title: The CUL3-KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms: disease-causing mutations in KLHL3 and WNK4 disrupt interaction.
abstract: The WNK (with no lysine kinase)-SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) signalling pathway plays an important role in controlling mammalian blood pressure by modulating the activity of ion co-transporters in the kidney. Recent studies have identified Gordon's hypertension syndrome patients with mutations in either CUL3 (Cullin-3) or the BTB protein KLHL3 (Kelch-like 3). CUL3 assembles with BTB proteins to form Cullin-RING E3 ubiquitin ligase complexes. To explore how a CUL3-KLHL3 complex might operate, we immunoprecipitated KLHL3 and found that it associated strongly with WNK isoforms and CUL3, but not with other components of the pathway [SPAK/OSR1 or NCC (Na(+)/Cl(-) co-transporter)/NKCC1 (Na(+)/K(+)/2Cl(-) co-transporter 1)]. Strikingly, 13 out of the 15 dominant KLHL3 disease mutations analysed inhibited binding to WNK1 or CUL3. The recombinant wild-type CUL3-KLHL3 E3 ligase complex, but not a disease-causing CUL3-KLHL3[R528H] mutant complex, ubiquitylated WNK1 in vitro. Moreover, siRNA (small interfering RNA)-mediated knockdown of CUL3 increased WNK1 protein levels and kinase activity in HeLa cells. We mapped the KLHL3 interaction site in WNK1 to a non-catalytic region (residues 479-667). Interestingly, the equivalent region in WNK4 encompasses residues that are mutated in Gordon's syndrome patients. Strikingly, we found that the Gordon's disease-causing WNK4[E562K] and WNK4[Q565E] mutations, as well as the equivalent mutation in the WNK1[479-667] fragment, abolished the ability to interact with KLHL3. These results suggest that the CUL3-KLHL3 E3 ligase complex regulates blood pressure via its ability to interact with and ubiquitylate WNK isoforms. The findings of the present study also emphasize that the missense mutations in WNK4 that cause Gordon's syndrome strongly inhibit interaction with KLHL3. This could elevate blood pressure by increasing the expression of WNK4 thereby stimulating inappropriate salt retention in the kidney by promoting activation of the NCC/NKCC2 ion co-transporters. The present study reveals how mutations that disrupt the ability of an E3 ligase to interact with and ubiquitylate a critical cellular substrate such as WNK isoforms can trigger a chronic disease such as hypertension.
authors: MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK.
authors: Akihito
authors: A
authors: Ohta
authors: Frances-Rose
authors: FR
authors: Schumacher
authors: Youcef
authors: Y
authors: Mehellou
authors: Clare
authors: C
authors: Johnson
authors: Axel
authors: A
authors: Knebel
authors: Thomas J
authors: TJ
authors: Macartney
authors: Nicola T
authors: NT
authors: Wood
authors: Dario R
authors: DR
authors: Alessi
authors: Thimo
authors: T
authors: Kurz
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins; D015778:Minor Histocompatibility Antigens; D020125:Mutation, Missense; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011546:Pseudohypoaldosteronism / Q000201:enzymology* / Q000235:genetics; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism; D064506:Solute Carrier Family 12, Member 2; D054875:Ubiquitination; D000076204:WNK Lysine-Deficient Protein Kinase 1
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C503645:CUL3 protein, human; D002352:Carrier Proteins; D044842:Cullin Proteins; D047908:Intracellular Signaling Peptides and Proteins; C489837:KLHL3 protein, human; D008840:Microfilament Proteins; D015778:Minor Histocompatibility Antigens; C578877:SLC12A2 protein, human; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; C118135:OXSR1 protein, human; D017346:Protein Serine-Threonine Kinases; C486409:STK39 protein, human; D000076204:WNK Lysine-Deficient Protein Kinase 1; C498743:WNK1 protein, human; C433806:WNK4 protein, human
references: Biochim Biophys Acta. 2004 Nov 29;1695(1-3):133-70
references: 15571813
references: Mol Cell Biol. 2004 Dec;24(24):10941-53
references: 15572695
references: Nature. 2003 Sep 18;425(6955):316-21
references: 13679922
references: Biochem J. 2008 Jan 15;409(2):321-31
references: 18092945
references: Am J Nephrol. 2012;35(6):515-9
references: 22627394
references: J Cell Biol. 2007 Jan 1;176(1):89-100
references: 17190791
references: Curr Opin Nephrol Hypertens. 2008 Mar;17(2):186-92
references: 18277153
references: Anal Biochem. 2012 Sep 1;428(1):64-72
references: 22705378
references: Trends Biochem Sci. 2004 Dec;29(12):634-7
references: 15544948
references: Am J Physiol Renal Physiol. 2009 Oct;297(4):F838-48
references: 19474192
references: Physiol Rev. 2005 Apr;85(2):423-93
references: 15788703
references: Nat Methods. 2006 Dec;3(12):1021-32
references: 17117155
references: J Biol Chem. 2011 Sep 23;286(38):33613-21
references: 21828050
references: Nat Cell Biol. 2006 Apr;8(4):348-57
references: 16547521
references: Biochem J. 2005 Oct 1;391(Pt 1):17-24
references: 16083423
references: J Am Soc Nephrol. 2010 Nov;21(11):1868-77
references: 20813865
references: Science. 2001 Aug 10;293(5532):1107-12
references: 11498583
references: Antioxid Redox Signal. 2010 Dec 1;13(11):1699-712
references: 20486766
references: EMBO J. 2004 Apr 21;23(8):1681-7
references: 15071497
references: Nature. 2003 Sep 18;425(6955):311-6
references: 13679921
references: EMBO J. 2006 Aug 9;25(15):3605-17
references: 16888629
references: J Cell Sci. 2011 May 1;124(Pt 9):1391-5
references: 21486947
references: Nature. 2012 Jan 22;482(7383):98-102
references: 22266938
references: Nat Genet. 2006 Oct;38(10):1124-32
references: 16964266
references: Mol Cell Proteomics. 2005 Dec;4(12):2010-21
references: 16249172
references: Adv Exp Med Biol. 2011;717:115-25
references: 21713671
references: Am J Hum Genet. 2009 Jun;84(6):792-800
references: 19520207
references: Cell Metab. 2007 May;5(5):331-44
references: 17488636
references: EMBO J. 2011 May 4;30(9):1730-41
references: 21423148
references: Biochem J. 2006 Jul 1;397(1):223-31
references: 16669787
references: Mol Cell. 2006 Mar 3;21(5):689-700
references: 16507366
references: J Cell Sci. 2008 Mar 1;121(Pt 5):675-84
references: 18270262
references: Mol Cell Biol. 2004 Oct;24(19):8477-86
references: 15367669
references: Nat Genet. 2012 Mar 11;44(4):456-60, S1-3
references: 22406640
references: J Cell Sci. 2011 Mar 1;124(Pt 5):789-800
references: 21321328
references: Annu Rev Biochem. 1998;67:425-79
references: 9759494
references: EMBO Mol Med. 2010 Feb;2(2):63-75
references: 20091762
references: Cell Div. 2008 Feb 18;3:7
references: 18282298