Ohta A, Schumacher FR, Mehellou Y, Johnson C, Knebel A, Macartney TJ, Wood NT, Alessi DR, Kurz T

The WNK [with no lysine (K) kinase]-SPAK/OSR1 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 hypertension Gordon's syndrome patients with mutations in either Cullin-3 (CUL3) or the BTB-protein Kelch-like 3 (KLHL3). CUL3 assembles ...

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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/NKCC1). Strikingly, 13 out of 15 dominant KLHL3 disease mutations analysed inhibited binding to WNK1 or CUL3. Recombinant wild type CUL3-KLHL3 E3 ligase complex, but not a disease CUL3:KLHL3[R528H] mutant complex ubiquitylated WNK1 in vitro. Moreover, siRNA-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 to 667). Interestingly, the equivalent region in WNK4 encompasses residues that are mutated in Gordon syndrome patients. Strikingly, we found that the Gordon's disease causing WNK4[E562K] and WNK4[Q565E] mutations as well as the equivalent mutation in WNK1[479-667] fragment, abolished 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. Our findings also emphasise 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.

Date: Feb. 06, 2013

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