title: Critical role of the SPAK protein kinase CCT domain in controlling blood pressure.
abstract: The STE20/SPS1-related proline/alanine-rich kinase (SPAK) controls blood pressure (BP) by phosphorylating and stimulating the Na-Cl (NCC) and Na-K-2Cl (NKCC2) co-transporters, which regulate salt reabsorption in the kidney. SPAK possesses a conserved carboxy-terminal (CCT) domain, which recognises RFXV/I motifs present in its upstream activator [isoforms of the With-No-lysine (K) kinases (WNKs)] as well as its substrates (NCC and NKCC2). To define the physiological importance of the CCT domain, we generated knock-in mice in which the critical CCT domain Leu502 residue required for high affinity recognition of the RFXI/V motif was mutated to Alanine. The SPAK CCT domain defective knock-in animals are viable, and the Leu502Ala mutation abolished co-immunoprecipitation of SPAK with WNK1, NCC and NKCC2. The CCT domain defective animals displayed markedly reduced SPAK activity and phosphorylation of NCC and NKCC2 co-transporters at the residues phosphorylated by SPAK. This was also accompanied by a reduction in the expression of NCC and NKCC2 protein without changes in mRNA levels. The SPAK CCT domain knock-in mice showed typical features of Gitelman Syndrome with mild hypokalaemia, hypomagnesaemia, hypocalciuria and displayed salt wasting on switching to a low-Na diet. These observations establish that the CCT domain plays a crucial role in controlling SPAK activity and BP. Our results indicate that CCT domain inhibitors would be effective at reducing BP by lowering phosphorylation as well as expression of NCC and NKCC2.
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland and j.c.zhang@dundee.ac.uk kmo22@medschl.cam.ac.uk d.r.alessi@dundee.ac.uk.
authors.forename: Jinwei
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Experimental Medicine and Immunotherapeutics, Department of Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Keith
authors.initials: K
authors.lastname: Siew
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland and.
authors.forename: Thomas
authors.initials: T
authors.lastname: Macartney
authors.affiliation: Experimental Medicine and Immunotherapeutics, Department of Medicine, University of Cambridge, Cambridge, UK j.c.zhang@dundee.ac.uk kmo22@medschl.cam.ac.uk d.r.alessi@dundee.ac.uk.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: O'Shaughnessy
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland and j.c.zhang@dundee.ac.uk kmo22@medschl.cam.ac.uk d.r.alessi@dundee.ac.uk.
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001794:Blood Pressure; D053579:Gitelman Syndrome / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008817:Mice, Mutant Strains; D020125:Mutation, Missense; D010766:Phosphorylation / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D064486:Solute Carrier Family 12, Member 3 / Q000096:biosynthesis / Q000235:genetics
chemical_list: C578875:Slc12a3 protein, mouse; D064486:Solute Carrier Family 12, Member 3; C486407:Stk39 protein, mouse; D017346:Protein Serine-Threonine Kinases; C486409:STK39 protein, human