CoChat
Sign Up
Cited by 418Ion Transport and Channel RegulationProtein Kinase Regulation and GTPase SignalingIon channel regulation and function

Chloride sensing by WNK1 involves inhibition of autophosphorylation

Open access
Piala, Alexander T., Moon, Thomas M., Akella, Radha, He, Haixia, Cobb, Melanie H., Goldsmith, Elizabeth J.·2014·Science Signaling
DOI 10.1126/scisignal.2005050PMC PMC4123527
Open this paper in CoChat — free account

Abstract

WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade, leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. We found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride-binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1126/scisignal.2005050

Cites

1 of 1 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • WNK1 interacts with chloride ions to alter its conformation, facilitating capture of SGK1, promoting mTORC2-SGK1 interaction and SGK1 phosphorylation while inhibiting WNK1 kinase activity.

    “Additionally, WNK1 interacts with chloride ions (Cl−) to alter its conformation, facilitating the capture of SGK1 (Piala et al., 2014; Terker et al., 2015). This conformational change promotes the interaction between Mammalian Target of Rapamycin Complex 2 (mTORC2) and SGK1 and enhances the phosphorylation of SGK1 while simultaneously inhibiting the kinase activity of WNK1.”

    From Serum/glucocorticoid regulated kinase 1 (SGK1) in neurological disorders: pain or gain (2024) · cites this paper as Piala et al., 2014

    The Abstract and Results report that chloride binds WNK1, stabilizes its inactive conformation, and inhibits its autophosphorylation and activation. The Introduction mentions that WNK1 activates SGK1, but our copy does not support the claimed chloride-dependent capture of SGK1, increased mTORC2–SGK1 interaction, or enhanced SGK1 phosphorylation. No page numbers are available in our copy. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 74

  1. [1]Russell JM Sodium-potassium-chloride cotransport Physiol Rev 80 211 276 2000 10617769
  2. [2]Kahle KT Rinehart J Lifton RP Phosphoregulation of the Na-K-2Cl and K-Cl cotransporters by the WNK kinases Biochim Biophys Acta 1802 1150 1158 2010 20637866 PMC3529164
  3. [3]Salomonsson M Gonzalez E Westerlund P Persson AE Chloride concentration in macula densa and cortical thick ascending limb cells Kidney Int Suppl 32 S51 54 1991 1881050
  4. [4]Pellegrino C Gubkina O Schaefer M Becq H Ludwig A Mukhtarov M Chudotvorova I Corby S Salyha Y Salozhin S Bregestovski P Medina I Knocking down of the KCC2 in rat hippocampal neurons increases intracellular chloride concentration and compromises neuronal survival J Physiol 589 2475 2496 2011 21486764 PMC3115820
  5. [5]Miyazaki H Shiozaki A Niisato N Marunaka Y Physiological significance of hypotonicity-induced regulatory volume decrease: reduction in intracellular Cl− concentration acting as an intracellular signaling Am J Physiol Renal Physiol 292 F1411 1417 2007 17244897
  6. [6]Ohsawa R Miyazaki H Niisato N Shiozaki A Iwasaki Y Otsuji E Marunaka Y Intracellular chloride regulates cell proliferation through the activation of stress-activated protein kinases in MKN28 human gastric cancer cells J Cell Physiol 223 764 770 2010 20205250
  7. [7]Guggino WB Stanton BA New insights into cystic fibrosis: molecular switches that regulate CFTR Nat Rev Mol Cell Biol 7 426 436 2006 16723978
  8. [8]Chebib M Johnston GA The ‘ABC’ of GABA receptors: a brief review Clin Exp Pharmacol Physiol 26 937 940 1999 10561820
  9. [9]Dorwart MR Shcheynikov N Yang D Muallem S The solute carrier 26 family of proteins in epithelial ion transport Physiology 23 104 114 2008 18400693
  10. [10]Dutzler R Campbell EB Cadene M Chait BT MacKinnon R X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity Nature 415 287 294 2002 11796999
  11. [11]Ogawa H Qiu Y Philo JS Arakawa T Ogata CM Misono KS Reversibly bound chloride in the atrial natriuretic peptide receptor hormone-binding domain: possible allosteric regulation and a conserved structural motif for the chloride-binding site Protein Sci 19 544 557 2010 20066666 PMC2866279
  12. [12]Tzakos AG Galanis AS Spyroulias GA Cordopatis P Manessi-Zoupa E Gerothanassis IP Structure-function discrimination of the N− and C− catalytic domains of human angiotensin-converting enzyme: implications for Cl− activation and peptide hydrolysis mechanisms Protein Eng 16 993 1003 2003 14983080
  13. [13]Pokhrel R McConnell IL Brudvig GW Chloride regulation of enzyme turnover: application to the role of chloride in photosystem II Biochemistry 50 2725 2734 2011 21366335
  14. [14]Tavoulari S Rizwan AN Forrest LR Rudnick G Reconstructing a chloride-binding site in a bacterial neurotransmitter transporter homologue J Biol Chem 286 2834 2842 2011 21115480 PMC3024779
  15. [15]Perutz MF Shih DT Williamson D The chloride effect in human haemoglobin. A new kind of allosteric mechanism J Mol Biol 239 555 560 1994 8006967
  16. [16]Li X Surguchev A Bian S Navaratnam D Santos-Sacchi J Extracellular chloride regulation of Kv2.1, contributor to the major outward Kv current in mammalian outer hair cells Am J Physiol Cell Physiol 302 C296 306 2012 21940671 PMC4054960
  17. [17]Sangan P Rajendran VM Geibel JP Binder HJ Cloning and expression of a chloride-dependent Na+-H+ exchanger J Biol Chem 277 9668 9675 2002 11773056
  18. [18]Gamba G Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporters Physiol Rev 85 423 493 2005 15788703
  19. [19]Lytle C Forbush B 3rd Regulatory phosphorylation of the secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl Am J Physiol 270 C437 448 1996 8779905
  20. [20]Lytle C Forbush B 3rd Na-K-Cl cotransport in the shark rectal gland. II. Regulation in isolated tubules Am J Physiol 262 C1009 1017 1992 1314482
  21. [21]Pacheco-Alvarez D Cristobal PS Meade P Moreno E Vazquez N Munoz E Diaz A Juarez ME Gimenez I Gamba G The Na+:Cl− cotransporter is activated and phosphorylated at the amino-terminal domain upon intracellular chloride depletion J Biol Chem 281 28755 28763 2006 16887815
  22. [22]Jennings ML Schulz RK Okadaic acid inhibition of KCl cotransport. Evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or N-ethylmaleimide J Gen Physiol 97 799 817 1991 1647439 PMC2216490
  23. [23]Haas M Forbush B 3rd The Na-K-Cl cotransporter of secretory epithelia Annu Rev Physiol 62 515 534 2000 10845101
  24. [24]Lytle C McManus T Coordinate modulation of Na-K-2Cl cotransport and K-Cl cotransport by cell volume and chloride Am J Physiol Cell Physiol 283 C1422 1431 2002 12372803
  25. [25]Haas M McBrayer D Lytle C [Cl−]i-dependent phosphorylation of the Na-K-Cl cotransport protein of dog tracheal epithelial cells J Biol Chem 270 28955 28961 1995 7499426
  26. [26]Xu BE English JM Wilsbacher JL Stippec S Goldsmith EJ Cobb MH WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II J Biol Chem 275 16795 16801 2000 10828064
  27. [27]Min X Lee BH Cobb MH Goldsmith EJ Crystal structure of the kinase domain of WNK1, a kinase that causes a hereditary form of hypertension Structure 12 1303 1311 2004 15242606
  28. [28]Wilson FH Disse-Nicodeme S Choate KA Ishikawa K Nelson-Williams C Desitter I Gunel M Milford DV Lipkin GW Achard JM Feely MP Dussol B Berland Y Unwin RJ Mayan H Simon DB Farfel Z Jeunemaitre X Lifton RP Human hypertension caused by mutations in WNK kinases Science 293 1107 1112 2001 11498583
  29. [29]Lemmink HH Knoers NV Karolyi L van Dijk H Niaudet P Antignac C Guay-Woodford LM Goodyer PR Carel JC Hermes A Seyberth HW Monnens LA van den Heuvel LP Novel mutations in the thiazide-sensitive NaCl cotransporter gene in patients with Gitelman syndrome with predominant localization to the C-terminal domain Kidney Int 54 720 730 1998 9734597
  30. [30]Piechotta K Lu J Delpire E Cation chloride cotransporters interact with the stress-related kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) J Biol Chem 277 50812 50819 2002 12386165
  31. [31]Dowd BF Forbush B PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na-K-Cl cotransporter (NKCC1) J Biol Chem 278 27347 27353 2003 12740379
  32. [32]Zagorska A Pozo-Guisado E Boudeau J Vitari AC Rafiqi FH Thastrup J Deak M Campbell DG Morrice NA Prescott AR Alessi DR Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress J Cell Biol 176 89 100 2007 17190791 PMC2063630
  33. [33]Anselmo AN Earnest S Chen W Juang YC Kim SC Zhao YM Cobb MH WNK1 and OSR1 regulate the Na+, K+, 2Cl(−) cotransporter in HeLa cells P Natl Acad Sci USA 103 10883 10888 2006 PMC1544143 16832045
  34. [34]Richardson C Rafiqi FH Karlsson HKR Moleleki N Vandewalle A Campbell DG Morrice NA Alessi DR Activation of the thiazide-sensitive Na+-Cl− cotransporter by the WNK-regulated kinases SPAK and OSR1 J Cell Sci 121 675 684 2008 18270262
  35. [35]Vitari AC Deak M Morrice NA Alessi DR The WNK1 and WNK4 protein kinases that are mutated in Gordon’s hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases Biochem J 391 17 24 2005 16083423 PMC1237134
  36. [36]Moriguchi T Urushiyama S Hisamoto N Iemura S Uchida S Natsume T Matsumoto K Shibuya H WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE20-related kinases, SPAK and OSR1 J Biol Chem 280 42685 42693 2005 16263722
  37. [37]Ring AM Cheng SX Leng Q Kahle KT Rinehart J Lalioti MD Volkman HM Wilson FH Hebert SC Lifton RP WNK4 regulates activity of the epithelial Na+ channel in vitro and in vivo Proc Natl Acad Sci U S A 104 4020 4024 2007 17360470 PMC1805455
  38. [38]Xu BE Stippec S Lazrak A Huang CL Cobb MH WNK1 activates SGK1 by a phosphatidylinositol 3-kinase-dependent and non-catalytic mechanism J Biol Chem 280 34218 34223 2005 16081417
  39. [39]Debonneville C Flores SY Kamynina E Plant PJ Tauxe C Thomas MA Munster C Chraibi A Pratt JH Horisberger JD Pearce D Loffing J Staub O Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression EMBO J 20 7052 7059 2001 11742982 PMC125341
  40. [40]Xu BE Stippec S Chu PY Lazrak A Li XJ Lee BH English JM Ortega B Huang CL Cobb MH WNK1 activates SGK1 to regulate the epithelial sodium channel Proc Natl Acad Sci U S A 102 10315 10320 2005 16006511 PMC1177404
  41. [41]Wang HR Liu Z Huang CL Domains of WNK1 kinase in the regulation of ROMK1 Am J Physiol Renal Physiol 295 F438 445 2008 18550644 PMC2519181
  42. [42]Kahle KT Wilson FH Leng Q Lalioti MD O’Connell AD Dong K Rapson AK MacGregor GG Giebisch G Hebert SC Lifton RP WNK4 regulates the balance between renal NaCl reabsorption and K+ secretion Nat Genet 35 372 376 2003 14608358
  43. [43]Ohta A Yang SS Rai T Chiga M Sasaki S Uchida S Overexpression of human WNK1 increases paracellular chloride permeability and phosphorylation of claudin-4 in MDCKII cells Biochem Biophys Res Commun 349 804 808 2006 16949040
  44. [44]Park HW Kim JY Choi SK Lee YH Zeng W Kim KH Muallem S Lee MG Serine-threonine kinase with-no-lysine 4 (WNK4) controls blood pressure via transient receptor potential canonical 3 (TRPC3) in the vasculature Proc Natl Acad Sci U S A 108 10750 10755 2011 21670282 PMC3127915
  45. [45]Ponce-Coria J San-Cristobal P Kahle KT Vazquez N Pacheco-Alvarez D de Los Heros P Juarez P Munoz E Michel G Bobadilla NA Gimenez I Lifton RP Hebert SC Gamba G Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases Proc Natl Acad Sci U S A 105 8458 8463 2008 18550832 PMC2448858
  46. [46]Kahle KT Rinehart J de Los Heros P Louvi A Meade P Vazquez N Hebert SC Gamba G Gimenez I Lifton RP WNK3 modulates transport of Cl− in and out of cells: implications for control of cell volume and neuronal excitability Proc Natl Acad Sci U S A 102 16783 16788 2005 16275911 PMC1283843
  47. [47]Cruz-Rangel S Gamba G Ramos-Mandujano G Pasantes-Morales H Influence of WNK3 on intracellular chloride concentration and volume regulation in HEK293 cells Pflugers Arch 464 317 330 2012 22864523
  48. [48]Rinehart J Vazquez N Kahle KT Hodson CA Ring AM Gulcicek EE Louvi A Bobadilla NA Gamba G Lifton RP WNK2 kinase is a novel regulator of essential neuronal cation-chloride cotransporters J Biol Chem 286 30171 30180 2011 21733846 PMC3191056
  49. [49]Pacheco-Alvarez D Gamba G WNK3 is a putative chloride-sensing kinase Cell Physiol Biochem 28 1123 1134 2011 22179001
  50. [50]Lenertz LY Lee BH Min X Xu BE Wedin K Earnest S Goldsmith EJ Cobb MH Properties of WNK1 and implications for other family members J Biol Chem 280 26653 26658 2005 15883153
  51. [51]Pantoliano MW Petrella EC Kwasnoski JD Lobanov VS Myslik J Graf E Carver T Asel E Springer BA Lane P Salemme FR High-density miniaturized thermal shift assays as a general strategy for drug discovery J Biomol Screen 6 429 440 2001 11788061
  52. [52]Xu BE Min X Stippec S Lee BH Goldsmith EJ Cobb MH Regulation of WNK1 by an autoinhibitory domain and autophosphorylation J Biol Chem 277 48456 48462 2002 12374799
  53. [53]Bunkoczi G Salah E Filippakopoulos P Fedorov O Muller S Sobott F Parker SA Zhang H Min W Turk BE Knapp S Structural and functional characterization of the human protein kinase ASK1 Structure 15 1215 1226 2007 17937911 PMC2100151
  54. [54]Dauter Z Dauter M Dodson E Jolly SAD Acta Crystallogr, Sect D: Biol Crystallogr 58 494 506 2002 11856836
  55. [56]Hanks SK Hunter T Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification FASEB J 9 576 596 1995 7768349
  56. [57]Jura N Zhang X Endres NF Seeliger MA Schindler T Kuriyan J Catalytic control in the EGF receptor and its connection to general kinase regulatory mechanisms Mol Cell 42 9 22 2011 21474065 PMC3175429
  57. [59]De Bondt HL Rosenblatt J Jancarik J Jones HD Morgan DO Kim SH Crystal structure of cyclin-dependent kinase 2 Nature 363 595 602 1993 8510751
  58. [60]Taylor SS Kornev AP Protein kinases: evolution of dynamic regulatory proteins Trends Biochem Sci 36 65 77 2011 20971646 PMC3084033
  59. [61]Kim C Cheng CY Saldanha SA Taylor SS PKA-I holoenzyme structure reveals a mechanism for cAMP-dependent activation Cell 130 1032 1043 2007 17889648
  60. [62]Kobe B Heierhorst J Feil SC Parker MW Benian GM Weiss KR Kemp BE Giant protein kinases: domain interactions and structural basis of autoregulation EMBO J 15 6810 6821 1996 9003756 PMC452506
  61. [63]Manning G Whyte DB Martinez R Hunter T Sudarsanam S The protein kinase complement of the human genome Science 298 1912 1934 2002 12471243
  62. [64]Pippig S Andexinger S Daniel K Puzicha M Caron MG Lefkowitz RJ Lohse MJ Overexpression of beta-arrestin and beta-adrenergic receptor kinase augment desensitization of beta 2-adrenergic receptors J Biol Chem 268 3201 3208 1993 8381421
  63. [65]Parker JC In defense of cell volume? Am J Physiol 265 C1191 1200 1993 8238472
  64. [66]Richardson C Alessi DR The regulation of salt transport and blood pressure by the WNK-SPAK/OSR1 signalling pathway J Cell Sci 121 3293 3304 2008 18843116
  65. [67]Darman RB Flemmer A Forbush B Modulation of ion transport by direct targeting of protein phosphatase type 1 to the Na-K-Cl cotransporter J Biol Chem 276 34359 34362 2001 11466303
  66. [68]Glover M Mercier Zuber A Figg N O’Shaughnessy KM The activity of the thiazide-sensitive Na(+)-Cl(−) cotransporter is regulated by protein phosphatase PP4 Can J Physiol Pharmacol 88 986 995 2010 20962898
  67. [69]Bukhtiyarova M Northrop K Chai X Casper D Karpusas M Springman E Improved expression, purification, and crystallization of p38alpha MAP kinase Protein Expr Purif 37 154 161 2004 15294293
  68. [70]Min XS Akella R He HX Humphreys JM Tsutakawa SE Lee SJ Tainer JA Cobb MH Goldsmith EJ The Structure of the MAP2K MEK6 Reveals an Autoinhibitory Dimer Structure 17 96 104 2009 19141286 PMC3689539
  69. [71]Perkins DN Pappin DJ Creasy DM Cottrell JS Probability-based protein identification by searching sequence databases using mass spectrometry data Electrophoresis 20 3551 3567 1999 10612281
  70. [72]Koenig T Menze BH Kirchner M Monigatti F Parker KC Patterson T Steen JJ Hamprecht FA Steen H Robust prediction of the MASCOT score for an improved quality assessment in mass spectrometric proteomics J Proteome Res 7 3708 3717 2008 18707158
  71. [73]Otwinowski Z Minor W Processing of X-ray diffraction data collected in oscillation mode Methods Enzymol 276 307 326 1997 27754618
  72. [74]N. Collaborative Computational Project The CCP4 suite: programs for protein crystallography Acta Crystallogr, Sect D: Biol Crystallogr 50 760 763 1994 15299374
  73. [75]Emsley P Lohkamp B Scott WG Cowtan K Features and development of Coot Acta Crystallogr, Sect D: Biol Crystallogr 66 486 501 2010 20383002 PMC2852313
  74. [76]Adams PD Afonine PV Bunkoczi G Chen VB Davis IW Echols N Headd JJ Hung LW Kapral GJ Grosse-Kunstleve RW McCoy AJ Moriarty NW Oeffner R Read RJ Richardson DC Richardson JS Terwilliger TC Zwart PH PHENIX: a comprehensive Python-based system for macromolecular structure solution Acta Crystallogr, Sect D: Biol Crystallogr 66 213 221 2010 20124702 PMC2815670

Cited by

Sorted by citations

Authors
Piala, A. T., Moon, T. M., Akella, R., He, H., Cobb, M. H., & Goldsmith, E. J.
Affiliations
The University of Texas Southwestern Medical Center (US)
Corresponding author
  • Piala, Alexander T.
  • Moon, Thomas M.
  • Goldsmith, Elizabeth J.
ORCID
Venue
Science Signaling
Publisher
American Association for the Advancement of Science (AAAS)
Published
2014-05-06
Volume
7
Issue
324
Pages
ra41-ra41
ISSN
  • 1937-9145 (ISSN-L)
  • 1945-0877
DOI
10.1126/scisignal.2005050
PMC
PMC4123527
OpenAlex
W2135394966
Cited by
418
References
1
Citations by year
2026: 34 · 2025: 21 · 2024: 37 · 2023: 49 · 2022: 42 · 2021: 35 · 2020: 32 · 2019: 32 · 2018: 44 · 2017: 19 · 2016: 36 · 2015: 28 · 2014: 9
FWCI
12.58
Type
journal article
Language
en
Open access
Locations
  • Science Signaling (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institute of General Medical Sciences (R01GM053032)
  • National Institute of Diabetes and Digestive and Kidney Diseases (R01DK092692)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion Transport and Channel Regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion channel regulation and function
Keywords
WNK1, chloride sensing, autophosphorylation inhibition, chloride binding site, catalytic lysine, kinase activation, NKCC, NCC, potassium-chloride cotransporter, cotransporters, familial hypertension, kinase cascade, crystal structure
MeSH terms
WNK Lysine-Deficient Protein Kinase 1, Chlorides, Humans, Models, Molecular, Phosphorylation, Minor Histocompatibility Antigens, Protein Serine-Threonine Kinases, Crystallography, X-Ray, Catalytic Domain, Intracellular Signaling Peptides and Proteins

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review