WNK1 Promotes PIP2 Synthesis to Coordinate Growth Factor and GPCR-Gq Signaling

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provided by Elsevier - Publisher Connector Current Biology 21, 1979–1987, December 6, 2011 ª2011 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2011.11.002 Article WNK1 Promotes PIP2 Synthesis to Coordinate Growth Factor and GPCR-Gq Signaling

Sung-Wan An,1,4 Seung-Kuy Cha,1,3,4 Joonho Yoon,1,4 example, WNK1 and WNK4 phosphorylate and activate Seungwoo Chang,2 Elliott M. Ross,2,* Ste20-related proline-alanine-rich kinase (SPAK) and oxidative and Chou-Long Huang1,* stress-responsive kinase-1 (OSR1), which in turn activate 1Department of Medicine cation-chloride cotransporters NCC and NKCC [5–7]. Through 2Department of Pharmacology kinase-independent events, WNK1 binds and somehow University of Texas Southwestern Medical Center, Dallas, activates SGK kinase, causing it to activate the epithelial Na+ TX 75390, USA channel ENaC [8]; and WNK1 and WNK4 interact with the 3Department of Physiology, Wonju College of Medicine, endocytic scaffold protein intersectin to enhance endocytosis Yonsei University, Wonju, 220-710 Republic of Korea of the renal K+ channel ROMK [9]. Dysregulation of ion trans- port in the kidney contributes to the hypertension and hyperkalemia of PHA2. Summary The functions of WNKs extend beyond regulation of ion homeostasis. WNK1 is ubiquitously expressed, and homozy- Background: PLC-b signaling is generally thought to be medi- gous gene inactivation in mice causes cardiovascular devel- ated by allosteric activation by G proteins and Ca2+. Although opmental arrest and embryonic death [10]. WNK1 activates

availability of the phosphatidylinositol-4,5-biphosphate (PIP2) ERK5, and knockdown of WNK1 decreases activation of substrate is limiting in some cases, its production has not ERK5 by epidermal growth factor receptors [11]. WNK2 binds been shown to be independently regulated as a signaling the small GTPases RhoA and Rac1. WNK2 knockdown mechanism. WNK1 protein kinase is known to regulate ion decreases RhoA activation but promotes Rac1 activation

homeostasis and cause hypertension when expression is [12]. The Gq-coupled angiotensin II receptor regulates the increased by gene mutations. However, its signaling functions activity of the NCC cotransporter through the WNK4 signaling remain largely elusive. pathway [13], indicating functional interactions between WNK Results: Using diacylglycerol-stimulated TRPC6 and inositol and G protein signaling pathways. trisphosphate-mediated Ca2+ transients as cellular biosen- Upstream regulation of the WNKs is incompletely under- sors, we show that WNK1 stimulates PLC-b signaling in cells stood, and it is likely that several distinct mechanisms of

by promoting the synthesis of PIP2 via stimulation of phospha- WNK activation are used in different cells [3, 4]. WNK1 is a tidylinositol 4-kinase IIIa. WNK1 kinase activity is not required. substrate of Akt kinase [14, 15], which is a target of phospha-

Stimulation of PLC-b by WNK1 and by Gaq are synergistic; tidylinositol 3-kinase (PI3K)-activating growth factors such as WNK1 activity is essential for regulation of PLC-b signaling insulin and insulin-like growth factors (IGFs). Insulin and IGFs by Gq-coupled receptors, and basal input from Gq is necessary activate PI3K and its phosphatidylinositol-1,4,5-triphosphate for WNK1 signaling via PLC-b. WNK1 further ampliﬁes PLC-b (PIP3) product recruits and activates Akt to phosphorylate signaling when it is phosphorylated by Akt kinase in response WNK1 at threonine-58. It has been reported that Akt-catalyzed to insulin-like growth factor. phosphorylation of WNK1 at T58 alters the ability of WNK1 to Conclusions: WNK1 is a novel regulator of PLC-b that acts by regulate membrane trafﬁcking of ENaC and ROMK channels controlling substrate availability. WNK1 thereby coordinates [8, 16]. signaling between G protein and Akt kinase pathways. Phospholipase C-bs (PLC-bs) are important effectors of

Because PIP2 is itself a signaling molecule, regulation of PIP2 G protein-coupled receptor (GPCR)-mediated signaling path- synthesis by WNK1 also allows the cell to initiate PLC signaling ways [17, 18]. They hydrolyze phosphatidylinositol-4,5-

while independently controlling the effects of PIP2 on other biphosphate (PIP2), itself a signaling molecule, to form two targets. These ﬁndings describe a new signaling pathway for other second messengers, diacylglycerol (DAG) and inositol-

Akt-activating growth factors, a mechanism for G protein- 1,4,5-trisphosphate (IP3). PLC-bs are stimulated by Ga growth factor crosstalk, and a means to independently control subunits of the Gq family, by Gbg subunits of the Gis, and, in PLC signaling and PIP2 availability. the case of PLC-b2, by Rac [17–19]. The concentration of PIP2 in the plasma membrane is limiting for PLC-b activity, and resynthesis of the substrate initiated by phosphatidylino- Introduction sitol-4-kinase (PI4K) after depletion is important for sustained PLC-b signaling [20, 21]. This dependence on substrate Mammalian WNK kinases are a family of four serine-threonine concentration agrees with in vitro studies showing that PLC protein kinases with an atypical kinase active site [1–3]. Muta- activity increases linearly with the concentration of PIP tions in WNK1 and WNK4 cause the autosomal-dominant 2 regardless of stimulation by G proteins or Ca2+ [22, 23]. disease pseudohypoaldosteronism type 2 (PHA2), which is However, it is not known whether PIP production can be inde- characterized by hypertension and hyperkalemia [2]. WNKs 2 pendently regulated as a signaling mechanism and, if so, by regulate various ion transporters through both kinase what physiological stimulus. activity-dependent and -independent mechanisms [3, 4]. For We show here that WNK1 stimulates PLC-b signaling by

promoting the synthesis of PIP2 via stimulation of PI4KIIIa. 4These authors contributed equally to this work Stimulation of PLC-b by WNK1 and by Gaq-GTP is thus *Correspondence: [email protected] (E.M.R.), chou-long. synergistic, and WNK1 activity is essential for regulation of [email protected] (C.-L.H.) PLC-b signaling by Gq-coupled receptors. Phosphorylation Current Biology Vol 21 No 23 1980

of WNK1 by the PI3K-Akt kinase cascade enhances the ability of WNK1 to synergistically regulate PLC-b signaling via synthesis of PIP2. Regulated control of substrate availability represents a novel mechanism for regulation of PLC-b activity and for crosstalk between Gq-coupled receptors and receptors that activate the Akt kinase pathway.

Results

WNK1 Activates TRPC6 Channels The TRPC6 channel is directly activated by DAG, a product of the PLC-catalyzed hydrolysis of PIP2 [24]. Inspired by a serendipitous observation that WNK1 knockdown altered Gq signaling, we used TRPC6 as a biosensor to examine the role of WNK1 in PLC-b signaling in native cell membranes. Full-length WNK1 is difﬁcult to purify, and we tested the effect of WNK1 on TRPC6 by intracellular application of a puriﬁed fragment of rat WNK1 consisting of residues 1–491 (WNK11-491), which contains the Akt-phosphorylation site, the protein kinase domain, and an N-terminal proline-rich domain (Figure 1A). As shown for HEK293 cells in Figure 1B, intracellular WNK11–491 increased TRPC6 current density, peaking in these cells at around 1–2 min and lasting for

>5 min. The half-maximal concentration of WNK11–491 for activation of TRPC6 was w50 nM (Figure 1C). WNK11–491 also activates TRPC6 in membranes of HeLa cells (see Fig- ure 6) and mouse embryo ﬁbroblasts (see Figures 2C and Figure 1. Activation of TRPC6 by WNK1 Requires PLC Activity and Is 2D). TRPC6 was also activated by GTPgS, consistent with Independent of WNK1 Kinase Activity b (A) Top shows domain structure of rat WNK1. Bottom shows whole-cell Gq-PLC- signaling to produce DAG, and by the muscarinic 2+ agonist carbachol in cells that express the G -coupled M3 patch-clamp recording of TRPC6. Intracellular free [Ca ] was 70 nM except q as indicated in (F). The following abbreviations are used: Akt, site of phos- muscarinic cholinergic receptor (Figure 1B). phorylation by Akt; PRD, proline-rich domain; KD, kinase domain; CCh: Application of WNK11–491 to the cytoplasmic face of de- carbachol (100 mM); M3R: M3 muscarinic cholinergic receptor. tached plasma membranes also activated TRPC6 single (B) Activation of TRPC6 by CCh via M3R, GTPgS, or WNK11–491. HEK cells channel activity in membranes of cells that express TRPC6 were transfected with TRPC6 with or without M3R. Data points (circles (see Figure S1A available online) but not in membranes from and error bars) are means 6 SEM of inward current density (pA/pF 2 control cells. Thus, the cellular components required for acti- at 100 mV; n = 5 for each). At the end of each recording, extracellular Na+ was replaced by the TRPC6-impermeable cation N-methyl-D-gluc- vation of TRPC6 by WNK1 are contained in or are bound to amine (NMDG) to gauge the background current. Inset shows current- the excised plasma membrane patch. Stimulation of TRPC6 voltage (I-V) relationships of activated TRPC6 currents. was selectively blocked by antibody against amino acid resi- (C) Concentration-response curve for activation of TRPC6 by WNK11–491. dues 160–176 of WNK1 (Figure S1B). Puriﬁed WNK11–491 (D) Effect of PLC inhibitor U73122 or inactive analog U73343 (2.5 mM each) 2 carrying a kinase-dead K233M mutation [1, 9] retained full on WNK11–491 activation of TRPC6. Inward current is at 100 mV from ability to activate TRPC6 (Figure S1C), indicating that kinase a representative experiment. Similar results were observed in six independent experiments. activity of WNK1 is dispensable. (E) Cells were preincubated with 15 mM edelfosine for 1 hr and washed

before recording. TRPC6 currents in response to 100 nM WNK11–491,5nM Activation of TRPC6 by WNK1 Requires PLC Activity PLC-b1, or both were measured. Several experimental results argue that the activation of (F) TRPC6 currents in response to WNK11–491 under 0 or 70 nM intracellular TRPC6 by WNK1 is mediated by PLC-b-catalyzed production calcium. 1-oleoyl-2-acetyl glycerol (OAG) was applied as indicated. of the known TRPC6 activator DAG. First, activation of (G) TRPC6 currents were measured in cells with or without intracellular application of 3 nM WNK11–491. OAG was applied to the extracellular TRPC6 by WNK11–491 was blocked by the PLC inhibitors solution at indicated concentrations. Although this concentration of

U73122 (Figure 1D) and edelfosine [25](Figure 1E). Application WNK11–491 by itself does not activate TRPC6, it potentiated M3R activation of 1-oleoyl-2-acetyl glycerol (OAG), a soluble and membrane- of the channel (see Figure 3B; Figure S1). permeant DAG, restored TRPC6 activity in the continued presence of U73122, which excludes effects of U73122 on the channel itself or on other components upstream of PLC. data above, WNK11–491 might activate TRPC6 by protecting Second, because edelfosine acts slowly [25], we could rescue DAG from hydrolysis by DAG lipase or phosphorylation by WNK1 signaling by addition of puriﬁed PLC-b1 after endoge- DAG kinase. However WNK11–491 did not alter the potency nous PLC was inhibited (Figure 1E). After edelfosine pretreat- with which OAG activates TRPC6 (Figure 1G), indicating that ment, application of both puriﬁed PLC-b1 and WNK11–491 its effect is at the level of PLC. Together, these results provide caused robust activation of TRPC6. Application of PLC-b1 compelling evidence that WNK1 activates TRPC6 by stimula- without WNK1 had no effect, indicating that both WNK1 and tion of PLC to produce DAG, the proximal activator. 2+ PLC-b1 are required for activation of TRPC6. Third, PLC We also measured intracellular Ca release triggered by IP3, 2+ activity is dependent on cytosolic Ca [17, 18], and removal another product of PIP2 hydrolysis, as a readout for WNK1’s 2+ of Ca blocked TRPC6 stimulation by WNK11–491, but not effects on PLC activity. Microinjection of anti-WNK1 antibody direct stimulation by OAG (Figure 1F). Based only on the or knocking down endogenous WNK1 by small interfering WNK1 Coordinates Growth Factor and GPCR Signaling 1981

Figure 2. Role of Gq in the Activation of TRPC6 by WNK1 (A) GDPbS (0.2 mM) inhibits WNK1 activation of TRPC6. (B) Anti-WNK1 antibody inhibits TRPC6 activated by GTPgS. TRPC6 single channel activity was recorded from inside-out patches from TRPC6-expressing HEK cells. Buffer, GTPgS, anti-WNK1 Ab and/or OAG was applied to the cytoplasmic face as indicated. Each data point is NPo (number x open probability) averaged over 30 s. Inset shows mean 6 SEM of peak open probability. *p < 0.01 versus buffer, and **p < 0.01 GTPgS versus GTPgS+anti-WNK1 antibody. (C) Whole-cell WNK1-activated TRPC6 currents were

measured in Gaq/11 null ﬁbroblasts transfected with wild-type Gaq (Gaq/WT) or empty vector. (D) Single channel activity was measured in inside-out

patches from TRPC6-expressing Gaq/11 null ﬁbroblasts that have endogenous WNK1 depleted by small interfering (siRNA). Data show responses to GTPgS-preacti-

vated Gaq (Gaq*) and/or WNK1 applied to the cytoplasmic face. Holding membrane potential is 280 mV. Inset shows mean 6 SEM of peak open probability. *p < 0.01 versus buffer, **p < 0.05 versus buffer, and ***p < 0.02 between indicated groups. (E and F) Single channel activity was recorded in inside- out patches from HEK cells expressing both TRPC6 and

wild-type Gaq (Gaq/WT) (E) or G188S mutant Gaq (Gaq/ G188S) (F). Buffer, WNK11–491, and/or RGS4 (0.5 mM) was applied to the cytoplasmic face as indicated. NPo values were averaged every 20 s. There was a 2 min time gap between application of buffer and WNK1 (see also Figure S2).

protein. To determine whether WNK1 requires activation of PLC-b by Gq/11 in cells, we measured TRPC6 regulation by WNK1 in murine 2/2 2/2 Gaq -Ga11 embryonic ﬁbroblasts [26]. In intact Gaq/11 null cells, WNK11–491 failed to activate TRPC6, and expression of wild-type 2+ RNA (siRNA) reduced the intracellular Ca increases in re- Gaq restored activation by WNK1 (Figure 2C). When the con- sponse to carbachol (Figures S1D–S1F). Endogenous WNK1 stitutively active Q209L-Gaq mutant was expressed, addition is thus required for normal PLC-b signaling in response to of WNK11–491 further activated TRPC6 (Figures S2C and stimulation of Gq. S2D), which argues that WNK1 and Gaq act in parallel rather than in series to stimulate DAG synthesis by PLC-b.

WNK1 Works in Parallel with Gq to Synergistically When WNK1 was depleted in the Gaq/11 null cells by siRNA, Stimulate TRPC6 neither WNK11–491 nor Gaq-GTPgS alone stimulated TRPC6 The PLC-bs are activated by Gaq [17, 18]. GDPbS, which substantially, only simultaneous addition of both activators blocks G protein activation, inhibited the stimulation of produced major TRPC6 opening (Figure 2D). Moreover, the TRPC6 by WNK11–491 (Figure 2A). GTPgS stimulated the Gq inhibitor RGS4 blocked activation of TRPC6 by WNK1 in TRPC6 current without added WNK1, and stimulation was cells and the N128G mutant of RGS4 [27] had no effect (Figures blocked by anti-WNK1 antibody (Figure 2B; Figures S2A S2E and S2F). In detached patches, inhibition by RGS4 was and S2B). These data suggest both that some G protein reduced and delayed by expression of the RGS-insensitive is required for WNK1 stimulation of TRPC6 and that en- G188S mutant of Gaq [28, 29](Figures 2E and 2F). Thus, dogenous WNK1 is required for TRPC6 stimulation by a G WNK1 and activated Gaq stimulate TRPC6 synergistically.

Figure 3. Role of WNK1 in Activation of TRPC6 by Gq- Coupled Receptor (A) Whole-cell inward TRPC6 current density in response to CCh with anti-WNK1 Ab, GDPbS, or control buffer applied via patch pipettes. *p < 0.01 for WNK1-Ab versus control. (B) Whole-cell CCh-induced TRPC6 currents were re-

corded in the presence of control buffer or WNK11–491 (3 nM), a concentration that does not appreciably stimulate TRPC6 by itself. *p < 0.01 for WNK1 versus buffer (see also Figure S3). Current Biology Vol 21 No 23 1982

too low to activate TRPC6 by itself, approximately doubled the TRPC6 current elicited by M3 muscarinic stimulation (Fig- ure 3B). Thus, some level of endogenous WNK1 activity is necessary for full Gq-PLC-b signaling and additional WNK1 potentiates Gq signaling in cells. Anti-WNK1 antibody did not signiﬁcantly inhibit activation

of TRPC6 by the Gi-coupled M2 muscarinic receptor (Fig- ure S3E). However, Gi is a less effective activator of PLC-bs [17, 18], and TRPC6 activation by the M2 receptor is much smaller than that by the Gq-coupled receptors (Figure S3E versus Figure 3A). This apparent selectivity of anti-WNK1 antibody for Gq-coupled receptors suggests that WNK1 is needed to support the higher flux of the Gq-PLC-b pathway but is not detectably required for Gi-PLC-b signaling. Although these data are consistent with the stimulation of PLC-b activity by WNK1, we did not find any effect of purified WNK11–491 on the activity of purified PLC-b1, 2, or 3; its regulation by Gaq or Gbg; or regulation of Gq by M1 muscarinic receptors (Figure S4).

Activation of TRPC6 by WNK1 via PI4K-Mediated PIP2 Synthesis The interdependence and mutual potentiation of TRPC6 acti-

vation by the Gaq and WNK1 pathways suggests that they each increase the activity of PLC-b by distinct mechanisms.

Gaq allosterically stimulates the catalytic activity of PLC-b. We therefore asked whether WNK1 acts by increasing the

concentration of the PIP2 substrate. Generation of phosphatidylinositol 4-P (PI4P) by PI4K is believed to be the ﬁrst step

in the synthesis of PIP2 [30]. Pretreatment of cells with the PI4K inhibitor wortmannin inhibited activation of TRPC6 by

WNK11–491 (Figure 4A) as did simultaneous application of an anti-PI4P antibody [31](Figure 4B). Last, intracellular application of PI4KIIIb activated TRPC6 even in the absence of added WNK1, and pretreatment with wortmannin prevented activation (Figure 4C). PI4P synthesis is thus involved in the stimulation of TRPC6 by WNK1, and WNK1 can be mimicked by addition of a PI4K. We used the GFP-tagged pleckstrin homology (PH) domain Figure 4. Role of PI4K in the Activation of TRPC6 by WNK1 of PLC-d1 and confocal imaging to examine the role of WNK1 (A) Effect of wortmannin (Wmn) on whole-cell TRPC6 currents activated by in the degradation and resynthesis of PIP2 in the plasma WNK11–491. Cells were pretreated with 10 mM wortmannin or vehicle membrane. The PLC-d1-PH domain binds both PIP and IP (dimethyl sulfoxide [DMSO]) for 10 min before recording. *p < 0.01 for vehicle 2 3 versus wortmannin. with relatively high affinity and specificity [32–34]. In cells that express M3 receptor, addition of carbachol decreased (B) Whole-cell TRPC6 currents in response to WNK11–491 coapplied with monoclonal anti-PI4P antibody or control mouse IgM. *p < 0.01 for control GFP-PLC-d1-PH fluorescence at the plasma membrane, IgM versus PI(4)P-Ab. indicating reduced PIP2 and increased fluorescence in the (C) Whole-cell TRPC6 current in response to intracellular delivery of 100 nM cytosol, indicating increased IP3 (Figure 5). Both effects PI4KIIIb preincubated with or without 10 mM wortmannin for 10 min. *p < 0.01 peak w45 s after carbachol addition and then decline to for vehicle versus wortmannin. Note that both type IIIa and IIIb PI4K can prestimulus levels, reflecting PLC-b-catalyzed hydrolysis of generate PI4P for synthesis of phosphatidylinositol 4,5-biphosphate (PIP2) in the membrane. Due to difficulties in purification of PI4KIIIa, we used PIP2 and its subsequent resynthesis. only PI4KIIIb for experiments here. In all panels, data point is mean 6 SEM Microinjection of anti-WNK1 antibody both delayed and of inward current density, n = 5–9 for each condition (see also Figure S4). decreased the recovery of GFP-PLC-d1-PH fluorescence at the plasma membrane after treatment with carbachol. Instead of the monophasic return to baseline in <3 min ob- WNK1 Activity Is Necessary for Activation of TRPC6 served in control cells, cells treated with anti-WNK1 antibody by Gq-Coupled Receptors displayed biphasic recovery of plasma membrane fluores- Intracellular application of anti-WNK1 antibody markedly cence that was only w50% complete after 15 min. The return blunted the activation of TRPC6 by agonist through the of cytosolic fluorescence to baseline was also biphasic, with expressed M3 muscarinic receptor (Figure 3A), the a1A-, a1B- a greatly prolonged and incomplete second phase. These adrenergic receptors, and H1 histaminergic receptor in HEK data indicate that anti-WNK1 antibody substantially inhibits cells (Figures S3A–S3C) or through endogenous muscarinic resynthesis of PIP2 after a burst of PLC activity. Taken receptor in HeLa cells (Figure S3D). Conversely, increased together with the effects of the anti-PI4P antibody, they

WNK1 activity potentiated activation of TRPC6 by M3 re- indicate that WNK1 is necessary to maintain cellular PIP2 ceptor. Intracellular application of 3 nM WNK11–491, which is synthesis. WNK1 Coordinates Growth Factor and GPCR Signaling 1983

Figure 5. Anti-WNK1 Antibody Delays and Inhibits

Plasma Membrane PIP2 Resynthesis Following Receptor-Stimulated Hydrolysis HeLa cells transfected with M3R and GFP-tagged PLC- d1-pleckstrin homology domain were microinjected with control IgG or anti-WNK1 antibody and stimulated by 1 mM CCh. (A) Changes in fluorescence intensity at the plasma membrane (decrease) and cytosol (increase) in response to carbachol were normalized by setting maximal change to 1.0. Data are means 6 SEM of 18 and 20 cells (seven separate experiments) that were microinjected with control rabbit IgG or anti-WNK1 antibody, respectively. (B) Ratio of fluorescence intensity in the plasma membrane to that in cytosol from (A). Lines indicate slopes of the initial recovery rates (0.032 6 0.002 and 0.014 6 0.002 for control IgG and anti-WNK1 antibody, respectively; p < 0.01). (C) Representative confocal images (obtained across the middle of cells) from one of seven experiments showing fluorescence at the plasma membrane and cytosol before addition of CCh and at 45 s (peak), 90 s, and 180 s after addition of CCh. Signal intensity is shown by pseudocolor.

WNK1 Potentiates PI4P Synthesis by PI4KIIIa enhanced carbachol-induced intracellular Ca2+ transients The PI4KIIIa isozyme is believed to be primarily responsible (Figure S5D), consistent with a ﬁnal common site of action at for resynthesis of PIP2 in the plasma membrane during PLC-b. IGF1 did not increase the cell-surface abundance of receptor stimulation of PLC [30]. To determine whether TRPC6 (Figure S5E).

WNK1 regulates PI4KIIIa, we studied effects of manipulating Several observations argue that IGF1 potentiates GPCR-Gq WNK1 activity in cells that express recombinant PI4KIIIa, signaling through a PI3K-Akt-WNK1-PI4KIIIa sequence. First, where this isoform is the predominant PI4K activity (w95%) wortmannin, at a low concentration selective for PI3K in a light membrane fraction, which includes plasma mem- (20 nM), blocked the effect of IGF1 on Gq signaling. Knock- branes (Figure 6A). Knockdown of WNK1 by siRNA decreased down of WNK1 also blocked the ability of IGF1 to potentiate both PI4K activity and the amount of immunoreactive PI4KIIIa carbachol’s effect (Figure 7C). Expression of wild-type in the light membrane fraction by 35%–40% (Figures 6B–6E) WNK1, but not the T58A mutant WNK1, restored the ability and increased the abundance of PI4KIIIa in cytosol (Figures of IGF1 to enhance carbachol activation of TRPC6 in WNK1- 6D and 6E). WNK1 knockdown did not alter the amount of knockdown cells (Figure 7D), indicating that the synergistic PI4KIIIa in the total postnuclear supernatant (PNS). These effect of IGF1 and M3 muscarinic stimulation depends on data suggest that WNK1 recruits PI4KIIIa from cytosol to the Akt-mediated phosphorylation of WNK1 at T58. Note that light membrane fraction, presumably the plasma membrane. both wild-type WNK1 and the T58A mutant can support carba- Recruitment may be indirect, because we did not detect phys- chol activation of TRPC6 in WNK1-knockdown cells. Thus, ical association between PI4KIIIa and puriﬁed WNK11–491 in T58-phosphorylation is not required for WNK1 ampliﬁcation pull-down assays. of Gq-PLC-b signaling, but is indispensable for its potentiation WNK1 failed to activate TRPC6 in PI4KIIIa-knockdown cells by IGF1. (Figures 6F and 6G) and expression of PI4KIIIa in the knock- Knockdown of PI4KIIIa also prevented enhancement of down cells rescued activation by WNK1. These results are carbachol activation of TRPC6 and intracellular Ca2+ tran- consistent with the idea that WNK1 increases PIP2 synthesis sients by IGF1 (Figure 7E; Figure S5F). These results argue by recruitment of PI4KIIIa to its site of action and that the that PI3K-dependent activation of Akt by IGF1 leads to phos- consequently increased availability of PIP2 synergistically phorylation of WNK1 and enhances the ability of WNK1 to enhances Gq-coupled receptor signaling through PLC-b. synergistically regulate PLC-b signaling by promoting PIP2 synthesis.

IGF1 Potentiates Gq-PLC-b Signaling via PI3K and Akt-Mediated Phosphorylation of WNK1 Discussion The physiological role of WNK1 in the regulation of Gq-PLC-b signaling was further supported by the ﬁnding that IGF1, Our data establish three new and distinct but interrelated a known upstream activator of WNK1, enhances the effect of signaling mechanisms: the mutual dependence and syner- WNK1. Pretreatment with IGF1 in serum-starved cells potenti- gistic interaction between the Gq-PLC-b and Akt-WNK1 ated the activation of TRPC6 by either carbachol (Figures 7A signaling pathways, the regulation of PLC-b activity by modu- and 7B) or GTPgS(Figures S5A and S5B), although IGF1 did lating the availability of its substrate, and the regulation of not alter basal TRPC6 current. Lack of effect on the basal PI4KIIIa by WNK1 to control the concentration of PIP2-derived current is probably due to a low basal output from Gq-PLC-b second messengers. in serum-starved cells, and IGF1 activated TRPC6 in these As summarized in Figure 7F, blocking WNK1 activity mark- cells when prestimulated by GTPgS(Figure S5C). IGF1 also edly inhibits PLC-b signaling by Gq-coupled receptors and, Current Biology Vol 21 No 23 1984

Figure 6. Role of WNK1 in the Production of PI4P by Expressed PI4KIIIa (A) Chromatogram showing PI4K activity in membranes prepared from HeLa cells transfected with vector or complementary DNA (cDNA) for PI4KIIIa. The following abbreviation is used: Std, PI4P standard. (B) Chromatogram showing PI4P production by membrane-associated recombinant PI4KIIIa from cells that were cotransfected with control oligonucleotide or WNK1 siRNA. Relative knockdown of WNK1 is shown in the western blot. (C) Mean 6 SEM of PI4P production from ﬁve separate experiments, each with triplicate samples, as shown in (B). *p < 0.01 for control versus knockdown. (D) Abundance of PI4KIIIa in the postnuclear supernatant (PNS), cytosol, and membrane fractions from control and WNK1 siRNA-transfected HeLa cells. A western blot at bottom shows relative WNK1 knockdown. (E) Mean 6 SEM (ﬁve separate experiments) of PI4KIIIa abundance in the postnuclear supernatant (PNS), cytosol, and membrane fraction in control (blue) and WNK1 knockdown cell (red), shown relative to the control. *p < 0.05 between indicated data.

(F) Whole-cell TRPC6 currents stimulated by WNK11–491 in cells cotransfected with control oligonucleotide or siRNA for human PI4KIIIa, with either empty vector or plasmid that encodes rat PI4KIIIa. (G) Peak WNK1-induced inward current density from (F). *p < 0.01 between indicated data.

must do more than just recruit PI4KIIIa to the plasma membrane because it remains necessary for PLC-b signaling in detached patches. We have no evidence that WNK1 either binds or phosphorylates PI4KIIIa, and additional factors may be involved. Regardless, our results indicate that the activity of PI4KIIIa is regulated and that its regulation by WNK1 is critical for PLC-b conversely, inhibiting Gq blocks PLC-b signaling by WNK1. signaling by Gq-coupled receptors. The basal activity of Inhibition could be exerted by several approaches and at WNK1 provides adequate tonic stimulation of PI4KIIIa to several steps in each pathway, both in cells and in detached provide PIP2 for normal PLC-b signaling. However, additional membrane patches. In each case, replenishment of the in- WNK1 markedly amplifies signaling by Gq-coupled receptor hibited or depleted species, or stimulation of a downstream even at a concentration that does not by itself activate component, restored normal activities of both WNK1 and TRPC6 (Figure 3B). In addition, PI3K-activating growth factors Gq-PLC-b. Importantly, WNK1 can elicit PLC signals without such as IGF1 activate WNK1 to further potentiate Gq-PLC-b added GPCR agonist, and WNK1 synergistically amplifies signaling, probably via Akt-mediated phosphorylation of the stimulation of PLC-b by Gq. WNK1 at T58 (Figure 7). These results suggest that WNK1 is In contrast to G proteins, WNK1 increases PLC-b activity by an important physiological mediator of the Akt kinase increasing the supply of the PIP2 substrate. Because the signaling pathway and coordinates Akt signaling with the concentration of PIP2 in the plasma membrane is below Km Gq-PLC-b signaling pathway. [22, 23], PLC-b displays first-order substrate kinetics and This action of WNK1 represents the first known instance of increase in available PIP2 linearly amplifies PLC-b output. acute control of PIP2 availability as a mechanism of regulation Note that WNK1 only modulates Gq signaling via PLC-b. Other of cellular DAG and IP3 concentrations without concurrent outputs, such as stimulation of a rho guanine nucleotide stimulation of the intrinsic activity of PLC. Application of exchange factor [35], are not involved. WNK1 thus alters the WNK1 alone stimulates the production of DAG measured spectrum of Gq outputs as well as its amplitude. using the TRPC6 biosensor by an amount comparable to WNK1 promotes synthesis of PIP2 by somehow activating that initiated by GTPgS(Figure 1B). Some low output of PI4KIIIa (Figure 6; Figure 7F). PI4KIIIa is important for the re- PLC-b, evidently dependent on basal input from Gaq,is synthesis of PIP2 following hydrolysis by receptor-stimulated adequate to support this WNK1 signaling. By promoting PIP2 PLC [30], but how the activity of PI4KIIIa is regulated is synthesis, WNK1 can also acutely modulate the amplitude of unknown. The amount of PI4KIIIa is decreased in membranes receptor-stimulated PLC-b signaling and/or its duration. Inhi- from WNK1 knockdown cells, suggesting that regulation of bition of endogenous WNK1 by anti-WNK1 antibody blocks

PI4KIIIa by WNK1 may involve its redistribution between both the DAG and IP3 limbs of PLC-b signaling stimulated by plasma membrane and intracellular pools. However, WNK1 multiple Gq-coupled receptors by R50% (Figure 3A; Figures WNK1 Coordinates Growth Factor and GPCR Signaling 1985

Figure 7. IGF1 Enhances Gq-Coupled Receptor Activa- tion of TRPC6 via WNK1 (A) Cells coexpressing TRPC6 and M3R were serum- starved for 18 hr and incubated for 10 min with 100 ng/ml IGF1, 20 nM wortmannin, both, or neither before ruptured whole-cell recording. Carbachol was applied to the bath where shown. (B) Peak CCh-induced current density from (A). *p < 0.01 between indicated groups. (C) Peak CCh-induced inward current density in cells transfected with control oligonucleotides or WNK1 siRNA and with or without IGF1 pretreatment. *p < 0.01 between indicated groups, and #, p < 0.01 versus cells transfected with control oligo and without IGF1 pretreatment. (D) Peak CCh-induced inward current density in cells transfected with WNK1 siRNA and cDNA for either wild-type WNK1 or T58A mutant WNK1 and with or without IGF1 pretreatment. *p < 0.01 between indicated data. (E) Peak CCh-activated TRPC6 currents in cells transfected with control oligonucleotides or PI4KIIIa siRNA and with or without IGF1 pretreatment. *p < 0.01 between indicated data.

(F) Interaction of WNK1, Gq-PLC-b, and PI4KIIIa signaling pathways. Components of the pathways are shown in black. Inhibitors are color-coded per their biochemical sites of action and placed where their inhibitory effects on cellular signaling were noted in the experiments. Pathways are indicated by black arrows. The experimental results indicate that the point at which

Gq and WNK1 interact is at the level of PLC activity (marked by dark green box) (see also Figure S5).

tone by virtually all vasoactive hormones [41], and potentiation of PLC-b signaling by WNK1 would promote vasoconstriction and cause hypertension. The role of WNK1 in the regulation of vascular tone is supported by the report that mice with heterozygous inactivation of Wnk1 display reduced blood S1D–S1F; Figure S3). Acute stimulation of WNK1 activity by pressure without renal Na+ wasting and hypovolemia [42]. IGF1 potentiates PLC-b signaling by w2-fold (Figures 7A and Finally, the similarity between the phenotypes of WNK1-

7B). WNK1 thus controls a major mode of desensitization of knockout and Gaq/Ga11-knockout mice (both lethal at embry- Gq signaling in addition to modulating acute responses. PIP2 onic day w10.5, with cardiac defects) supports the idea that itself is a signaling molecule that regulates many plasma interaction between the WNK1 and Gaq/11 pathways is impor- membrane targets [36]. Increasing PIP2 synthesis by WNK1 tant for embryonic development [10, 26]. is thus an important mechanism for increasing cellular DAG In conclusion, WNK1 potentiates Gq-PLC-b signaling by and IP3 concentrations without causing untoward effects of stimulating the activity of PI4KIIIa to increase synthesis of decreases in membrane PIP2 on other targets. PIP2. Phosphorylation of WNK1 by Akt kinase increases its PLC-b enzymes function as coincidence detectors that activity in this pathway and allows tyrosine kinase receptors synergistically integrate multiple upstream inputs including or other activators of the Akt kinase cascade to regulate G 2+ Gaq,Gbg,Ca , and Rac [37, 38, 39]. These mechanisms of protein-mediated PLC-b signaling. The extent and physiolog- synergism are results of combined superadditive stimulation ical role of WNK1 in the regulation of PLC-b signaling will of the catalytic activity of PLC-b. In contrast, synergism probably differ among specialized cells. Our understanding between WNK1 and Gaq depends on the ability of WNK1 to of these interactions should expand as we learn more ways enhance substrate supply for PLC-b. This system is analogous in which WNK1 activity is regulated. to the interaction between PLCs and inositol phosphate kinases in which PLC provides the IP3 starting material that Experimental Procedures the cell-type-specific kinases then phosphorylate to the appropriate inositol polyphosphates [40]. Detailed experimental procedures are in the Supplemental Information. HEK293 and HeLa cells and mouse embryonic fibroblasts were cultured The interactive control of PLC-b by WNK and Gq may have important implications in the pathogenesis of hypertension and cotransfected with complementary DNAs (cDNAs) for TRPC6-Flag WNK1 with or without human M3 muscarinic receptor [43]. Ruptured whole-cell, in patients with PHA2 that is caused by mutations of cell-attached, and inside-out excised patch-clamp recording were per- that increase WNK1 expression [2]. Gq/11-mediated PLC-b formed as described [9, 43]. Intracellular Ca2+ transients were examined 2+ signaling plays a central role in the regulation of vascular using ratiometric fluorescent Ca images of fura-2-loaded cells. PIP2 and Current Biology Vol 21 No 23 1986

IP3 were localized according to fluorescence of a GFP-tagged PLC-d1-PH 14. Vitari, A.C., Deak, M., Collins, B.J., Morrice, N., Prescott, A.R., Phelan, domain by confocal microscopy [34]. PLC activity was measured by moni- A., Humphreys, S., and Alessi, D.R. (2004). WNK1, the kinase mutated 3 toring hydrolysis of [ H]PIP2 on large unilamellar phospholipid vesicles in an inherited high-blood-pressure syndrome, is a novel PKB (protein [22, 37]. PI4KIIIa activity from HeLa cells was measured by phosphorylation kinase B)/Akt substrate. Biochem. J. 378, 257–268. of phosphatidylinositol (PI) in PI/Triton X-100-mixed micelles [44]. 15. Jiang, Z.Y., Zhou, Q.L., Holik, J., Patel, S., Leszyk, J., Coleman, K., Chouinard, M., and Czech, M.P. (2005). Identification of WNK1 as a substrate of Akt/protein kinase B and a negative regulator of insulin- Supplemental Information stimulated mitogenesis in 3T3-L1 cells. J. Biol. Chem. 280, 21622– 21628. Supplemental Information includes five figures and Supplemental Experi- mental Procedures and can be found with this article online at doi:10.1016/ 16. Cheng, C.J., and Huang, C.L. (2011). Activation of phosphatidylinositol j.cub.2011.11.002. 3-kinase stimulates endocytosis of ROMK channel via Akt1/SGK1- dependent phosphorylation of WNK1. J. Am. Soc. Nephrol. 22, 460–471. 17. Rebecchi, M.J., and Pentyala, S.N. (2000). Structure, function, and Acknowledgments control of phosphoinositide-specific phospholipase C. Physiol. Rev. 80, 1291–1335. This study was supported by the National Institutes of Health (GM30355 to 18. Rhee, S.G. (2001). Regulation of phosphoinositide-specific phospholi- E.M.R. and DK59530, DK85726, and DK-079328 to C.-L.H), the Welch Foun- pase C. Annu. Rev. Biochem. 70, 281–312. dation (I-0982 to E.M.R.), and Genzyme (GRIP grant to C.-L.H.). We are 19. Hicks, S.N., Jezyk, M.R., Gershburg, S., Seifert, J.P., Harden, T.K., and particularly indebted to Barbara Barylko and Joseph Albanesi for purifica- Sondek, J. (2008). General and versatile autoinhibition of PLC isozymes. tion of PI4KIIIb and for continuing help and advice. We thank Melanie Mol. Cell 31, 383–394. Cobb for anti-WNK1 antibody, Tamas Balla for GFP-PLC-d1-PH, Melvin 20. Creba, J.A., Downes, C.P., Hawkins, P.T., Brewster, G., Michell, R.H., Simon for Ga -null mouse fibroblasts, and Anna Dugas for technical q/11 and Kirk, C.J. (1983). Rapid breakdown of phosphatidylinositol 4-phos- assistance. phate and phosphatidylinositol 4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+-mobilizing hormones. Received: August 25, 2011 Biochem. J. 212, 733–747. Revised: October 4, 2011 21. Nakanishi, S., Catt, K.J., and Balla, T. (1995). A wortmannin-sensitive Accepted: November 1, 2011 phosphatidylinositol 4-kinase that regulates hormone-sensitive pools Published online: November 23, 2011 of inositolphospholipids. Proc. Natl. Acad. Sci. USA 92, 5317–5321. 22. Biddlecome, G.H., Berstein, G., and Ross, E.M. (1996). Regulation of

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