Disease-causing mutations in KLHL3 impair its effect on WNK4 degradation - PubMed (original) (raw)

Disease-causing mutations in KLHL3 impair its effect on WNK4 degradation

Guojin Wu et al. FEBS Lett. 2013.

Abstract

Mutations in with-no-lysine (K) kinase 4 (WNK4) and a ubiquitin E3 ligase complex component kelch-like 3 (KLHL3) both cause pseudohypoaldosteronism II (PHAII), a hereditary form of hypertension. We determined whether WNK4 or its effector is regulated by KLHL3 in Xenopus oocytes. KLHL3 inhibited the positive effect of WNK4 on Na(+)-Cl(-) cotransporter (NCC) by decreasing WNK4 protein abundance without decreasing that of NCC and the downstream kinase OSR1 directly. Ubiquitination and degradation of WNK4 were induced by KLHL3. The effect of KLHL3 on WNK4 degradation was blocked by a dominant negative form of cullin 3. All five PHAII mutations of KLHL3 tested disrupted the regulation on WNK4. We conclude that KLHL3 is a substrate adaptor for WNK4 in a ubiquitin E3 ligase complex.

Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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Figures

Fig. 1

Fig. 1

KLHL3 inhibited the effect of WNK4 on NCC-mediated Na+ uptake by decreasing WNK4 protein abundance. (A) KLHL3 inhibited NCC-mediated Na+ uptake and blocked the effect of WNK4 on NCC when expressed in X. laevis oocytes. Data from 33–45 oocytes/group from 4 independent experiments are shown as means ± S.E. * indicates P < 0.05; NS, not significant (_P_ > 0.05). (B) The effects of KLHL3 on the protein abundance of NCC, WNK4, and OSR1. HA-tagged NCC, WNK4, or OSR1 was expressed with HA- or FLAG-tagged KLHL3 in X. laevis oocytes and their protein abundance and that of phosphorylated serine 325 (p-S325) of OSR1 and β-actin (loading control) were assessed by Western blot analysis two days later. The levels of WNK4 and p-S325, but not those of NCC and total OSR1, were reduced by KLHL3.

Fig. 2

Fig. 2

The role of CUL3 in WNK4 stability. (A) HA-CUL3 moderately inhibited Na+ uptake of HA-NCC without reducing its protein abundance. Data from 33–35 oocytes/group from 4 independent experiments are shown as means ± S.E. * indicates P < 0.05. (B) Left panel, oocytes were injected with HA-WNK4 cRNA and 12 hrs later, HA-KLHL3 cRNA (or water in the control group) was injected. After 4 hrs, oocytes were treated with protein synthesis blocker cycloheximide (CHX) at 100 µg/ml. Proteins of oocytes were extracted at 12, 16, and 20 hrs after injection of HA-WNK4 cRNA. Upper panel shows the time points of experiment, and lower panel shows the level of HA-WNK4 and HA-KLHL3 at these points. Right panel, co-injection of HA-CUL3 1–400 partially blocked HA-KLHL3 induced reduction in HA-WNK4 abundance. (C) HA-CUL3 1–400 dose-dependently increases the level of HA-WNK4 protein in some batch of oocytes. (D) When the lysate of oocytes expressing GST-CUL3, GST-WNK4, or GST alone was mixed with lysate of oocytes expressing HA-KLHL3, HA-KLHL3 was pulled down by either GST-CUL3 or GST-WNK4 but not by GST alone.

Fig. 3

Fig. 3

KLHL3 increased WNK4 ubiquitination. cRNAs for HA-ubiquitin and GST-WNK4 (or GST as control) were injected into oocytes. After 12 hrs, oocytes were injected with cRNA for FLAG-KLHL3 or water as control. GST-WNK4 (A) and GST (B) proteins were pulled down at 0, 3, and 6 hrs after injection of FLAG-KLHL3 or water and the ubiquitinated proteins were detected by HA-antibody (upper panel). The levels of GST-WNK4 (or GST) in pulled down samples and FLAG-KLHL3 in oocytes lysate were detected with GST antibody and FLAG antibody, respectively.

Fig. 4

Fig. 4

PHAII-causing mutants of KLHL3 were less effective in reducing WNK4 protein abundance in X. laevis oocytes. (A) Representative PHAII-causing mutations examined in this study. (B) HA-WNK4 protein abundance assessed by Western blot analysis in the presence or absence of wild-type (WT) or mutants of HA-KLHL3. HA antibody detected both HA-WNK4 and HA-KLHL3 at different molecular weights. A summary of the band intensity of HA-WNK4 from three experiments is shown in the upper panel. (C) NCC-mediated Na+ uptake in the presence of WNK4 and WT or mutant HA-KLHL3. Similar to the Western blot experiments in (B), uptake experiments were performed at 36 hrs after cRNA injection. Data from 18 oocytes/group derived from 2 frogs are shown as means ± S.E. * indicates P < 0.05 vs. WT HA-KLHL3 group; # indicates P < 0.05 vs. the group without HA-KLHL3.

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