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 - PubMed (original) (raw)

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 et al. Biochem J. 2013.

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.

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Figures

Figure 1

Figure 1. Evidence that KLHL3 associates with WNK isoforms and interaction is impaired by Gordon's syndrome mutations

(A) Control 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. Cells were lysed and KLHL3 was immunoprecipitated from 5 mg of extract using an anti-FLAG antibody covalently coupled to agarose. The immunoprecipitates were electrophoresed on an SDS polyacrylamide gel and protein bands visualized following Colloidal Blue staining. Molecular masses are shown on the left-hand side in kDa. (B) The labelled Colloidal Blue-stained bands identified in (A) were excised from the gel and digested with trypsin, and their identities were determined by tryptic peptide mass-spectral fingerprinting, as described in the Materials and methods section. Accession numbers are for the NCBI Entrez Protein database. Mascot protein scores >67 were considered significant (P<0.05). N.P.D., no significant protein identity determined. MW, molecular mass. (C) As (A), except that the immunoprecipitates were immunoblotted with the indicated antibodies. Similar results were obtained in three separate experiments. Molecular masses are shown on the left-hand side in kDa.

Figure 2

Figure 2. Evidence that dominant KLHL3 Gordon's syndrome mutations impair binding to CUL3 or WNK

(A) Schematic representation of the domain structure of KLHL3 with the positions of the dominant Gordon's syndrome-associated mutations illustrated. The amino acid boundaries of the domains are indicated. PHAII, pseudohypoaldosteronism type II. (B) Control HEK-293 cells or HEK-293 cells stably expressing the indicated forms of wild-type (WT) and mutant KLHL3 possessing an N-terminal FLAG tag were cultured in the presence of 1 μg/ml tetracyclin to induce expression of KLHL3. Cells were lysed and KLHL3 was immunoprecipitated (IP) from 0.2 mg of extract using an anti-FLAG antibody covalently coupled to agarose. The immunoprecipitates (upper panel) as well as whole-cell extracts (lysates, lower panel) were immunoblotted with the indicated antibodies. All gels were immunoblotted in parallel. The broken lines indicate that samples were run on separate gels. Similar results were obtained in three separate experiments. Molecular masses are shown on the left-hand side in kDa.

Figure 3

Figure 3. Evidence that wild-type or dominant KLHL3 Gordon's syndrome mutations do not interact with SPAK/OSR1 or NKCC1 or NCC

(A) HEK-293 stably expressing the wild-type (WT) and indicated mutants of KLHL3 possessing an N-terminal FLAG epitope were cultured in the presence of 1 μg/ml tetracyclin to induce the expression of KLHL3. WNK1, SPAK/OSR1 and NKCC1 were separately immunoprecipitated (IP) from 0.2 mg of extract using previously characterized immunoprecipitating antibodies covalently coupled to Protein G–Sepharose. Whole-cell extracts (lysates, upper panel) as well as immunoprecipitates (lower panels) were immunoblotted with the indicated antibodies. (B) N-terminal GFP-tagged wild-type KLHL3 was transiently transfected into previously characterized HEK-293 cells stably expressing FLAG–NCC [4]. At 24 h post-transfection cells were cultured for a further 24 h with 1 μg/ml tetracyclin to induce NCC expression before lysis. GFP–KLHL3 was immunoprecipitated from 0.2 mg of extract employing anti-GFP antibody covalently coupled to agarose. Whole-cell extracts (lysates, left-hand panel) as well as immunoprecipitates (right-hand panel) were immunoblotted with the indicated antibodies. Similar results were obtained in three separate experiments. Molecular masses are shown on the left-hand side in kDa.

Figure 4

Figure 4. WNK1 is a substrate for the CUL3–KLHL3 complex in vitro

(A) Coomassie Blue-stained SDS/PAGE gel of Cul3–Rbx1–KLHL3 wild-type, Cul3–Rbx1–KHLH3[R528H], ubiquitin (Ub), E1 and UBE2D3 used in the_in vitro_ reactions in which all tags used for affinity purification of each protein has been removed with the rTEV protease and the proteins were re-purified. The line indicates that samples were run on separate gels. (B) Immunoprecipitated WNK1 from HEK-293 cells was incubated at 30°C for 30 min with purified E1, E2 (UBE2D3), Cullin3–Rbx1, KLHL3 and ubiquitin in the presence of 1 mM ATP. Control reactions were performed whereby one of the proteins required for ubiquitylation was omitted from the reaction as indicated. Samples were subjected to immunoblot (IB) analysis with an anti-WNK1 antibody. (C) Ubiquitylation reactions were performed as in (B) and the ability of KLHL3 R528H to promote WNK1 ubiquitylation was analysed. Independent duplicate reactions are shown. (D) Ubiquitylation reactions were performed as in (B) and the ubiquitylation of KLHL3 was analysed by immunoblot detection with anti-KLHL3 total antibody. Similar results were obtained in three separate experiments. Molecular masses are shown on the left-hand side in kDa.

Figure 5

Figure 5. Evidence that CUL3 knockdown enhances WNK1 expression and activity

HeLa cells were treated with 50 nM scrambled siRNA or CUL3-directed siRNA (probe 1) for 5 days and cells were lysed. (A) Cell lysates were subjected to immunoblotting with the indicated antibody. Each lane contains cell extract from an independent dish of cells from an identical experiment undertaken on the same day. (B–D) Quantitative LI-COR immunoblot analysis was undertaken and the ratio of CUL3 (B), KLHL3 (C) and WNK1 (D) to GAPDH was quantified. Results are means±S.E.M. for two independent samples each assayed in duplicates with P values indicated. (E) Total mRNA was isolated from cells and WNK1 mRNA levels were determined by qRT-PCR. Data were normalized to internal GAPDH and RPL13A controls as described in the Materials and methods section. Results are means±S.D. for three independent samples each assayed in triplicate. (F) WNK1 was immunoprecipitated from 0.25 mg of cell extracts and assayed for its ability to phosphorylate kinase inactive SPAK expressed in E. coli cells. The top panel displays activity in 32P radioactivity incorporated into kinase inactive SPAK (c.p.m.) as mean±S.D. The lower panels display representative immunoblot analysis of WNK1, autorad of phosphorylated SPAK and Coomassie Blue staining of kinase-inactive SPAK employed in the kinase assay. Similar findings were made with an independent CUL3-directed siRNA (probe 2); see data in Supplementary Figure S1 (at

http://www.biochemj.org/bj/451/bj4510111add.htm

). Cont, control; KD, kinase-dead; IP, immunoprecipitation; RNAi, RNA interference.

Figure 6

Figure 6. Evidence that WNK4 Gordon's syndrome missense mutations impair binding to KLHL3

(A) Schematic diagram of WNK1 and WNK4 isoform structures and the relative placement of their kinase domain, auto-inhibitory domain (AID) and predicted coiled-coiled domains (CCD). Also highlighted in WNK4 is the location of the E562K and Q565E mutations found in Gordon's hypertension patients. CCD, coiled-coil; CT, C-terminal. (B) HEK-293 cells stably expressing GFP–KLHL3 were transfected with GST-tagged constructs encoding the indicated fragments of WNK1. At 24 h post-transfection the cells were treated with tetracyclin (1 μg/ml) to induce KLHL3 expression and cells were lysed after a further 24 h. Cell extracts were subjected to affinity purification on glutathione–Sepharose and immunoblotted with an anti-GFP antibody (top panel) or subjected to affinity purification with an anti-GFP antibody and immunoblotted with an anti-GST antibody (upper middle panel). Cell lysates were also subjected to immunoblot analysis with an anti-GST (lower middle panel) or anti-GFP (bottom panel) antibody. Similar results were obtained in two independent experiments. (C) HEK-293 cells stably expressing the wild-type (WT) and indicated mutant forms of FLAG-tagged WNK4 were transfected with wild-type GFP-tagged KLHL3. At 24 h post-transfection cells were treated with tetracyclin (1 μg/ml) to induce WNK4 expression and cells were lysed after 24 h. Extracts were subjected to affinity purification using an anti-FLAG antibody (to immunoprecipitate WNK4; upper panels) or an anti-GFP antibody (to immunoprecipitate KLHL3; lower panels) and immunoblotted with the indicated antibodies. Cell lysates were also immunoblotted with the indicated antibodies. Similar results were obtained in two independent experiments. (D) As (B) except that the wild-type and indicated mutants of the WNK1[479–667] fragment were analysed for their ability to interact with KLHL3. IP, immunoprecipitation. Molecular masses are shown on the left-hand side of the gels in kDa.

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