Fig 2: WNK1 promotes interaction between mTORC2 and SGK1 to enhance SGK1 phosphorylation in a Cl−-dependent K+-stimulated manner. (A) Western blot analysis of Flag immunoprecipitates derived from Flag–SGK1, Myc–WNK1 (WT and kinase-dead mutant, K233M) and SIN1–V5-transfected WNK1−/− SIN1−/− (double knockout) HEK-293 cells. Cells were transfected and serum starved overnight, then preincubated in 1 mM [K+], after which [K+] was shifted to 5 mM for 1 h prior to processing for IP with anti-Flag antibody and western blotted with antibodies as shown. Results are representatives of n=3 biological replicates. (B) Quantification of western blots from A demonstrating SGK1–SIN1 (left panel) and SGK1–WNK1 (right panel) interactions. All values are means±s.e.m. from n=3 biological replicates. (C) Western blot analysis of whole-cell extracts (WCEs) derived from cells in A. Upper panel, western blots were stained with antibodies as shown. pSGK1 was detected using anti-phospho-SGK1 S422 (SAB4503834, Sigma), antibody. Lower panel, quantification of bands in western blots. Note selective effect of 5 mM K+ on pSGK1 but not pAkt. All values are means±s.e.m. from n=3 biological replicates. t, total. (D) Western blot analysis of Myc–WNK1 IPs from whole-cell lysates as in A. Lysates were immunoprecipitated with anti-Myc antibody and stained for Myc, Rictor and SGK1. Note 5 mM K+ stimulation of Myc–WNK1 co-IP of both SGK1 (top row) and Rictor (middle row). Results are representatives of n=2 biological replicates. (E) K+ stimulated mTORC2-SGK1 interaction is Cl− dependent. Western blot analysis for V5 (SIN1) of Flag (SGK1) immunoprecipitates showing the effect of extracellular K+ on SIN1 and SGK1 interaction under normal or low extracellular Cl− conditions in Flag–SGK1, and SIN1–V5-transfected WNK1+/+ SIN1−/− HEK-293 cells. Results demonstrate that extracellular K+-stimulated interaction between SGK1 and mTORC2 requires Cl− (compare with Fig. 6A). In D and E, results are representatives of n=3 biological replicates. *P≤0.05; **P≤0.01; ns, not significant [unpaired two-tailed t-test (B); two-way ANOVA with Bonferroni's multiple-comparison test. (C)].

Fig 3: The kinase activity of WNK1 is not required for K+-stimulated SGK1 phosphorylation and ENaC activity in HEK-293 cells. (A) Single-channel ENaC current measured by patch-clamp in WNK1−/− HEK-293 cells, showing the effect of WT and kinase-dead WNK1 on K+-stimulated ENaC activity. Cells were transfected with ENaC, Nedd4-2, Myc-tagged WT (aa 1–491) or kinase-dead (aa 1–491)/K233M WNK1, and WT SGK1 (left panel) or kinase-dead SGK1 (K127M) (right panel). Cells were adapted to 1 mM [K+], and then bath [K+] was either raised to 5 mM or kept at 1 mM for 1 h prior to measurement of amiloride-sensitive current by patch-clamp. Note that extracellular K+ stimulated ENaC current in cells expressing either WT or kinase-dead WNK1 and WT SGK1. However, in cells expressing kinase-dead SGK1 (K127M), raising extracellular [K+] to 5 mM failed to stimulate ENaC current. All values are means±s.e.m.; n=5 in each group. ****P<0.001 (two-way ANOVA with Bonferroni's multiple-comparison test). (B) Western blot showing the effect of WT and kinase-dead full length (L-WNK1) and truncated (WNK1 1–491) WNK1 on SGK1 phosphorylation. Kinase active (WT) and kinase-dead (K233M) forms similarly stimulated pSGK1 (S422). WNK1-deficient HEK-293 cells were transfected with empty vector, WT HA-L-WNK1 or kinase-dead HA–L-WNK1/K233M and WT Myc–WNK1 (aa 1–491) or kinase-dead Myc–WNK1 (aa 1-491)/K233M along with WT SGK1. Cells were adapted to 1 mM extracellular [K+] and then transferred to 5 mM K+ media or maintained in 1 mM for 1 h prior to processed for western blot analysis. pSGK1 protein was detected using anti–phospho-SGK1 S422 (sc-16745, Santa Cruz Biotechnology) antibody. t, total. Results are representatives of n=3 biological replicates.

Fig 4: Effects of polyphenols and rapamycin on mitochondrial respiration and expression of mTOR downstream genes. (a) Rapamycin inhibits the effects of the polyphenol combination on mitochondrial respiration. Cells were pretreated with vehicle (DMSO) or 5 μM rapamycin (Rap) for 1 h followed by the addition of vehicle or 7 μM CUR and 5 μM CA (CUR + CA). Data are presented as mean ± SEM (n = 3) relative to DMSO. Statistically significant differences for the indicated groups vs. DMSO (* p < 0.05 and ** p < 0.01). § p < 0.05, significant differences between the indicated groups (Student’s t-test). (b) Changes in mTOR downstream transcriptional profile upon treatment of DU145 cells with 5 μM rapamycin or 7 μM CUR, 5 μM CA or CUR + CA) for 24 h, relative to DMSO. (c) Quantitative analysis of SGK1 gene expression. Data are presented as fold regulation vs. DMSO (mean ± SEM; n = 3), where DMSO is set as 0. Statistically significant differences for treatments vs. control (DMSO): * p < 0.05 and ** p < 0.01.

Fig 5: WNK1 is required for K+-induced ENaC activity and SGK1 phosphorylation. (A) Western blot analyses of WNK1 protein in WT and WNK1−/− mpkCCD clones. WNK1 was deleted from mpkCCD cells using CRISPR/Cas9 (see Materials and Methods for details). WNK1 protein was detected using anti-WNK1 antibody. Results are representatives of n=2 biological replicates. (B) Effect of extracellular K+ on ENaC current in WT and WNK1−/− mpkCCD cells. WT and KO cells (two independent clones) were grown on Transwell filters, adapted to 1 mM [K+] on the basolateral side, and then the medium [K+] was either raised to 5 mM or kept at 1 mM for 1 h prior to measurement of amiloride-sensitive current. All values are means±s.e.m. from n=3 biological replicates. µA, microamperes. AZD8055, mTOR inhibitor. n=3 independent experiments. (C) Effect of extracellular K+ on SGK1 phosphorylation in WT and WNK1−/− mpkCCD cells. Cells as in B, were harvested and subjected to western blotting for various downstream targets of mTORC2 and WNK1 as indicated. Fl, full length; Cl, cleaved; t, total. pSGK1 and WNK1 proteins were detected using anti-phospho-SGK1 S422 (sc-16745, Santa Cruz Biotechnology), and anti-WNK1 (AF2849, R&D Systems) antibody, respectively. Results are representatives of n=3 biological replicates. (D–F) Quantification of bands in western blots from C. Note that stimulatory effects of 5 mM [K+] on pSGK1 (direct substrate), and pNEDD4-2 (SGK1 substrate) are observed in WT but not in WNK1-deleted cells. Phospho-SPAK was inhibited by 5 mM [K+] in WT cells but not in WNK1−/− cells consistent with prior literature (see text for details). All values are means±s.e.m. from n=3 biological replicates. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant (one-way ANOVA with Bonferroni's multiple-comparison test).