Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action Supplemental Data and Methods

The thiazide-sensitive NaCl cotransporter (NCC) is important for renal salt handling and blood-pressure homeostasis. The canonical NCC-activating pathway consists of With-No-Lysine (WNK) kinases and their downstream effector kinases SPAK and OSR1, which phosphorylate NCC directly. The upstream mechanisms that connect physiological stimuli to this system remain obscure. Here, we have shown that aldosterone activates SPAK/OSR1 via WNK1. We identified 2 alternatively spliced exons embedded within a proline-rich region of WNK1 that contain PY motifs, which bind the E3 ubiquitin ligase NEDD4-2. PY motif-containing WNK1 isoforms were expressed in human kidney, and these isoforms were efficiently degraded by the ubiquitin proteasome system, an effect reversed by the aldosterone-induced kinase SGK1. In gene-edited cells, WNK1 deficiency negated regulatory effects of NEDD4-2 and SGK1 on NCC, suggesting that WNK1 mediates aldosterone-dependent activity of the WNK/SPAK/OSR1 pathway. Aldosterone infusion increased proline-rich WNK1 isoform abundance in WT mice but did not alter WNK1 abundance in hypertensive Nedd4-2 KO mice, which exhibit high baseline WNK1 and SPAK/OSR1 activity toward NCC. Conversely, hypotensive Sgk1 KO mice exhibited low WNK1 expression and activity. Together, our findings indicate that the proline-rich exons are modular cassettes that convert WNK1 into a NEDD4-2 substrate, thereby linking aldosterone and other NEDD4-2-suppressing antinatriuretic hormones to NCC phosphorylation status.

. L-WNK1 and KS-WNK1 contain a complete assortment of exon 11 and 12 combinations.
(A.) RT-PCR reactions were performed on adult human kidney RNA using methods similar to those in Figure S1. For these reactions, forward primers specific for L-WNK1 (exon 3) and KS-WNK1 (exon 4a) were used in combination with reverse primers designed to anneal to exon junctions 10/11, 11/12, 12/13, 10/12, 10/13, and 11/13 (see Table S1). Annealing sites for these primers are depicted with arrows, and predicted amplicons sizes are listed next to each isoform.
(B.) Agarose gel electrophoresis of RT-PCR products from human kidney RNA. The PCR products in rows A-K of the gel were generated using primer sets corresponding to isoforms A-K in panel A. 1kb and 100bp ladders (New England Biolabs) are indicated with asterisks and a dot, respectively. In each case, a doublet was identified, and sequencing of the shorter major band identified additional alternative splicing events in exon 9. These data match the findings of a recent report (3). Representative of 2 experiments. iv Figure S3. WNK1 transcript abundance is not increased by 100nM aldosterone in mpkCCD c14 cells.
(A.) mpkCCD c14 cells were treated with 100nM aldosterone for up to 16h, and miR-192 abundance was assessed by quantitative real-time PCR (qRT-PCR) using the SYBRgreen intercalation method. Parallel quantitative analysis of miR-19a, a control microRNA that is not affected by aldosterone, was also performed. miRNA abundance at each time point was normalized to the abundance at time zero. (B.) Quantification of miR-192 abundance following 1, 4, and 16h aldosterone treatment, normalized to the abundance at time zero. P values reflect standard unpaired T-test comparisons to the zero hour time point. (C.) Top, Schematic showing the location of primer set specific for L-WNK1 (M4/5), KS-WNK1 (M4a/5) and Exon 12 (M12/13) WNK1 isoforms, indicated with arrows. Bottom, Representative PCR reactions demonstrating single WNK1 amplicons of expected size, using the indicated primer sets, during a 24h 100nM aldosterone timecourse. (D.) Quantification of WNK1 isoform abundance following 6, 14, and 24h of 100nM treatment. An increase in SGK1 transcription was detected, indicating that the cells were aldo responsive. (n=4; *P<0.05 vs the zero time point by Kruskal-Wallis test with Dunn's multiple comparison post-hoc test).
v Figure S4. Nedd4-2 mediated ubiquitylation of the WNK1 C-terminus is PY-motifdependent and requires intact catalytic activity.
(A.) In vivo ubiquitylation assay of Halo-HA-L-WNK1 C-terminus containing exons 11 and 12 (L-WNK1 782-2374). Lysates of MG-132 pretreated HEK-293T cells expressing WNK1 fragments with WT or catalytically inactive dominant negative (DN; C938S) Nedd4-2 were covalently attached to HaloLink resin. Following pulldown, fragments were washed with SDS to remove autoubiquitylated Nedd4-2 and other ubiquitylated coprecipitants. The fragments were then cleaved from the resin with TEV protease and probed with the indicated antibodies. A stronger immunoreactive smear is noted when L-WNK1 is coexpressed with Nedd4-2; this smear is absent in samples where L-WNK1 is coexpressed with DN Nedd4-2. Representative of 4 experiments. (B.) Top, Schematic representation of C-terminal Halo HA-tagged constructs used for the experiments: Wild type C-terminus containing exons 11 and 12, a "PY-null" mutant lacking Nedd4-2 binding sites, and L-WNK1 1031-2374, a construct encoding exon 13 to the C-terminal end of the protein ("∆11-12"). Bottom, the above constructs were coexpressed with WT Nedd4-2 in HEK-293T cells and subjected to the ubiquitylation assay following MG-132 pretreatment. A stronger immunoreactive smear was observed with the "Wild Type" L-WNK1 C-terminus, compared to the double PY motif mutant or the ∆11-12 construct. Representative of 3 experiments. A variant serine at the C-terminal end of the original L-WNK1 clone (AAF74258.1) that replaces a phylogenetically conserved glycine residue was recently reported to confer reduced L-WNK1 activity toward NCC (4). This residue (serine 2368 based on the numeration for RefSeq XP_008761429.1, corresponding to the full length rat L-WNK1 amino acid sequence encoded by the cDNA construct used for these studies) was mutated back to the conserved glycine in both WT and PY-Null (Y829A/Y945A) L-WNK1 constructs. Cycloheximide chases were then performed as outlined in the Methods. (n=4; *P<0.01 by Student's T-tests for the indicated time points). (A.) WNK1 exon 12, SPAK/OSR1, and NCC abundance in kidney homogenates from Nedd4-2 Pax8/LC1 mice and single transgenic controls treated with high salt diet. (B.) WNK1 exon 12 and phospho-SPAK abundance in Nedd4-2 knockouts and single transgenic controls treated with 3d of aldosterone of vehicle by subcutaneous osmotic minipump. In both exon 12 blots, full length L-WNK1 ("L") and lower molecular weight species (brackets) are shown. In the blot for (B), the membrane was cut above ~150kDa. Control values were normalized to a mean of 100%, and densitometry values from Nedd4-2 Pax8/LC1 mice were expressed relative to the mean of the control. Actinnormalized data were pooled with the immunoblotting studies in

Molecular Methods
All L-WNK1 clones used in this study were derived from the original rat L-WNK1 cDNA, isolated from rat forebrain by Xu et al (AAF74258.1, (5)). This cDNA encodes a splice isoform that lacks exons 11 and 12, and is termed "L-WNK1 ∆11-12" in this study. Site directed mutagenesis (QuickChange XL kit, Agilent) was performed to  Table S1.

Antibodies and Reagents
The WNK1 exon 12 antisera were generated by immunizing rabbits to a keyhole limpet hemocyanin-conjugated peptide epitope located within exon 12 of rat WNK1

Immunoprecipitation studies
For coimmunoprecipitation studies in HEK-293T cells, cells transiently expressing the indicated constructs were lysed in detergent solution (13) as described above. 500µg lysate was diluted in detergent solution to a volume of 500µl and precleared with 50µl of Sepharose CL-6B slurry by end over end rotation at 4°C for 2 h.
xiii The cleared lysates were rotated in fresh 25µl aliquots of anti-myc-conjugated agarose resin (Millipore, clone 4A6) overnight at 4 °C. Samples were centrifuged at low speed, and the beads were washed five times in 500µl of PBS supplemented with protease inhibitors. Immunoprecipitated proteins were eluted by incubating the beads at 90 o C for 5 min in 5X Laemmli buffer, separated by SDS-PAGE, and analyzed by immunoblotting.
For coimmunoprecipitation of endogenous proteins in mpkCCD c14 cells, 500µg detergent solution-solubilized lysates were diluted to 500µl and precleared using 30µl of protein A/G beads mixed by rotation for 1hr at 4°C. Following low-speed centrifugation, 1µg of rabbit anti-Nedd4 antibody and 35µl of prewashed protein A/G beads was added to the supernatants and rotated overnight at 4°C. The samples were then centrifuged at 3000 x g, and the beads were washed three times for 5 minutes in 500µl PBS supplemented with protease inhibitors. Complexes were eluted from the beads with 5X Laemmli buffer by heating the samples at 90˚C for 5 min, and the samples were subsequently analyzed by immunoblotting.

Reverse Transcriptase PCR (RT-PCR) studies in human kidney
Total adult kidney RNA was purchased from Clontech (Cat #636584). RNA was treated with DNAse I, amplification grade (Life Technologies) and reverse-transcribed using oligodT primers and the Superscript III First-Strand Synthesis Supermix (Life Technologies). Detection of WNK1 transcripts was performed using exon-exon junction specific primers (Supplemental Table 1) and standard PCR conditions.

RNA Isolation and qRT-PCR Analysis
xiv RNA from cultured mpkCCD c14 cells was isolated using the miRNeasy RNA isolation kit (Qiagen) according to the manufacturer's protocol. The kit facilitated isolation of both miRNA and total RNA from each sample for use in qRT-PCR and RT-PCR. Total RNA (containing miRNAs) concentration and quality was evaluated for inclusion in subsequent in vitro transcription assays based on a spectrophotometric absorption ratio of 260/280 >1.8. For quantitative RT-PCR the nCode Express SYBR-Green miRNA with ROX qRT-PCR kit was used for reverse transcriptase and first-strand DNA synthesis for the miRNA and SuperScript VILO kit for mRNA (Invitrogen). For miRNA qRT-PCRs the miRNA-specific forward primers were paired to a universal reverse primer per the manufacture's protocol, and primer pairs were used as listed in Table S1 for Table S1.

Kinase Inhibitor assays in mpkCCD c14 cells
Hormone deprived mpkCCD c14 cells were treated with 10µM GSK650394, a SGK1 specific inhibitor, 50µM LY294002, a P13K inhibitor, for 2 hours prior to a 5h treatment with 100nM aldosterone. Control samples were treated with DMSO for 2h prior to aldosterone treatment. Cells were pelleted, lysed and analyzed by immunoblotting.