Zambrowicz","year":"2003","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2021112217133360200_B5","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1016\/j.amjhyper.2003.12.020","article-title":"Three novel missense mutations of WNK4, a kinase mutated in inherited hypertension, in Japanese hypertensives: implication of clinical phenotypes","volume":"17","author":"Kamide","year":"2004","journal-title":"Am. J. Hypertens."},{"key":"2021112217133360200_B6","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.tem.2005.02.012","article-title":"Regulation of diverse ion transport pathways by WNK4 kinase: a novel molecular switch","volume":"16","author":"Kahle","year":"2005","journal-title":"Trends Endocrinol. Metab."},{"key":"2021112217133360200_B7","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/j.pharmthera.2004.11.010","article-title":"WNK kinases and the control of blood pressure","volume":"106","author":"Cope","year":"2005","journal-title":"Pharmacol. Ther."},{"key":"2021112217133360200_B8","doi-asserted-by":"crossref","first-page":"F245","DOI":"10.1152\/ajprenal.00311.2004","article-title":"Role of WNK kinases in regulating tubular salt and potassium transport and in the development of hypertension","volume":"288","author":"Gamba","year":"2005","journal-title":"Am. J. Physiol. Renal Physiol."},{"key":"2021112217133360200_B9","doi-asserted-by":"crossref","first-page":"372","DOI":"10.1038\/ng1271","article-title":"WNK4 regulates the balance between renal NaCl reabsorption and K+ secretion","volume":"35","author":"Kahle","year":"2003","journal-title":"Nat. Genet."},{"key":"2021112217133360200_B10","doi-asserted-by":"crossref","first-page":"1039","DOI":"10.1172\/JCI17443","article-title":"WNK kinases regulate thiazide-sensitive Na\u2013Cl cotransport","volume":"111","author":"Yang","year":"2003","journal-title":"J. Clin. Invest."},{"key":"2021112217133360200_B11","doi-asserted-by":"crossref","first-page":"4690","DOI":"10.1073\/pnas.0306924101","article-title":"Disease-causing mutant WNK4 increases paracellular chloride permeability and phosphorylates claudins","volume":"101","author":"Yamauchi","year":"2004","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2021112217133360200_B12","doi-asserted-by":"crossref","first-page":"741","DOI":"10.1016\/j.molcel.2004.07.018","article-title":"WNK1 phosphorylates synaptotagmin 2 and modulates its membrane binding","volume":"15","author":"Lee","year":"2004","journal-title":"Mol. Cell"},{"key":"2021112217133360200_B13","article-title":"Antibodies: a Laboratory Manual","author":"Harlow","year":"1988"},{"key":"2021112217133360200_B14","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1042\/bj3550597","article-title":"The kinase DYRK1A phosphorylates the transcription factor FKHR at Ser329 in vitro, a novel in vivo phosphorylation site","volume":"355","author":"Woods","year":"2001","journal-title":"Biochem. J."},{"key":"2021112217133360200_B15","doi-asserted-by":"crossref","first-page":"3551","DOI":"10.1002\/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2","article-title":"Probability-based protein identification by searching sequence databases using mass spectrometry data","volume":"20","author":"Perkins","year":"1999","journal-title":"Electrophoresis"},{"key":"2021112217133360200_B16","doi-asserted-by":"crossref","first-page":"E9","DOI":"10.1093\/nar\/30.2.e9","article-title":"High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells","volume":"30","author":"Durocher","year":"2002","journal-title":"Nucleic Acids Res."},{"key":"2021112217133360200_B17","first-page":"121","article-title":"Identification of protein phosphorylation sites by a combination of mass spectrometry and solid phase Edman sequencing","volume":"13","author":"Campbell","year":"2002","journal-title":"J. Biomol. Techn."},{"key":"2021112217133360200_B18","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1006\/abbi.1998.0736","article-title":"Molecular cloning and characterization of a novel Ste20-related protein kinase enriched in neurons and transporting epithelia","volume":"355","author":"Ushiro","year":"1998","journal-title":"Arch. Biochem. Biophys."},{"key":"2021112217133360200_B19","doi-asserted-by":"crossref","unstructured":"Gagnon\n              K.\u00a0B.\n            \n            \n              England\n              R.\n            \n            \n              Delpire\n              E.\n            \n          \n          Volume sensitivity of cation\u2013chloride cotransporters is modulated by the interaction of two kinases: SPAK and WNK4\n          Am. J. Physiol. Cell Physiol.\n          2005\n          \n            doi:10.1152\/ajpcell.0037.2005","DOI":"10.1152\/ajpcell.00037.2005"},{"key":"2021112217133360200_B20","first-page":"4","article-title":"Humoral immunity to human breast cancer: antigen definition and quantitative analysis of mRNA expression","volume":"1","author":"Scanlan","year":"2001","journal-title":"Cancer Immun."},{"key":"2021112217133360200_B21","doi-asserted-by":"crossref","first-page":"1379","DOI":"10.1172\/JCI22452","article-title":"Mechanisms of WNK1 and WNK4 interaction in the regulation of thiazide-sensitive NaCl cotransport","volume":"115","author":"Yang","year":"2005","journal-title":"J. Clin. Invest."},{"key":"2021112217133360200_B22","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1007\/s100380050121","article-title":"Isolation and characterization of a novel serine threonine kinase gene on chromosome 3p22\u201321.3","volume":"44","author":"Tamari","year":"1999","journal-title":"J. Hum. Genet."},{"key":"2021112217133360200_B23","doi-asserted-by":"crossref","first-page":"3062","DOI":"10.1093\/emboj\/cdg292","article-title":"Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD","volume":"22","author":"Baas","year":"2003","journal-title":"EMBO J."},{"key":"2021112217133360200_B24","doi-asserted-by":"crossref","first-page":"5102","DOI":"10.1093\/emboj\/cdg490","article-title":"MO25\u03b1\/\u03b2 interact with STRAD \u03b1\/\u03b2 enhancing their ability to bind, activate and localize LKB1 in the cytoplasm","volume":"22","author":"Boudeau","year":"2003","journal-title":"EMBO J."},{"key":"2021112217133360200_B25","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1042\/bj20031692","article-title":"WNK1, the kinase mutated in an inherited high-blood-pressure syndrome, is a novel PKB (protein kinase B)\/Akt substrate","volume":"378","author":"Vitari","year":"2004","journal-title":"Biochem. J."},{"key":"2021112217133360200_B26","doi-asserted-by":"crossref","first-page":"27347","DOI":"10.1074\/jbc.M301899200","article-title":"PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na\u2013K\u2013Cl cotransporter (NKCC1)","volume":"278","author":"Dowd","year":"2003","journal-title":"J. Biol. Chem."},{"key":"2021112217133360200_B27","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1016\/j.molcel.2004.08.024","article-title":"Regulation of protein kinases; controlling activity through activation segment conformation","volume":"15","author":"Nolen","year":"2004","journal-title":"Mol. Cell"},{"key":"2021112217133360200_B28","doi-asserted-by":"crossref","first-page":"52848","DOI":"10.1074\/jbc.M309436200","article-title":"Characterization of the interaction of the stress kinase SPAK with the Na+-K+-2Cl\u2212 cotransporter in the nervous system: evidence for a scaffolding role of the kinase","volume":"278","author":"Piechotta","year":"2003","journal-title":"J. Biol. Chem."},{"key":"2021112217133360200_B29","doi-asserted-by":"crossref","first-page":"50812","DOI":"10.1074\/jbc.M208108200","article-title":"Cation chloride cotransporters interact with the stress-related kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1)","volume":"277","author":"Piechotta","year":"2002","journal-title":"J. Biol. Chem."},{"key":"2021112217133360200_B30","doi-asserted-by":"crossref","first-page":"26653","DOI":"10.1074\/jbc.M502598200","article-title":"Properties of WNK1 and implications for other family members","volume":"280","author":"Lenertz","year":"2005","journal-title":"J. Biol. Chem."},{"key":"2021112217133360200_B31","doi-asserted-by":"crossref","first-page":"C1184","DOI":"10.1152\/ajpcell.1999.277.6.C1184","article-title":"Characterization of a phosphorylation event resulting in upregulation of the salivary Na+-K+-2Cl\u2212 cotransporter","volume":"277","author":"Kurihara","year":"1999","journal-title":"Am. J. Physiol."},{"key":"2021112217133360200_B32","doi-asserted-by":"crossref","first-page":"25438","DOI":"10.1016\/S0021-9258(19)74060-5","article-title":"The Na\u2013K\u2013Cl cotransport protein of shark rectal gland. II. Regulation by direct phosphorylation","volume":"267","author":"Lytle","year":"1992","journal-title":"J. Biol. Chem."}],"container-title":["Biochemical Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/391\/1\/17\/638874\/bj3910017.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/391\/1\/17\/638874\/bj3910017.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,11,22]],"date-time":"2021-11-22T19:27:46Z","timestamp":1637609266000},"score":1,"resource":{"primary":{"URL":"https:\/\/portlandpress.com\/biochemj\/article\/391\/1\/17\/78759\/The-WNK1-and-WNK4-protein-kinases-that-are-mutated"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2005,9,26]]},"references-count":32,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2005,10,1]]}},"URL":"https:\/\/doi.org\/10.1042\/bj20051180","relation":{"has-review":[{"id-type":"doi","id":"10.3410\/f.1027484.328569","asserted-by":"object"}]},"ISSN":["0264-6021","1470-8728"],"issn-type":[{"value":"0264-6021","type":"print"},{"value":"1470-8728","type":"electronic"}],"subject":[],"published":{"date-parts":[[2005,9,26]]}}}