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+/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.
BTB domain; Cullin–RING E3 ligase (CRL); Kelch-like domain (KLHL domain); Na+/Cl− co-transporter (NCC); Na+/K+/2Cl− co-transporter 2 (NKCC2); SPS1-related proline/alanine-rich kinase/oxidative stress-responsive kinase 1 (SPAK/OSR1); ubiquitin; CUL3, Cullin-3; CRL, Cullin–RING E3 ligase; DCT, distal convoluted tubule; DTT, dithiothreitol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GST, glutathione transferase; HEK, human embryonic kidney; HRP, horseradish peroxidase; KEAP1, Kelch-like ECH-associated protein 1; KLHL3, Kelch-like 3; LC, liquid chromatography; NCC, Na+/Cl− co-transporter; NKCC, Na+/K+/2Cl− co-transporter; NRF2, NF-E2-related factor 2; OSR1, oxidative stress-responsive kinase 1; qRT-PCR, real time quantitative reverse transcription PCR; RBX1, RING-box 1, E3 ubiquitin protein ligase; RPL13A, ribosomal protein L13a; RT, reverse transcription; rTEV, recombinant tobacco etch virus; siRNA, small interfering RNA; SPAK, SPS1-related proline/alanine-rich kinase; TAL, thick ascending limb; TTBS, Tris-buffered saline containing Tween 20; UBE1, ubiquitin-like modifier-activating enzyme 1; UBE2D3, ubiquitin-conjugating enzyme E2 D3; WNK, with no lysine kinase
The proteins from the UBA-UBX family interact with ubiquitylated proteins via their UBA domain and with p97 via their UBX domain, thereby acting as substrate-binding adaptors for the p97 ATPase. In particular, human UBXN7 (also known as UBXD7) mediates p97 interaction with the transcription factor HIF1α that is actively ubiquitylated in normoxic cells by a CUL2-based E3 ligase, CRL2. Mass spectrometry analysis of UBA-UBX protein immunoprecipitates showed that they interact with a multitude of E3 ubiquitin-ligases. Conspicuously, UBXN7 was most proficient in interacting with cullin-RING ligase subunits. We therefore set out to determine whether UBXN7 interaction with cullins was direct or mediated by its ubiquitylated targets bound to the UBA domain.
We show that UBXN7 interaction with cullins is independent of ubiquitin- and substrate-binding. Instead, it relies on the UIM motif in UBXN7 that directly engages the NEDD8 modification on cullins. To understand the functional consequences of UBXN7 interaction with neddylated cullins, we focused on HIF1α, a CUL2 substrate that uses UBXD7/p97 as a ubiquitin-receptor on its way to proteasome-mediated degradation. We find that UBXN7 over-expression converts CUL2 to its neddylated form and causes the accumulation of non-ubiquitylated HIF1α. Both of these effects are strictly UIM-dependent and occur only when UBXN7 contains an intact UIM motif. We also show that HIF1α carrying long ubiquitin-chains can recruit alternative ubiquitin-receptors, lacking p97's ATP-dependent segregase activity.
Our study shows that independently of its function as a ubiquitin-binding adaptor for p97, UBXN7 directly interacts with neddylated cullins and causes the accumulation of the CUL2 substrate HIF1α. We propose that by sequestering CUL2 in its neddylated form, UBXN7 negatively regulates the ubiquitin-ligase activity of CRL2 and this might prevent recruitment of ubiquitin-receptors other than p97 to nuclear HIF1α.
cullin; NEDD8; p97; ubiquitin-dependent degradation; UBXD7