Activation of PI3-Kinase Stimulates Endocytosis of ROMK Via Akt1/SGK1-Dependent Phosphorylation of WNK1

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Activation of PI3-Kinase Stimulates Endocytosis of ROMK via Akt1/SGK1-Dependent Phosphorylation of WNK1

Chih-Jen Cheng*† and Chou-Long Huang*

*Division of Nephrology, Department of Medicine University of Texas Southwestern Medical Center, Dallas, Texas; and †Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan

ABSTRACT WNK kinases stimulate endocytosis of ROMK channels to regulate renal Kϩ handling. Phosphatidylino- sitol 3-kinase (PI3K)-activating hormones, such as insulin and IGF 1, phosphorylate WNK1, but how this affects the regulation of ROMK abundance is unknown. Here, serum starvation of ROMK-transfected HEK cells led to an increase of ROMK current density; subsequent addition of insulin or IGF1 inhibited ROMK currents in a PI3K-dependent manner. Serum and insulin also increased phosphorylation of the downstream kinases Akt1 and SGK1 as well as WNK1. A biotinylation assay suggested that insulin and IGF1 inhibit ROMK by enhancing its endocytosis, a process that WNK1 may mediate. Knockdown of WNK1 with siRNA or expression of a phospho-deficient WNK1 mutant (T58A) both prevented insulin- induced inhibition of ROMK currents, suggesting that phosphorylation at Threonine-58 of WNK1 is important to mediate the inhibition of ROMK by PI3K-activating hormones or growth factors. In vitro and in vivo kinase assays supported the notion that Akt1 and SGK1 can phosphorylate WNK1 at this site, and we established that Akt1 and SGK1 synergistically inhibit ROMK through WNK1. We used dominant- negative intersectin and dynamin constructs to show that SGK1-mediated phosphorylation of WNK1 inhibits ROMK by promoting its endocytosis. Taken together, these results suggest that PI3K-activating hormones inhibit ROMK by enhancing its endocytosis via a mechanism that involves phosphorylation of WNK1 by Akt1 and SGK1.

J Am Soc Nephrol 22: 460–471, 2011. doi: 10.1681/ASN.2010060681

The concentration in the blood of the potassium ion channel at the cell surface, thus controlling renal Kϩ ϩ (K ), an important determinant of cell membrane secretion.3 Recently, WNK (with-no-lysine [K]) ki- potential, is tightly regulated within a narrow range. nases have been identified as important regulators of The excretion of Kϩ occurs mainly in the kidney the cell surface abundance of ROMK. WNKs are ser- through processes involving glomerular filtration, tu- ine-threonine protein kinases with an unusual posi- bular reabsorption, and secretion. The transepithelial tion of the catalytic lysine in subdomain I instead of Kϩ secretion in the kidney takes place predominantly in the aldosterone-sensitive distal nephron and in- Received June 28, 2010. Accepted September 26, 2010. volves Kϩ uptake into cells by the basolateral sodium- potassium pump and exit into lumen through apical Published online ahead of print. Publication date available at www.jasn.org. Kϩ channels, which include the Ca2ϩ-activated Correspondence: Dr. Chou-Long Huang, UT Southwestern Med- maxi-K channel and the renal outer medullary potas- ical Center, Department of Medicine, 5323 Harry Hines Boule- sium channel, ROMK (also known as Kir1.1).1,2 vard, Dallas, TX 75390-8856. Phone: 214-648-8627; Fax: 214- ROMK channel undergoes constitutive clathrin- 648-2071; E-mail: [email protected] dependent endocytosis, which regulates the density of Copyright © 2011 by the American Society of Nephrology

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ϩ subdomain II.4 Mammalian WNK family includes four members, Under the condition of symmetrical 140 mM [K ], ROMK- WNK1-4, which share 85 to 90% sequence identity in their kinase mediated Kϩ currents showed characteristic weak inward rec- domain.4–6 Mutations in WNK1 and WNK4 in humans cause an tification (Figure 1A, left). No currents were observed in autosomal-dominant disease called pseudohypoaldosteronism mock-transfected cells. To allow for testing the effect of insulin type 2 (PHA2), featuring hypertension and hyperkalemia.5 Stud- and IGF1 on ROMK, we first examined the effect of serum ies have shown that WNK1 and WNK4 regulate renal Naϩ and deprivation. After culturing in the serum-containing media for Kϩ transporters, and dysregulation of these transporters contrib- 48 hours to allow maximal expression of the channel, ROMK- utes to hypertension and hyperkalemia in PHA2. transfected cells were deprived of serum for 3 to 25 hours be- WNKs regulate renal Naϩ transport through both catalytic fore ruptured whole-cell recording. As shown, ROMK current and noncatalytic mechanisms. With respect to the catalytic mech- density (normalized to capacitance, pA/pF) increased progres- anism of regulation, WNK1 and 4 phosphorylate and activate sively with increasing duration of serum deprivation (Figure OSR1 (oxidative stress-responsive kinase-1) and its related kinase 1A, right). Six hours after serum deprivation, current density SPAK (Ste20-related proline-alanine-rich kinase), which in turn was significantly higher than that before deprivation. ROMK phosphorylate and activate the thiazide-sensitive sodium-chlo- currents continued to increase and reached a plateau at 16 to 20 ride co-transporter NCC and the bumetanide-sensitive sodium- hours (Figure 1A). Addition of insulin thereafter caused a sig- potassium-2 chloride cotransporter NKCC.7,8 WNK1 and 4 can nificant inhibition of ROMK currents in 30 minutes (Figure also regulate ENaC and NCC via noncatalytic mechanisms that 1B). ROMK currents reached the nadir at approximately involve protein–protein interaction with serum- and glucocorti- 2-hour incubation with insulin (Figure 1B). The dose–re- coid-induced kinase-1 (SGK1) for the regulation of ENaC and sponse relationship for inhibition of ROMK was examined by with transporter directly for the regulation of NCC.9,10 With re- incubating insulin ranging from 1 to 100 nM for 2 hours. In- spect to Kϩ transport, WNK1 and 4 stimulate endocytosis of sulin significantly inhibited ROMK at a concentration as low as ROMK via a kinase-independent mechanism that involves a di- 1 nM (Figure 1C). The concentration for half-maximal inhibi- 11 rect interaction with an endocytic scaffold protein, intersectin. tion (IC50) of ROMK for insulin was estimated at 3.2 nM (Fig- Compared with the downstream effects of WNKs, the physio- ure 1C). For reference, the plasma concentration of insulin in logic upstream regulators of WNKs are less understood. Vitari et normal individuals is 10 to 150 pM at fasting but may reach 300 al.12 showed that IGF1 induces phosphorylation of endogenous to 800 pM after carbohydrate meals.16 IGF and insulin act on WNK1 in cultured human embryonic kidney (HEK) cells at thre- similar membrane receptors and elicit overlapping cellular re- onine-60 (equivalent to threonine-58 of rat WNK1). The effect of sponses.17 Accordingly, we found a similar inhibition of

IGF1 is through activation of phosphatidylinositol 3-kinase ROMK by IGF1, with an IC50 for IGF1 estimated at 18.5 ng/ml (PI3K), leading to activation of the 3-phosphoinositide–depen- (ϭ2.4 nM; Figure 1D). The normal basal plasma concentra- dent protein kinase-1 (PDK1) and protein kinase B (PKB)/Akt1. tion of IGF1 in humans is around 50 to 100 ng/ml.18 Phosphorylation of WNK1 by Akt1 does not affect its kinase activity or subcellular distribution.12 Jiang et al.13 reported that in- Effect of Insulin and IGF1 Is Dependent on PI3K and sulin induces a similar phosphorylation of WNK1, which under- WNK1-T58 Phosphorylation scores the inhibition of cell proliferation of 3T3-L1 preadipocytes To study whether the inhibition of ROMK by insulin and IGF1 by insulin. Xu et al.14 reported that WNK1 activates SGK1 requires PI3K, ROMK-transfected cells were incubated with through direct protein–protein interactions independently of insulin or IGF1 with or without a specific PI3K inhibitor, wort- WNK1 kinase activity. Xu et al.9 further showed that phosphory- mannin. In these experiments, we also compared the effects of lation of rat WNK1 at threonine-58 by the IGF1–Akt1 pathway insulin and IGF1 on ROMK with or without serum. We found enhances the ability of WNK1 to stimulates SGK1 kinase activity, that 100 nM of insulin caused a significant inhibition of leading to activation of ENaC. Others have also shown that insulin ROMK even in the presence of serum (Figure 2A), indicating stimulates ENaC via Akt1, although the role of WNK1 was not that the receptors are not maximally occupied by insulin pres- studied.15 The mechanism for WNK1 regulation of ROMK and ent in the serum. For comparison, IGF1 at 100 ng/ml did not ENaC are fundamentally distinct, raising an interesting question cause further inhibition of ROMK in the presence of serum. As as to whether phosphorylation of WNK1 by PI3K-activating hor- before, serum deprivation increased ROMK currents, and ap- mones, such as insulin and IGF1, affects its regulation of ROMK. plication of insulin or IGF1 thereafter inhibited the currents. We investigate this question in this study. Co-application of wortmannin (WM, 100 nM) completely abolished the effect of insulin and IGF1 on ROMK (Figure 2A), indicating that the effect of insulin and IGF1 depends on PI3K. RESULTS Stimulation of PI3K by hormones or growth factors pro- duces 3-phosphoinositides in the plasma membrane and leads Effects of Serum Deprivation, Insulin, and IGF1 on to activation of downstream AGC kinases, Akt1 and SGK1, via ROMK multiple concerted actions.19 First, it stimulates the mamma- Barium-sensitive Kϩ currents were measured by ruptured lian target of rapamycin complex-2 (mTORC2) to phosphor- whole-cell recording from HEK cells transfected with ROMK. ylate Akt1 or SGK1 at the hydrophobic motif. Phosphorylation

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serine-473 (S473) of the hydrophobic motif, respectively (Figure 2B, lane 4). Serum and/or insulin increased phosphorylation of Akt1 at both residues (lanes 1, 2, and 5). Wortmannin abrogated basal and serum or insulin-stimulated phosphorylation of Akt1 (lanes 3 and 6). Phosphorylation of WNK1 at threonine-60 (T60 ϭ T58 of rat WNK1) was not detectable in the absence of serum (lane 4) but was enhanced by serum and/or insulin in a wortmannin-sensitive manner (lanes 1, 2, and 5). Serum and insulin also stimulated wortmannin-sensitive phosphorylation of overexpressed SGK1 at the T- loop (threonine-256) (Figure 2B, bottom two gels highlighted in box). Overexpressed SGK1 was used because endogenous SGK1 was below detection by our antibody. Cell surface abundance of ROMK was examined using a biotinylation assay. Se- rum deprivation increased ROMK surface abundance (Figure 2C, lane 2 versus 3). Insu- Figure 1. Insulin and IGF1 inhibit ROMK current. (A) Effect of serum deprivation on lin and IGF1 decreased ROMK surface abun- ROMK current. (Left) Configuration of whole-cell recording, voltage-clamp protocol Ϫ dance in a wortmannin-sensitive manner (from 100 to 100 mV), representative currents from ROMK- and mock-transfected (lanes 4 to 6). These results support the no- cells are shown. (Right) ROMK current density (pA/pF at Ϫ100 mV; normalized to the tion that insulin and IGF1 inhibit ROMK cell surface area) at different time of serum deprivation are shown (mean Ϯ SEM, n Ն 6 for each) and analyzed by nonlinear regression curve. Inset shows current-voltage by enhancing its endocytosis, in which (I-V) relationship curve of ROMK with serum deprivation for 0, 13, and 25 hours. Data WNK1 may play a role. in each time point was compared with serum-containing group (0-hour serum depri- To confirm the role of WNK1 in the vation). **P Ͻ 0.01. All time points beyond 6 hours are significant compared with the PI3K-mediated regulation of ROMK, we serum-containing group (not indicated by asterisk). All time points between 16 and 25 knocked down endogenous WNK1 using hours are not significantly different (not indicated). In all experiments throughout this small interference RNA (siRNA). Effi- study, ROMK currents shown are after subtracting residual currents in the presence of cacy of WNK1 siRNA was validated by 5 mM barium. (B) Time course of effect of insulin on serum-deprived ROMK current. blotting endogenous WNK1 in HEK cells Cells were cultured in serum-free medium at least 16 hours before addition of insulin Ϯ Ն (Figure 2D). Cells transfected with (100 nM) for different time periods. Data points are mean SEM (n 6 for each), WNK1 siRNA (“WNK1 siRNA”, white compared with serum-deprived (0-hour insulin incubation), and analyzed by nonlinear bar) or control oligonucleotides (“Con- regression curve. Inset shows I-V curve of ROMK current before and after 2-hour insulin. (C and D) Dose–response curve of insulin and IGF1 on serum-deprived ROMK. trol oligo”, black bar) had similar ROMK ROMK current density (mean Ϯ SE, n Ն 6) at Ϫ100 mV was measured in cells cultured current in the absence of serum (“serum- in serum-containing medium (SC), serum-free medium (SF), and 2-hour incubation of free”). Addition of insulin inhibited different concentration of insulin or IGF1. Data of each insulin or IGF1 treatment group ROMK currents in cells transfected with were compared with the SF group. Dose–response curve and IC50 of insulin or IGF1 on control oligonucleotides but not in cells ROMK resulted from nonlinear regression analysis. *P Ͻ 0.05 versus designated group whose endogenous WNK1 was knocked by unpaired two-tailed t test. **P Ͻ 0.01. down by siRNA. Next, we used a phospho-deficient of Akt1 or SGK1 by mTORC2 enhances its binding with PDK1, T58A mutant to examine the role of T58 phosphorylation on which phosphorylates Akt1 or SGK1 at the T-loop to activate WNK1 (Figure 2E). Insulin inhibited ROMK in cells express- its catalytic activity. Finally, the recruitment and binding of ing endogenous (“vector”) or exogenous WNK1 [“WNK1(1- PDK1 to Akt1 is also facilitated by the production of 3-phos- 491)/WT”] but not in cells expressing exogenous T58A mu- phoinositides in the plasma membrane. To understand the tant of WNK1 [“WNK1(1-491)/T58A”]. Overexpression of role of PI3K in WNK1 regulation of ROMK, we examined the WNK1-T58A exerted a dominant-negative effect on endoge- phosphorylation status of endogenous Akt1 and WNK1 in nous WNK1 (data not shown). These results strongly support HEK cells using respective residue-specific phospho-antibod- the notion that phosphorylation on T58 of WNK1 is important ies. In the absence of serum, there was a basal level of phos- for inhibition of ROMK by hormones or growth factors that phorylation of Akt1 at threonine-308 (T308) of the T-loop and activate PI3K.

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control for specific phosphorylation at T58. As shown, PDK1 by itself did not phosphorylate WNK1 (Figure 3A, lane 1). For Akt1, both wild-type Akt1 and myristoylated Akt1, but not their kinase dead mutant, phosphorylated WNK1 (lane 2 to 4). Although myristoylated Akt1 is reported catalytically more active than wild type toward certain substrates, we did not find this using WNK1 as a substrate. Co- expression of PDK1 did not further en- hance the in vitro kinase activity of Akt1 (lane 8 to 10). For SGK1, only S422D (not wild-type or kinase-dead mutant) phosphorylated WNK1 in the absence of PDK1 (lanes 5 to 7). The SGK1-S422D mutant with serine-422 in the hydrophobic motif substituted by aspartate is constitutively active because it does not require phos- Figure 2. Insulin and IGF1 inhibit ROMK through PI3K and WNK1–T58 phosphoryla- phorylation by mTOR for activation by tion. (A) Effect of insulin and IGF1 on ROMK is blocked by wortmannin. Cells cultured PDK1.20 Co-expression of PDK1 enhanced with or without serum were treated by DMSO, insulin 100 nM, or insulin 100 nM plus the kinase activity of WT and SGK1-S422D wortmannin (WM, 100 nM), respectively, for 2 hours (mean Ϯ SEM, n Ն 6). (B) Effect of (lanes 11 to 13). The increase in the kinase serum, insulin, and wortmannin on phosphorylation of endogenous WNK1, Akt1, and activity on the S422D mutant was so much overexpressed SGK1. Phosphorylation on specific residues was determined by specific that phosphorylation on WNK1 also oc- anti-phospho antibodies. Shown is representative of three separate experiments of curred at residue(s) other than T58 [see similar results. (C) Effect of insulin and IGF1 on surface abundance of ROMK. Cells were lane 11, on WNK1(1-119)/T58A mu- serum-deprived for 16 hours and treated with insulin (100 nM), IGF1 (100 ng/ml), tant]. The averaged relative kinase activ- and/or WM (100 nM) for 2 hours as indicated before biotinylation (Biotin). ROMK in ity of Akt1 and SGK1 (normalized to total cell lysates (Lysate ROMK) and elute from avidin beads (Biotin ROMK) were detected by Western blot. (D) Insulin inhibits ROMK through WNK1. Cells transfected “Akt1-Myr” in lane 2, which is given as with control oligonucleotide or WNK1 siRNA (200 nM each) were deprived of serum for 1), representing specific phosphorylation 16 hours (serum-free group). Insulin (100 nM) was added for 2 hours (ϩInsulin group). on WNK1-T58 [i.e., after subtracting sig- Successful knockdown of endogenous WNK1 by siRNA is evident by Western blot nal on T58A mutant from signal on wild analysis. Mean Ϯ SEM (n Ն 6 each). (E) Insulin inhibits ROMK through phosphorylation type of WNK1(1-119)] from three inde- on T58 of WNK1. Cells transfected with empty vector, wild-type (WT), or T58A mutant pendent experiments is shown in bar WNK1(1-491) were cultured in serum-free medium for 16 hours and received insulin graph in the bottom. (100 nM) or not for 2 hours. Equal amount of WT or T58A WNK1(1-491) expression is We further examined the activity of evident by Western blot analysis. Mean Ϯ SEM (n Ն 6 each). In A, D, and E, *P Ͻ 0.05 Ͻ these kinases acting in vivo (i.e., in intact between indicated groups by unpaired two-tailed t test. **P 0.01. NS, not statisti- cells). WNK1(1-220) (which lacks kinase cally significant. domain thus avoiding autophosphoryla- tion) was co-expressed with epitope-tagged Akt1 and SGK1 Phosphorylate WNK1 at Threonine-58 Akt1, SGK1, and/or PDK1 as indicated. Phosphorylation at In Vitro and In Vivo T58 was probed by anti-WNK1-T58 phospho-antibody. T58A Although WNK1 has been reported as a substrate for Akt1 and mutant of WNK1(1-220) was used as a negative control SGK1, the efficacy of WNK1 phosphorylation by different sta- (Figure 3B, lane 14, labeled as “M” for mutant). As shown, tus of Akt1 and SGK1 has not been clearly clarified yet. In this there was a low basal level of phosphorylation on WNK1(1- study, we decided to examine kinase activity of Akt1 and SGK1 220) (lane 1), presumably from the endogenous Akt1. Myr- systematically with the goal to guide our physiologic studies. istoylated and wild-type Akt1, but not kinase-dead mutant, We first examined the ability of exogenously expressed Akt1 caused phosphorylation of WNK1 above the basal level and SGK1 to phosphorylate WNK1 in vitro with or without (lanes 2 to 4). Coexpression with PDK1 slightly enhanced coexpressed PDK1. Expressed epitope-tagged Myc-PDK1, kinase activity of myristoylated Akt1 but not wild-type Akt1 HA-Akt1, or Flag-SGK1 was immunoprecipitated from HEK (lanes 8 to 10). The increase in the kinase activity of myris- cell lysates by respective antibodies. Purified bacterial His- toylated Akt1 by PDK1 in vivo, but not in vitro, may be tagged fragment of WNK1 consisting of amino acids 1 to 119 caused by preferential targeting of the myristoylated Akt1 to was used as the substrate. T58A mutant WNK1 was used as the the cell membrane, thus increasing co-localization with

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Figure 3. Akt1 and SGK1 phosphorylate WNK1 at threonine-58. (A) In vitro kinase assay of PDK1, Akt1, and SGK1. Epitope-tagged (Myc-, HA-, or Flag-) wild-type SGK1 (SGK1-WT), kinase-dead SGK1 (SGK1-KD), constitutively active mutant SGK1 (SGK1–S422D), WT Akt1 (Akt1-WT), myristoylated-Akt1 (Akt1-Myr), and kinase-dead Akt1 (Akt1-KD) were expressed in HEK cells with or without wild-type PDK1. Kinase activity of immunoprecipitated PDK1, Akt1, and SGK1 was assayed using wild-type or T58A WNK1(1-119) as a substrate. Immunoblots of precipitated proteins by respective epitope antibody (labeled “IB” on the right) and autoradiograph of kinase assay analyzed by phosphoimager (labeled “KA” on the right) are shown. Bar graph in the bottom is the relative kinase activity (normalized to lane 2, “Akt1-Myr”) specific for phosphorylation at T58 [i.e., subtracting non-T58 phosphorylation signal on WNK1(1-119)/T58A mutant from signal on wild-type WNK1(1-119)]. Mean Ϯ SEM from three separate experiments is shown on top of each bar. (B) In vivo phosphorylation on T58 of WNK1 by Akt1 and SGK1. Cells were transfected with epitope-tagged Akt1, SGK1, or PDK1 with wild-type WNK1(1-220) (lanes 1 to 13) or with WNK1(1-220)/T58A mutant (lane 14 labeled “M”). Protein expressions were blotted by specific antibodies. Phosphorylation on T58 of WNK1(1-220) was detected by anti-phospho-T58 WNK1 antibody. Doublet bands detected by anti-WNK1 and anti-phospho-T58 WNK1 antibodies were always found in WNK1(1-119) and WNK1(1-220) but not WNK1(1-491). The doublets probably represent different conformational forms of eukaryotic WNK1 proteins because they are not observed for purified His-tagged WNK1(1-119) proteins produced in the bacteria (see A). The abundance of each band in the gel reflecting kinase activity of Akt1 or SGK1 was measured by densitometry by the Image J program available at the NIH website. Basal level of T58 phosphorylation on WNK1(1-220) without exogenous Akt1 or SGK1 (lane 1) was defined as 1 for relative kinase activity measurement. Mean Ϯ SEM from three separate experiments is shown on top of each bar.

PDK1. As in vitro studies, S422D and wild-type SGK1 phos- Akt1 Inhibits ROMK through WNK1 phorylated WNK1 in vivo (lanes 5 and 6). PDK1 slightly The finding that a supraphysiologic concentration of insulin enhanced SGK1 kinase activity but not as much as the effect can further inhibit ROMK in the presence of serum (Figure found in the in vitro experiments (lanes 11 and 12). Differ- 2A) suggests that normally the endogenous Akt1 and WNK1 ences in the ratio of substrate relative to kinase and/or the are not maximally activated. Consistent with the idea, exoge- efficiency of kinase may account for the discrepancy. To our nous WNK1 inhibits ROMK (Figure 2E; see also ref. 21). We surprise, expression of kinase-dead SGK1 caused some thus asked whether overexpression of Akt1 may inhibit ROMK phosphorylation of WNK1 above the basal level, although by enhancing WNK1-T58 phosphorylation. We used myris- the activity was less compared with wild-type or S422D mu- toylated Akt1 because it can be enhanced by PDK1 in intact tant (lanes 7 and 13). At the moment, we do not have a good cells (Figure 3B). Myristoylated Akt1 (“Akt1-Myr”) inhibited explanation for the finding except to speculate that it may be ROMK but did not cause additional effect when WNK1(1- caused by altered activity of endogenous Akt1. Overall, 491) was co-transfected (Figure 4A). Kinase dead mutant of these results from in vitro and in vivo kinase assays support Akt1 (Akt1-KD) did not cause inhibition of ROMK but re- the idea that Akt1 and SGK1 can phosphorylate WNK1 at versed WNK1(1-491)–mediated inhibition. Thus, Akt1 and T58. WNK1 act on the same pathway.

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Figure 4. Akt1 inhibits ROMK in a WNK1–T58 phosphorylation-dependent manner. (A) Effect of Akt1 on ROMK. Cells were transfected with ROMK and with myristoylated Akt1 (Akt1-Myr), kinase-dead Akt1 (Akt1-KD), and/or WNK1(1-491) as indicated. (B) Effect of myristoylated Akt1 (Akt1-Myr) on ROMK in the presence of wild-type (WT) or T58A mutant of WNK1(1-491). (C) Effect of Akt1 on ROMK with or without endogenous WNK1. Cells were transfected with control oligonucleotide or WNK1 siRNA (200 nM each) and with empty vector or myristoylated Akt1 (Akt1-Myr). In each panel, ROMK current density (pA/pF at Ϫ100 mV) was represented as mean Ϯ SEM (n Ն 6). *P Ͻ 0.05 between indicated groups by unpaired two-tailed t test. **P Ͻ 0.01. NS, not statistically significant. Equal protein expression was confirmed by Western blot. (D) Effect of insulin on membrane abundance of ROMK with or without endogenous Akt1. Cells were transfected with ROMK and control oligonucleotide or Akt1 siRNA (200 nM each) and deprived of serum for 16 hours.

To confirm the role of Akt1 is from phosphorylation on T58 WNK1(1-491), even the constitutively active form of SGK1 did of WNK1, we showed that WNK1(1-481)/T58A had no effect not inhibit ROMK (Figure 5B). Knocking down endogenous on ROMK but reversed myristoylated Akt1-mediated inhibi- WNK1 by siRNA also abrogated the effect of SGK1 on ROMK tion of ROMK (Figure 4B). Knocking down endogenous (Figure 5C). Thus, exogenous Akt1 and SGK1 showed a similar WNK1 by siRNA prevented the inhibition of ROMK by myr- inhibitory effect on ROMK, and both effects depend on istoylated Akt1 (Figure 4C). Knocking down Akt1 blunted the WNK1-T58 phosphorylation. Next, we examined potential effect of insulin to decrease cell surface abundance of ROMK in synergistic effects of Akt1 and SGK1 on ROMK by silencing serum-free conditions (Figure 4D), linking the effect of Akt1- endogenous Akt1 and/or SGK1 using siRNA in the absence WNK1 on ROMK to insulin. (“vector”) or presence of exogenous WNK1 [“WNK1(1- 491)”]. Knocking down Akt1 or SGK1 individually increased SGK1 Inhibits ROMK through WNK1 and Works ROMK current significantly with or without exogenous Together with Akt1 WNK1 (Figure 5D). The effect of knocking down both Akt1 We next examined the potential role of SGK1, another mem- and SGK1 is greater than knocking down each individually in ber of the AGC kinase family that can also mediate down- the “WNK1(1-491)”–transfected but not in the “vector”- stream effect of PI3K, in regulating WNK1 inhibition of transfected group. Differences in the abundance of WNK1 ROMK. Similar to Akt1, constitutively active SGK1 mutant, substrate likely account for the different results. In summary, S422D, inhibited ROMK but did not cause an additional effect both Akt1 and SGK1 phosphorylate WNK1 and contribute to when WNK1(1-491) was co-expressed. Kinase-dead SGK1 did its regulation of ROMK. The importance of Akt1 versus SGK1 not inhibit ROMK but reversed the inhibition caused by in vivo will depend on the relative abundance of each respective WNK1 (Figure 5A). In the presence of the T58A mutant of kinase and WNK1 in the setting.

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Figure 5. SGK1 inhibits ROMK through the same pathway as Akt1. (A) Effect of SGK1 on ROMK. Cells were co-transfected with ROMK and constitutively active SGK1 (SGK1–S422D) or kinase-dead SGK1 (SGK1-KD) with or without WNK1(1-491). (B) Effect of SGK1 on ROMK in the presence of wild-type or T58A mutant of WNK1(1-491). SGK1–S422D was co-transfected with wild-type or T58A mutant WNK1(1-491). (C) Effect of SGK1 on ROMK with or without endogenous WNK1. Cells were transfected with control oligonucleotide or WNK1 siRNA (200 nM each) and with empty vector or SGK1–S422D. (D) Effect of siRNA of Akt1 and/or SGK1 on ROMK. Cells were transfected with siRNA for Akt1 (siAkt1) and/or SGK1 (siSGK1, 200 nM each) and with vector or WNK1(1-491). In each panel, ROMK current density was measured and presented as mean Ϯ SEM (n Ն 6 for each group). *P Ͻ 0.05 between indicated groups by unpaired two-tailed t test. **P Ͻ 0.01. NS, not statistically significant. Efficacy of Akt1 siRNA and equal expression of protein were confirmed by Western blot.

Inhibition of ROMK by SGK1 via Enhanced specific WNK1 (KS-WNK1) that lacks most of the kinase domain Endocytosis and Not by Phosphorylation of ROMK and preceding amino acids in the N terminus.24 Long WNK1 con- WNK1 inhibits ROMK by enhancing endocytosis through a tains T58, the target of Akt1/SGK1. In contrast, KS-WNK1 lacks dynamin-dependent, clathrin-mediated pathway.3,21,22 This T58. We showed that KS-WNK1 binds and antagonizes long effect of WNK1 requires an interaction with intersectin.11 It WNK1–induced inhibition of ROMK.21 Here, we asked whether has also been reported that SGK1 can directly phosphorylate KS-WNK1 antagonizes the effect of long WNK1 in the presence of on ROMK1 at serine-44, although this effect is believed to re- Akt1/SGK1. As reported previously, KS-WNK1 reversed sult in an increase of the cell surface abundance of ROMK.23 WNK1(1-491)–induced inhibition of ROMK (Figure 7A, left We found that co-expression of a dominant-negative (“DN”) three bars). The ability of KS-WNK1 to antagonize WNK1(1- intersectin (“ITSN”) or dynamin prevented inhibition of ROMK 491)–induced inhibition of ROMK was unaltered in the presence by SGK1-S422D (Figure 6A). These results, together with our pre- of exogenous constitutively active SGK1 (SGK1-S422D) (Figure vious reports that these experimental maneuvers abolish inhibi- 7A, last bar on the right). KS-WNK1 may antagonize long WNK1 tion of ROMK by WNK1, support that phosphorylation of inhibition of ROMK by interfering with its phosphorylation by WNK1-T58 by SGK1 leads to inhibition of ROMK by increasing Akt1/SGK1 (Figure 7B, mechanism “1”) or interfering with its endocytosis of ROMK. In further support of this idea, we found interaction with downstream effectors of endocytosis, such as in- that SGK1-S422D inhibited ROMK bearing a mutation of ser- tersectin (mechanism “2”). To distinguish between these two pos- ine-44 (S44D) and wild-type ROMK (Figure 6B). sibilities, we examined the effect of KS-WNK1 on serum-induced phosphorylation of WNK1(1-491) in HEK cells coexpressed with Kidney-Specific WNK1 Blocks SGK1 Effect on ROMK SGK1-S422D. Serum deprivation decreased T58 phosphorylation without Interfering with Phosphorylation on WNK1 on WNK1, which was enhanced by SGK1-S422D (lanes 1 to 3; WNK1 has multiple alternatively spliced isoforms, including the Figure 7C). Coexpression of KS-WNK1 did not affect phosphor- full-length WNK1 (also known as long WNK1) and a kidney- ylation of WNK1 at T58 (lanes 4 to 6). KS-WNK1 alone had no

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physiologic relevancies. One of these is the maintenance of Kϩ homeostasis during chronic (approximately 1 week) Kϩ deficiency. IGF1 is produced in the kidney, and production is upregulated by chronic dietary ϩ K restriction.25 Upregulation of IGF1 is believed to play a role in the Kϩ deficiency– induced renal hypertrophy. An increase in the level of IGF1, nonetheless, can decrease renal Kϩ secretion via ROMK, contribut- ing to Kϩ conservation during Kϩ deficiency. In support of the idea that IGF1 inhibits renal Kϩ secretion in vivo, intravenous administration of IGF1 in humans decreases renal Kϩ excretion without significant changes in the filtered load of Figure 6. Effect of SGK1 on ROMK is dynamin and intersectin (ITSN)-dependent, but ϩ 26 not dependent on phosphorylation of ROMK at S44. (A) Effect of SGK1 on ROMK in K . Conversely, mice with liver-specific the presence of dominant-negative intersectin (ITSN-DN) or dynamin (dynamin-DN). deletion of IGF1 have approximately 80% Cells were transfected with SGK1–S422D or without (“vector”) and with ITSN-DN or reduction in the circulating IGF1 and in- ϩ dynamin-DN or without (“control”). (B) Effect of SGK1 on wild-type or S44D mutant creased renal K excretion despite a nor- ϩ ROMK. Cells were co-transfected with SGK1–S422D and with wild-type ROMK (ROMK- mal filtered load of K .27 Finally, inhibition WT) or S44D mutant of ROMK (ROMK-S44D). In each panel, ROMK current density was of PI3K increases the density of native measured and presented as mean Ϯ SEM (n Ն 6 for each group). *P Ͻ 0.05 between ROMK channels in mouse CCD,28 provid- Ͻ indicated groups by unpaired two-tailed t test. **P 0.01. NS, not statistically ing further support for the physiologic role significant. Equal protein expression was confirmed by Western blot. of PI3K in the inhibition of ROMK and renal Kϩ secretion. effect on WNK1 phosphorylation (lane 7). Thus, KS-WNK1 Insulin also activates PI3K and thus may be another up- likely affects WNK1 interaction with downstream effectors. stream signal that uses the Akt1/SGK1-WNK1 pathway to inhibit renal Kϩ excretion. A physiologic role of insulin in de- creasing renal Kϩ excretion in vivo, however, is not universally DISCUSSION accepted. Although it is known that insulin decreases urinary Kϩ excretion, some suggested that the effect is entirely from ROMK undergoes constitutive clathrin-mediated endocy- the decrease in the plasma Kϩ caused by the intracellular tosis.3 WNK kinases including WNK1 and WNK4 inhibit shift.29 In contrast, others showed that the decrease in urinary ROMK by increasing endocytosis.21,22 We previously reported that WNK1 and 4 interact with intersectin, an endocytic scaffold protein that binds dynamin and other endocytic accessory proteins.11 The interaction with intersectin leads to stimulation of endocytosis of ROMK, probably by enhancing the recruitment and assembly of endocytic ma- chinery. In this study, we showed that activation of PI3K by insulin, IGF1, and likely by other serum growth factors enhances endocytosis of ROMK through phosphorylation of T58 on WNK1. This effect on ROMK via WNK1 depends on two members of AGC kinases: Akt1 and SGK1. As summarized in Figure 8, activation of PI3K stimulates mTORC2 to phosphorylate Akt1 and SGK1 at S473 and S422 in the hydrophobic motif, respectively. Phosphorylation by mTORC2 allows binding of PDK1, which phosphorylates Akt1 and SGK1 at T308 and T256 in the T-loop, respectively, to activate their catalytic activity. Activated Akt1 and SGK1 phosphorylate WNK1 at T58, leading to enhanced endocytosis of ROMK via an intersectin-dependent mechanism. Figure 8. A working model for regulation of ROMK by PI3K- This mechanism of regulation of ROMK by Akt1 and/or activating hormones via Akt1/SGK1 and WNK1 and by aldoste- SGK1 via WNK1 has several potential physiologic or patho- rone. See texts for details and for abbreviations.

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ies. The abundance of ROMK in the apical membrane of distal nephron in mice ho- mozygous for Sgk1 deletion is higher than that in the wild-type littermates.34 The increase in the ROMK abundance in Sgk1 knockout mice was suggested to be caused by a compensatory response to hyperkalemia caused by reduced Naϩ reabsorption and thus reduced electrical driving force for Kϩ secretion. However, in mice with double knockout of Sgk1 and Sgk3 (in which Naϩ wasting is evident in normal Naϩ diets), the fractional urinary Kϩ excretion is higher than that in wild-type littermates, despite a normal blood Kϩ level and an impairment in Naϩ reabsorption in the double knockout mice.35 These results support that SGK1 (perhaps together with SGK3 or isoforms) inhibits ROMK in vivo. Figure 7. Effect of SGK1 on ROMK is reversed by kidney-specific WNK1 (KS-WNK1). It is possible that SGK1 can also exert a (A) Effect of SGK1 on ROMK in the presence of KS-WNK1. Cells were transfected with stimulatory effect on ROMK under differ- SGK1–S422D, WNK1(1-491), and/or KS-WNK1(1-253) as indicated. KS-WNK1(1-253) ent physiologic contexts or presence of cer- consists of amino acids 1 to 253 of KS-WNK1. ROMK current density was measured tain co-regulators. Our present cell-based Ϯ Ն Ͻ and presented as mean SEM (n 6 for each group). **P 0.01 between indicated study, nonetheless, provides strong evi- groups by unpaired two-tailed t test. NS, not statistically significant. Equal protein dence to support the hypothesis that Akt1/ expression was confirmed by Western blot. (B) Possible mechanisms for KS-WNK1 to SGK1-mediated phosphorylation of block SGK1 effect on ROMK. Mechanism 1: Interfering with WNK1 phosphorylation by WNK1 can mediate inhibition of ROMK SGK1. Mechanism 2: Interfering with WNK1 interaction with downstream effectors, ϩ such as intersectin (“ITSN”) and dynamin (“Dyn”). (C) Effect of KS-WNK1 on WNK1– and renal K secretion by PI3K-activating T58 phosphorylation by SGK1. Cells were all transfected with WNK1(1-491), KS- hormones. Future experiments will exam- ϩ WNK1(1-253) (at DNA amount from 0 to 0.9 ␮g), and/or SGK1–S422D and incubated ine the effect of insulin and IGF1 on K with or without serum as indicated. Basal level of WNK1(1-491) phosphorylation is homeostasis in mice with WNK1, SGK1, shown in lane 1. For experiments shown in lanes 2 to 7, cells were incubated in and/or Akt1 deletions. serum-free media for 16 hours. Protein expression was detected by specific antibodies. Aldosterone is a positive regulator of Phosphorylation on WNK1–T58 was determined using anti-phospho-T58 WNK1 anti- SGK1.36 Our finding that SGK1 inhibits body. ROMK may seem to be counterintuitive to the fact that aldosterone stimulates renal Kϩ Kϩ excretion caused by insulin infusion in humans is more secretion. However, besides SGK1, aldosterone also stimulates than the decrease in the filtered load, supporting that insulin KS-WNK1,37 which antagonizes the effect of SGK1 phosphoryla- ϩ inhibits renal K excretion in vivo.30,31 In support of this idea, tion of (long) WNK1 on ROMK (see Figures 7 and 8). Thus, application of insulin to the basolateral bath of isolated per- whereas insulin and IGF1 activate the Akt1/SGK1-WNK1 signal- fused rabbit CCD inhibits the net transepithelial Kϩ secre- ing cascade to inhibit ROMK, aldosterone activates two opposing ϩ 32 tion. The IC50 for inhibition of K secretion in the CCD is effects on the signaling cascade (the negative and positive effect via 500 pM, which is higher than the normal fasting level of insulin SGK1 and KS-WNK1, respectively) and therefore may not have a (10 to 150 pM) but within the normal postprandial level (300 net effect on ROMK. The principal mechanism for aldosterone ϩ to 800 pM).16 The minimal concentration of insulin required stimulation of K secretion is likely the increase in the electrical for inhibition of ROMK in our studies is 1 nM. This value is not driving force for Kϩ secretion secondary to enhanced Naϩ reab- far from the effective concentration of insulin in the CCD and sorption, rather than via the SGK1-WNK1 cascade. the postprandial levels in vivo, considering that different experimental systems are compared. The inhibition of ROMK-me- ϩ diated renal K secretion by insulin, if it occurs in vivo, will CONCISE METHODS help to maintain the plasma Kϩ level in the postprandial state ϩ during which a very active intracellular K shift occurs. DNA Constructs and Reagents Our finding that SGK1 inhibits ROMK is different from pEGFP-ROMK1, pCMV5-Myc-WNK1, pIRES-Flag-KS-WNK1, and other reports that SGK1 stimulates ROMK.23,33 The in vivo dominant-negative intersectin and dynamin have been described pre- importance of our finding is supported by several animal stud- viously.21 The plasmids encoding N-terminal 60 amino acids trun-

468 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 460–471, 2011 www.jasn.org BASIC RESEARCH cated and Flag-tagged wild-type, S422D mutant and kinase-dead mu- bated with respective antibodies (anti-rabbit anti-Myc for PDK1, an- tant SGK1 (pCMV7–3xFlag-‚SGK1), HA-tagged wild-type, ti-mouse anti-HA for Akt1, and anti-mouse anti-Flag for SGK1 each myristoylated and kinase-dead mutant Akt1 (pCMV-HA-Akt1), and at 1:300 dilution) and 30 ␮l of 50% slurry of protein A (for immuno- myc-tagged PDK1 (pcDNA3-Myc-PDK1) were generous gifts from precipitating PDK1) or protein G (for immunoprecipitating Akt1 and M. Cobb (UT Southwestern Medical Center at Dallas).38 Point muta- SGK1) at 4°C overnight. Then beads were washed three times with 1 tions were generated by site-directed mutagenesis (QuickChange kit; ml wash buffer (0.25 M Tris, pH 7.4, 1 M NaCl, 0.1% Triton X-100, Stratagene) and confirmed by sequencing. Sense and anti-sense oli- proteinase, and phosphatase inhibitor cocktails) for 10 minutes and gonucleotides (Dharmacon RNA Technology) for human WNK1 followed by one wash with 1 ml kinase wash buffer (10 mM HEPES, Ј Ј Ј siRNA were 5 -UGUCUAACGAUGGCCGCUUdTdT-3 and 5 - pH 7.6, and 10 mM MgCl2). After removing the kinase wash buffer, AAGCGGCCAUCGUUAGACAdTdT-3Ј. Sense and anti-sense oligo- beads were incubated with 2 ␮g WNK1(1-119) wild-type or T58A nucleotides for human SGK1 were 5Ј-GUCCUUCUCAGCAA- mutant proteins purified from Escherichia coli in 30 ␮l of kinase buffer AUCAAUU-3Ј and 5Ј-UUGAUUUGCUGAGAAGGACUU-3Ј. Sense (20 mM HEPES, pH 7.6, 5 ␮M ATP [5 ␮Ci of ␥-32P ATP], 10 mM Ј ␤ and anti-sense oligonucleotides for human Akt1 were 5 -GACCGC- MgCl2, and 10 mM -glycerol phosphate) at 30°C for 45 minutes. The CUCUGCUUUGUCAdTdT-3Ј and 5Ј-UGACAAAGCAGAGGCG- samples were boiled with 7.5-␮l fivefold sampling buffer at 90°C for 5 GUCdTdT-3Ј. Insulin from bovine pancreas and IGF1 were minutes. Supernatants were separated by SDS-PAGE for Western blot purchased from Sigma. The following antibodies were used: anti- and phosphoimager analysis. The radioisotope intensity of the bands WNK1 antibody (Q256) (1:5000 dilution; described previously),21 was determined by phosphoImager, Storm 860 (GE Healthcare, Pis- anti-Flag antibody (M2) (1:5000 dilution; Sigma), anti-c-Myc (1: cataway, NJ), and ImageQuant 5.2 software (GE Healthcare, Little 5000 dilution; Sigma), anti-HA antibody (12CA5) (1:5000 dilu- Chalfont, Buckinghamshire, UK). tion; Berkeley Antibody), anti-WNK1 phospho-Thr-58 (1:1000 dilution; Abcam), anti-Akt1 (AW24; 1:1000 dilution; Millipore), anti-Akt1 phospho-Thr-308 (C31E5E; 1:1000 dilution; Cell Sig- Whole-Cell Patch-Clamp Recording of ROMK Channels After 48-hour transfection, cells were trypsinized and plated on poly- naling), anti-Akt1 phospho-Ser-473 (D9E; 1:1000 dilution; Cell L-lysine–coated coverslips. Whole-cell ROMK currents were re- Signaling), anti-SGK1 phospho-Thr-256 (1:1000 dilution; Santa corded using an Axopatch 200B amplifier (Axon Instruments, Foster Cruz), and anti-GFP horseradish peroxidase conjugate (1:1000 di- City, CA) as described previously.11 The pipette resistance was around lution; Invitrogen). 1.5 to 3 M⍀. Green fluorescence of GFP-ROMK in transfected cells was identified by epifluorescent microscopy. The pipette solution Cell Culture, Transfection, Preparation of Cell Lysates, contained 140 mM KCl and 10 mM HEPES (pH 7.2); the bath solu- Immunoblotting, and Kinase Assays tion contained 140 mM KCl, 1 mM MgCl , 1 mM CaCl , and 10 mM HEK-293 cells were co-transfected with cDNA (0.3 ␮g per six wells) 2 2 HEPES (pH 7.4). The cell membrane capacitance and series resistance for GFP-ROMK1 plus other indicated cDNAs using Fugene HD were monitored and compensated (Ͼ75%) electronically. The volt- (Roche) as described.11 In each experiment, the total amount of DNA age protocol consisted of 0-mV holding potential and 500-ms steps for transfection was balanced by using empty vectors. Transfected from Ϫ100 to 100 mV in 25-mV increments. ClampX 9.2 software cells were identified by green fluorescence. Approximately 36 to 48 (Axon Instruments) was used for data acquisition. Current density hours after transfection, cells were dissociated and placed in a cham- was calculated by dividing current at Ϫ100 mV (pA; measured at ber for ruptured whole-cell recordings. For knockdown by siRNA, 25°C) by capacitance (pF). Results were shown as mean Ϯ SEM (n ϭ oligonucleotides (200 nM each) were mixed with cDNAs for ROMK1 6–10). Each experiment (i.e., set of results shown in each panel of a and other indicated constructs for co-transfection by PolyFect (Qua- figure) was repeated two to four times. gen). For serum deprivation studies, cells were washed with PBS two times and cultured in serum-free DMEM for different time periods as indicated. Surface Biotinylation Assay Cultured cells were incubated with lysis buffer (50 mM HEPES, For biotinylation of cell surface ROMK, cells were washed with ice- pH 7.6, 150 mM sodium chloride, 0.5% Triton X-100, 10% glycerol, cold PBS and incubated with 0.75 ml PBS containing 1.5 mg/ml EZ- protease inhibitor cocktail [Mini EDTA-free, Roche], and phospha- link-NHS-SS-biotin (Thermo Scientific) for 1 hour at 4°C. After tase inhibitor cocktail [PhosSTOP, Roche]). After shaking for 30 min- quenching with glycine (100 mM), cell were lysed in a RIPA buffer utes on a rotator at 4°C, extracts were cleared by centrifugation. Pro- (150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1% Triton X-100, tein concentrations of supernatant were measured by the Bradford 0.5% DOC, and 0.1% SDS) containing protease inhibitor cocktail. assay using BSA as a standard. Equal amounts of lysates were boiled in Biotinylated proteins were precipitated by streptavidin-agarose beads Laemmli sample buffer and separated by SDS-PAGE under reducing (Thermo Scientific). Beads were subsequently washed four times with conditions. For Western blotting, proteins were transferred to nitro- PBS containing 1% Triton X-100. Biotin-labeled proteins were eluted cellulose membranes, blocked using 5% nonfat milk, incubated with in sample buffer, separated by SDS-PAGE, and transferred to nitro- the appropriate antibodies, and detected using enhanced chemilumi- cellulose membranes for Western blotting. ROMK proteins on the nescence detection reagent (Pierce). membrane were detected using anti-GFP horseradish peroxidase con- For kinase assays, epitope-tagged PDK1, Akt1, and/or SGK1 were jugate antibody. The biotinylation experiment was performed three co-transfected in HEK cells as indicated. Lysates (500 ␮g) were incu- times with similar results.

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Statistical Analysis cation-chloride-coupled cotransporters via the STE20-related kinases, Data analysis and curve fitting were performed with Prism (v5.03) SPAK and OSR1. J Biol Chem 280: 42685–42693, 2005 software (GraphPad Software, San Diego, CA). Data are presented as 9. Xu BE, Stippec S, Chu PY, Lazrak A, Li XJ, Lee BH, English JM, Ortega Ϯ B, Huang CL, Cobb MH: WNK1 activates SGK1 to regulate the epi- mean SEM. Statistical comparisons between two groups of data thelial sodium channel. Proc Natl Acad Sci USA 102: 10315–10320, were made using the two-tailed unpaired t test. Multiple comparisons 2005 were determined using one-way ANOVA. Time course and dose– 10. Yang CL, Angell J, Mitchell R, Ellison DH: WNK kinases regulate response curves were fitted by nonlinear regression analysis. Statisti- thiazide-sensitive Na-Cl cotransport. J Clin Invest 111: 1039–1045, cal significance was defined as P Ͻ 0.05 for single comparison and P Ͻ 2003 11. He G, Wang HR, Huang SK, Huang CL: Intersectin links WNK kinases 0.01 for multiple comparisons. to endocytosis of ROMK1. J Clin Invest 117: 1078–1087, 2007 12. Vitari AC, Deak M, Collins BJ, Morrice N, Prescott AR, Phelan A, Humphreys S, Alessi DR: WNK1, the kinase mutated in an inherited high-blood-pressure syndrome, is a novel PKB (protein kinase B)/Akt ACKNOWLEDGMENTS substrate. Biochem J 378: 257–268, 2004 13. Jiang ZY, Zhou QL, Holik J, Patel S, Leszyk J, Coleman K, Chouinard We thank Dr. Melanie Cobb and Aileen Klein at UT Southwestern at M, Czech MP: Identification of WNK1 as a substrate of Akt/protein Dallas for reagents and for comments and advice on the in vitro kinase kinase B and a negative regulator of insulin-stimulated mitogenesis in 3T3–L1 cells. J Biol Chem 280: 21622–21628, 2005 assay; our colleague Dr. Seung-Kuy Cha for advice on patch-clamp 14. Xu BE, Stippec S, Lazrak A, Huang CL, Cobb MH: WNK1 activates experiments; and Drs. Yuh-Feng Lin, Shih-Hua Lin, and Pauling Chu SGK1 by a phosphatidylinositol 3-kinase-dependent and non-catalytic at Tri-Service General Hospital, Taipei, Taiwan for support and en- mechanism. J Biol Chem 280: 34218–34223, 2005 couragement. This study is supported by National Institutes of Health 15. Lee IH, Dinudom A, Sanchez-Perez A, Kumar S, Cook DI: Akt mediates Grants RO1DK-59530 and P30DK079328. C.L.H. holds the Jacob Le- the effect of insulin on epithelial sodium channels by inhibiting Nedd4–2. J Biol Chem 282: 29866–29873, 2007 mann Professorship in Calcium Transport of University of Texas 16. Ahmed M, Gannon MC, Nuttall FQ: Postprandial plasma glucose, Southwestern Medical Center. C.-J.C. is supported by a scholarship insulin, glucagon and triglyceride responses to a standard diet in grant from the Ministry of Defense, Taiwan. normal subjects. Diabetologia 12: 61–67, 1976 17. Rechler MM, Nissley SP: The nature and regulation of the receptors for insulin-like growth factors. 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