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.
Highlights
SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 are closely related protein kinases, which play key roles in regulating cellular ion homeostasis and blood pressure (BP) [1,2]
Our results indicate that conserved carboxy-terminal (CCT) domain inhibitors would be effective at reducing BP by lowering phosphorylation as well as expression of NCC and NKCC2
In vitro fluorescence polarisation studies confirmed that mutation of this Leu residue in the SPAK CCT domain reduced binding to an RFXV-motif-possessing peptide derived from WNK4 by ∼100-fold (Fig. 1C)
Summary
SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) are closely related protein kinases, which play key roles in regulating cellular ion homeostasis and blood pressure (BP) [1,2]. SPAK/OSR1 substrates comprise the SLC12A (solute carrier family 12) family of electroneutral CCCs (cation–Cl co-transporters) [6,7,8,9,10,11] These transporters regulate intracellular chloride concentration critical in controlling BP and cell volume homoeostasis [12,13]. SPAK/OSR1 protein kinases drive chloride influx by phosphorylation and activating sodium-driven CCC members. These include the NCC (Na–Cl co-transporter) in the distal convoluted tubule of the kidney [10], the NKCC2 (Na–K–2Cl co-transporter 2) in the thick ascending limb (TAL) of the kidney [9] and the ubiquitously expressed NKCC1 [6,7,8]. SPAK/OSR1 phosphorylate and inhibit potassium-driven CCCs that drive chloride efflux [11], 4546 | Human Molecular Genetics, 2015, Vol 24, No 16
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