Fig 2: Renal kidney-specific with no lysine kinase 1 (KS-WNK1) expression is induced by a low-K+ diet (LKD). Total kidney lysates from wild-type (WT) and Kelch-like protein 3 (KLHL3)+/R528H mice maintained on a normal-K+ diet (NKD) or LKD were analyzed by Western blot to assess the expression of KS-WNK1. Compared with WT mice, moderately higher expression was observed in KLHL3+/R528H mice. In addition, a robust increase in KS-WNK1 expression was observed in WT mice but also in KLHL3+/R528H mice, suggesting that KLHL3-targeted degradation was further affected under this condition. Results of quantitation of the band corresponding to KS-WNK1 are shown at the bottom. Band intensity values of mice on a NKD were normalized to 100%. A WNK4 blot is also shown. The expected increase in WNK4 expression was observed in KLHL3+/R528H samples. Expression was further increased when mice were placed on a LKD, as previously reported (10). n = 9 for WT mice on the LKD and NKD, n = 3 for KLHL3+/R528H mice on the NKD, and n = 4 for KLHL3+/R528H mice on the LKD. *P < 0.05 versus the NKD. WNK1, with no lysine kinase 1.

Fig 3: WNK1 silencing reduced FOXO4 phosphorylation at different sites from those phosphorylated by Akt. (A) Immunoblots showing that FOXO4 phosphorylation at T32, S197, or S262, which promotes FOXO4 nuclear exclusion and inactivates the protein, was not decreased by WNK1 silencing in C2C12 cells. (B) Domain structure of human FOXO4 protein. (C) Phos-tag SDS-PAGE revealed that the abundance of upper smear bands representing phosphorylated full-length FOXO4 [1–505] was decreased in WNK1-silenced HEK 293 T cells compared with that in control cells (Fig. S3B,C). The abundance of phosphorylated bands of FOXO4 [1–312] was unchanged in WNK1-silenced cells, whereas that of FOXO4 [313–505] was decreased. FH/DBD, forkhead box/DNA-binding domain; NLS, nuclear localization sequence, NLS; NES, nuclear export sequence; WNK1, with-no-lysine (K); FOXO4, Forkhead box protein O4. Full-length blots are presented in Supplementary Fig. 11.

Fig 4: Kidney-specific with no lysine kinase (WNK)1 (KS-WNK1), WNK3, and WNK4, but not full-length WNK1 (L-WNK1), are degraded when coexpressed with Kelch-like protein 3 (KLHL3) in oocytes. A: thiazide-sensitive 22Na+ uptake was assessed in Xenopus laevis oocytes injected with the indicated cRNAs. Uptake values observed in the control group (NCC only) were set to 100%, and the other groups were normalized accordingly. While both L-WNK1 and KS-WNK1 increased Na+ uptake, KLHL3 coexpression prevented NaCl cotransporter (NCC) activation by KS-WNK1 but not by L-WNK1. Dots represent uptake values for individual oocytes. At least three independent experiments were performed with >10 oocytes per group (****P < 0.0001 vs. NCC; three points outside graphic limits). B: representative Western blots showing the effect of KLHL3 coexpression on L-WNK1, KS-WNK1, WNK3, and WNK4 levels. Oocytes were injected with cRNAs encoding for c-Myc-tagged L-WNK1, KS-WNK1, or WNK3 or Flag-tagged WNK4 with or without Flag-tagged KLHL3. All kinases except L-WNK1 were degraded in the presence of KLHL3. C: densitometric analysis of the Western blots presented in B. Two independent experiments were performed with similar results. c-Myc-tagged L-WNK1, KS-WNK1, or WNK3 or Flag-tagged WNK4 were normalized to 100% and compared with those observed in groups expressing KLHL3.

Fig 5: The segment encoded by exon 4a in kidney-specific with no lysine kinase 1 (KS-WNK1) is needed to activate NaCl cotransporter (NCC) and to be targeted for degradation by cullin-3 (CUL3)–Kelch-like protein 3 (KLHL3) E3. A: thiazide-sensitive Na+ uptake of NCC cRNA-injected oocytes was set to 100%, and uptake values of additional groups were normalized accordingly. KS-WNK1 coexpression increased NCC activity, but the KS-WNK1-Δ4a mutant failed to activate (n = 3 transport assays, ****P < 0.0001 vs. NCC; one point outside graphic limits). B: representative Western blots showing KS-WNK1 and KS-WNK1-Δ4a expression in the absence or presence of KLHL3. CUL3-KLHL3 E3-induced degradation of KS-WNK1 is observed regardless of proteosome inhibition, whereas KS-WNK1-Δ4a is expressed but resistant to CUL3-KLHL3 E3-induced degradation. C: densitometric analysis of the Western blots presented in B. Results from four different experiments were included. Expression of KS-WNK1 or KS-WNK1-Δ4a in the absence of KLHL3 were arbitrarily set to 100% and compared with expression levels observed in the presence of KLHL3 (n = 4 Western blots, ***P > 0.001 vs. control without KLHL3).