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

Akihito Ohta,Nicola T Wood,Thimo Kurz,Clare Johnson,Thomas J Macartney,Axel Knebel,Frances-Rose Schumacher,Dario R Alessi,Youcef Mehellou

doi:10.1042/bj20121903

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.

Highlights

Mutations that increase expression of the with no lysine kinase (WNK) 1 gene result in an inherited hypertension syndrome termed Gordon’s syndrome or PHAII [1]
MS analysis revealed that this region of the gel derived from wild-type Kelch-like 3 (KLHL3) immunoprecipitate contained significant levels of WNK1, WNK2 and WNK3 isoforms emphasized by very high Mascot scores (WNK1-3228, Figure 1 Evidence that KLHL3 associates with WNK isoforms and interaction is impaired by Gordon’s syndrome mutations (A) Control human embryonic kidney (HEK)-293 cells or HEK-293 cells stably expressing the indicated forms of wild-type and mutant KLHL3 possessing an N-terminal FLAG tag were cultured in the presence of 1 μg/ml tetracyclin to induce expression of KLHL3
These data, together with the fact that we were able to successfully reconstitute the KLHL3-dependent ubiquitylation of WNK1 in vitro and to increase WNK1 expression following CUL3 knockdown in vivo, suggest that WNK1 is a substrate for CUL3–KLHL3 and defines a new substrate adaptor/substrate module for a CUL3 complex implicated in human disease

Summary

Introduction

Mutations that increase expression of the WNK (with no lysine kinase) 1 gene result in an inherited hypertension syndrome termed Gordon’s syndrome or PHAII (pseudohypoaldosteronism type II) [1]. Missense mutations in the related WNK4 gene that alter three close-by non-catalytic residues (Glu562, Asp564 and Gln565) cause Gordon’s syndrome [1]. How these mutations influence WNK4 function is unknown. Most evidence points towards the WNK1 and WNK4 isoforms exerting their effects on blood pressure through their ability to phosphorylate and activate two highly related protein kinases termed SPAK [SPS1related proline/alanine-rich kinase; known as STK39 (serine threonine kinase 39)] and OSR1 (oxidative stress-responsive kinase 1) [2,3,4]. SPAK and OSR1 interact with MO25 (mouse protein-25) isoform subunits to form a maximally activated complex [5]. The SPAK and OSR1 kinases, once activated by WNK kinases, phosphorylate and activate members of the electroneutral cation-coupled chloride co-transporters [SLC12 (solute carrier family 12)], including the NCC (Na+ /Cl− cotransporter) and NKCC (Na+ /K+ /2Cl− co-transporter) 1 and 2, that are targets for the blood-pressure-lowering thiazide diuretic and loop diuretic drugs [4,6,7,8,9,10]

Methods

Results

Conclusion