Regulation of OSR1 and the sodium, potassium, two chloride cotransporter by convergent signals

Significance

With no lysine [K] (WNK) protein kinases are sensitive to changes in osmotic stress. Through the downstream protein kinases oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-, alanine-rich kinase, WNKs regulate a family of ion cotransporters and thereby modulate a range of processes including cell volume homeostasis, blood pressure, hearing, and kidney function. We found that a major phosphoinositide 3-kinase target pathway, the mammalian target of rapamycin complex 2, also phosphorylates OSR1, coordinating with WNK1 to enhance OSR1 and ion cotransporter function.

Abstract

The Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and the STE20/SPS1-related proline-, alanine-rich kinase directly regulate the solute carrier 12 family of cation-chloride cotransporters and thereby modulate a range of processes including cell volume homeostasis, blood pressure, hearing, and kidney function. OSR1 and STE20/SPS1-related proline-, alanine-rich kinase are activated by with no lysine [K] protein kinases that phosphorylate the essential activation loop regulatory site on these kinases. We found that inhibition of phosphoinositide 3-kinase (PI3K) reduced OSR1 activation by osmotic stress. Inhibition of the PI3K target pathway, the mammalian target of rapamycin complex 2 (mTORC2), by depletion of Sin1, one of its components, decreased activation of OSR1 by sorbitol and reduced activity of the OSR1 substrate, the sodium, potassium, two chloride cotransporter, in HeLa cells. OSR1 activity was also reduced with a pharmacological inhibitor of mTOR. mTORC2 phosphorylated OSR1 on S339 in vitro, and mutation of this residue eliminated OSR1 phosphorylation by mTORC2. Thus, we identify a previously unrecognized connection of the PI3K pathway through mTORC2 to a Ste20 protein kinase and ion homeostasis.

The protein kinases oxidative stress-responsive 1 (OSR1) and its homolog the STE20/SPS1-related proline-, alanine-rich kinase (SPAK or PASK) are the mammalian members of the germ-cell kinase VI subgroup of the large Ste20 branch of the mammalian kinome. OSR1 and SPAK directly regulate the solute carrier 12 family of cation-chloride cotransporters which modulate ion homeostasis throughout the body (1, 2). OSR1/SPAK kinase domains lie close to their N-termini and they contain two additional conserved regions named “PF1” and “PF2” [PASK and Fray (Drosophila homolog)] (3). PF1 is a C-terminal extension to the kinase domain and is required for enzyme activity (4). PF2 binds the consensus motif [(R/K)FX(V/I)] (5) in substrates including ion cotransporters and in regulators. OSR1 and SPAK are activated by with no lysine [K] (WNK) protein kinases, which phosphorylate the essential activation loop regulatory site as well as a second site in the PF1 region with an undefined function (6–9).

The four WNK protein kinases are large enzymes notable for the alternative placement of the essential ATP-binding lysine residue in their catalytic domains, distinguishing them from other members of the protein kinase superfamily (10, 11). Initial attention was focused on these enzymes because certain mutations in two family members cause pseudohypoaldosteronism type II, a heritable form of hypertension (12). WNKs are activated by changes in tonicity. Cellular reconstitution studies and mouse genetics demonstrated the importance of WNK function in cell volume regulation and maintenance of blood pressure (13–19). Control of cation-chloride cotransporters through OSR1 and SPAK is among the best-documented actions of WNKs in diverse tissues (5, 20–22).

WNKs also regulate serum- and glucocorticoid-inducible protein kinases (SGKs) through a noncatalytic mechanism leading to increased sodium influx through the epithelial sodium channel (ENaC) (23, 24). SGKs and the related Akt enzymes are activated by phosphorylation on multiple sites, most prominently a residue in the activation loop by the phosphoinositide-dependent protein kinase and on a second site in a C-terminal hydrophobic motif (25). The kinase that phosphorylates the hydrophobic motif site under many circumstances is the mammalian target of rapamycin complex 2 (mTORC2), which provides an additional phosphatidylinositol-3 kinase (PI3K)-dependent input to these kinases (26–33).

In this study, we show that OSR1 is phosphorylated not only by WNKs but also on a C-terminal site, conserved in SPAK, by mTORC2. These studies reveal a link between WNK-OSR1/SPAK and the PI3K-mTORC2 cascade that suggests that OSR1 and SPAK integrate signals from osmosensing and survival pathways.

Results

OSR1 Is Regulated by a PI3K-Dependent Mechanism.

In response to changes in tonicity, OSR1 is activated through phosphorylation of its activation loop by WNKs. Previously, we failed to find activation of OSR1 by serum or epidermal growth factor (4). A recent report, however, indicates that it participates in insulin-regulated events in a PI3K-sensitive manner (34). To confirm the PI3K sensitivity and retest potential regulation by growth factors, we examined effects of the PI3K inhibitor wortmannin on OSR1 activity in HeLa cells stimulated with sorbitol or serum. The sodium, potassium, two chloride cotransporters 1 and 2 (NKCC1 and NKCC2) are related ion cotransporters of the solute carrier 12 family that are phosphorylated and activated by OSR1/SPAK (2, 15). The activity of immunoprecipitated OSR1 was measured using a recombinant N-terminal fragment of NKCC2, residues 1–175, as substrate (9). Sorbitol stimulated OSR1 activity, but serum did not, consistent with earlier results (Fig. 1A). Wortmannin decreased serum-stimulated phosphorylation of Akt on its hydrophobic motif site and also reduced sorbitol-stimulated OSR1 kinase activity, indicating an effect of PI3K on this WNK-dependent pathway. Unexpectedly, an increase in Akt phosphorylation was also noted with sorbitol (Figs. 1A and 2A).

Fig. 1.

PI3K pathways influence OSR1 activation and function in response to sorbitol. (A) HeLa cells were pretreated with 50 nM wortmannin (Wort) and then stimulated with 0.5 M sorbitol or 20% FBS to stimulate Akt. Endogenous OSR1 was immunoprecipitated and its activity measured using recombinant NKCC2 1–175. Four such experiments were quantified (Top). A representative autoradiogram is shown with immunoblots of OSR1 and Akt phosphorylated on S473. Phosphorylation of Akt at S473 measured as an indicator of mTORC2 activity and pAkt as a positive control of stimulation conditions. n = 4, one way ANOVA, P < 0.0001; ***P = 0.0003 (adjusted from Tukey’s test). Error bars show standard deviation. (B) Effect of depletion of endogenous mTORC2 by knockdown of Sin1 on OSR1 autophosphorylation (white bars) and activity toward substrate (gray bars) upon sorbitol stimulation. n = 5, **P < 0.05. Error bars show standard error. Immunoblots show efficiency of Sin1 knockdown. ERK1/2 was used as the loading control. Expression of Sin1 was normalized to ERK1/2 expression (Bottom). (C) HeLa cells were transfected with siRNA for Sin1 and scrambled siRNA as control (siC). Bumetanide sensitive uptake of ⁸⁶Rb was measured as an assay of NKCC1 activity. The fold change in NKCC1 activity was normalized to control and average of three separate experiments each performed in triplicate was plotted. Error bars are standard error. **P < 0.05.

Fig. 2.

Inhibition of mTORC2 reduces OSR1 activity. (A) HeLa cells were pretreated for 15 min with either 100 nM rapamycin (Rapa) or 300 nM KU-0063794 (KU) and then stimulated with 0.5 M sorbitol (Sorb). Endogenous OSR1 was immunoprecipitated and its autophosphorylation and phosphorylation of recombinant NKCC2 1–175 were measured by immune-complex kinase assay. The incorporated radioactivity was measured by scintillation counting and normalized to the amount of OSR1 immunoprecipitated in each case. Cells stimulated with sorbitol without any drugs and without any other treatment were used as controls. Four experiments with 10 replicates were used to calculate standard error (error bars). *P < 0.05. Immunoblots of Akt and pS473 Akt are shown (Lower). (B) After depletion of Sin1, HeLa cells were stimulated with sorbitol in duplicate and endogenous WNK1 was immunoprecipitated and detected by blotting. Duplicate samples of WNK1 were used in immune complex kinase assays using OSR1 K46R as substrate. Incorporated radioactivity was detected by autoradiography and the Coomassie blue-stained gel of the substrate is immediately below. Sorbitol-stimulated activity is shown as percent of unstimulated activity (Upper); error bars show standard deviation. Sin1 depletion was assessed by immunoblotting lysates (lanes in Lower). Sin1 is the upper band, indicated by the tick mark. n = 4. CTRL, control.

OSR1 Is Regulated by WNK1 and mTORC2.

Because of the input of mTORC2 to SGK (27), we tested the possibility that mTORC2 might be responsible for the PI3K-dependence of OSR1 activation by sorbitol. To evaluate the contribution of mTORC2, we inhibited its activity by depleting Sin1, a required component of complex 2 (26). Knockdown of Sin1 reduced activation of OSR1 by sorbitol (Fig. 1B), supporting the conclusion that mTORC2 is a PI3K-dependent input to OSR1. In contrast to NKCC2 which displays tissue-restricted expression in kidney, NKCC1 is expressed in most cell types including HeLa. To test the consequences of Sin1 depletion on cation-chloride cotransporter function, we measured the activity of endogenous NKCC1 in HeLa cells. Disruption of mTORC2 by Sin1 knockdown caused a substantial decrease in endogenous NKCC1 activity, measured as rubidium uptake sensitive to the loop diuretic bumetanide, an NKCC inhibitor (Fig. 1C). Taken together, these data suggest that mTORC2 regulates OSR1 activity and function.

To determine whether OSR1 is regulated by other mTOR complexes, we treated sorbitol-stimulated cells with rapamycin which inhibits mTORC1 better than mTORC2 and with KU0063794 (KU) which inhibits both mTORC1 and mTORC2. OSR1 was immunoprecipitated from cells and assayed with NKCC. We observed that treatment with KU0063794 for 15 min reduced OSR1 activity (Fig. 2A), although the effect of rapamycin was not statistically significant, suggesting that the regulation of OSR1 is primarily by mTORC2. Both compounds inhibited phosphorylation of Akt, although rapamycin was less effective. mTORC2 activity toward both OSR1 and Akt is evident in sorbitol-treated cells. Whether mTORC2 activity increases under these conditions or if its access to substrates is enhanced is not known. The fact that brief exposure to KU0063794 was sufficient to impair OSR1 activation implies that this is a rapid regulatory mechanism. For comparison, we examined the effects of the depletion of WNK1 without or with KU0063794 to block mTORC2. Depletion of WNK1 inhibited OSR1 activity strongly (Fig. S1), consistent with previous results from multiple groups indicating that phosphorylation of the activation loop by WNK1 is essential for activation of OSR1 (6, 7, 9).

WNK1 Is Not Regulated by mTORC2.

Another mechanism by which mTORC2 might regulate OSR1 is through direct effect on WNK1 itself. Several mTOR sites have been reported on WNK1 (35), although all lie in the C-terminal region not near the kinase domain. To test this hypothesis, we depleted HeLa cells of Sin1 to disrupt mTORC2, immunoprecipitated endogenous WNK1 from these cells, and tested its activity using an in vitro kinase assay with kinase-dead OSR1K46R as a substrate. No difference in WNK1 activity in cells depleted of Sin1 was detected compared with WNK1 from control cells (Fig. 2B), consistent with previous results showing no effects of wortmannin on WNK1 activity (36). Furthermore, mTORC2 and WNK1 do not coimmunoprecipitate (Fig. S2A).

mTORC2 Directly Phosphorylates OSR1 on S339.

To determine if OSR1 and SPAK are mTORC2 substrates, we tested whether mTORC2 could directly phosphorylate OSR1. We used antibodies against Sin1 to immunoprecipitate mTORC2 from HeLa cells (Fig. 3B, Bottom) and then measured phosphorylation of wild type OSR1 and the inactive mutant OSR1K46R in immune-complex kinase assays. Like immunoprecipitated WNK1, mTORC2 phosphorylated both forms of OSR1 in vitro (Fig. 3A). Recombinant WNK1 kinase domain (which has higher activity than the endogenous protein) caused a marked stimulation of recombinant OSR1 kinase activity in vitro; a smaller increase was detected with immunoprecipitated mTORC2, and the combination of both caused a greater increase in OSR1 kinase activity than either alone (Fig. 3B).

Fig. 3.

WNK1 and mTORC2 phosphorylate OSR1 and increase its activity. (A) Phosphorylation of OSR1 wild type (wt) and the inactive K46R (KR) mutant by Sin1 and WNK1 immunoprecipitates was measured in vitro. Immunoprecipitates with nonimmune serum (N) and no added enzyme (−) were used as controls. n = 3. The Coomassie blue-stained gel shows the relative amounts of OSR1 proteins present. Incorporated radiolabel was quantified using scintillation counting and normalized to the amount of OSR1 in each reaction. Results are plotted (Lower). (B) Wild type OSR1 was incubated for 30 min with recombinant WNK1 1–490 alone, with Sin1 immunoprecipitate alone (IP Sin1), with beads alone or with both enzyme preparations. OSR1 kinase activity was then measured with NKCC (Top); error bars show standard deviation. A representative assay is shown (Middle) with immunoblots indicating the amounts of enzymes present. Bottom shows that mTOR is present in Sin1 immunoprecipitates (IP sin1), but the mTOR substrate p70 S6 kinase is not.

In vitro phosphorylation by WNK1 on an additional site, S325, in the OSR1 PF1 segment was reported by Alessi and coworkers (6). To rule out an involvement of this residue as well as the adjacent serine S324 in phosphorylation by mTORC2, we again immunoprecipitated mTORC2 and tested phosphorylation of OSR1 S324A and SS324/325AA (SASA) mutants. Both mutants were phosphorylated as well as wild type OSR1, indicating that neither of these sites is phosphorylated by mTORC2 (Fig. S2B).

Mass spectrometry (MS) was used to determine the site phosphorylated by mTORC2. OSR1 was phosphorylated by recombinant WNK1 kinase domain alone or in combination with immunoprecipitated mTORC2 or a control serum immunoprecipitate. We found that T183 and S325 were phosphorylated by WNK1, as reported previously (6). A single site, S339, also within the PF1 region, was phosphorylated by mTORC2 (Fig. 4A). This region is nearly identical in SPAK and the comparable residue was identified as a phosphorylation site previously and associated with increased kinase activity (37). We mutated S339 to alanine and confirmed that phosphorylation of this OSR1 mutant by mTORC2 was lost. The OSR1 truncation mutant, residues 1–323, produced in bacteria can be activated by WNK1, but is unstable (9). Without S339, this fragment is not phosphorylated by mTORC2 (Fig. 4B).

Fig. 4.

mTORC2 phosphorylates OSR1 on S339. (A) Recombinant OSR1K46R was phosphorylated using Sin1 immunoprecipitates (IP Sin1) with or without recombinant WNK1-194-483 (Inset) and samples were analyzed using MS. Representative spectrum and sequence of OSR1 shown identifying the residue phosphorylated specifically by mTORC2, S339. (B) Recombinant GST-tagged OSR1 wild type (wt), S339A (SA), and 1-323 (323) were phosphorylated with Sin1 immunoprecipitates (IP) as the kinase. Incorporated radioactivity was measured and plotted relative to the intensity of the substrate band; error bars show standard deviation. (C) Model suggesting interconnections of the WNK-OSR1-NKCC pathway with the PI3K-mTORC2-Akt pathway.

Discussion

Changes in tonicity regulate the WNK-OSR1/SPAK pathway to control ion cotransporters for volume and ion homeostasis. We find that mTORC2 also contributes to enhanced OSR1 activity. Inhibiting mTORC2 does not inhibit WNK1 activity, indicating that mTORC2 regulates OSR1 activation independently of the effects of WNK1. The intersection of the WNK-OSR1/SPAK pathway with the mTORC2 pathway suggests that mTORC2, which is active in a normal growing cell with ample nutrients, is also important to muster ubiquitous responses to osmotic change mediated by WNK protein kinases (Fig. 4C). OSR1/SPAK appear to be coincidence detectors, requiring a real-time, regulated input from mTORC2 to enable optimum WNK-dependent homeostasis. Our findings add two members of the Ste20 branch of the kinome to the list of mTORC2 targets apparently regulated in this manner.

In contrast to the p21-activated kinase-like Ste20 kinases, in OSR1 and SPAK the kinase domain lies near the N terminus of the proteins and OSR1 and SPAK contain the additional C-terminal PF1 and PF2 regions. The PF1 segment is essential for their activity (4). Interaction of an N- or C-terminal extension, such as PF1, with its protein kinase catalytic core is a common stabilizing and regulatory event in the protein kinase family (38, 39). Protein kinase C and Akt, for example, are regulated by phosphorylation on residues C-terminal to their catalytic cores; the best understood of these sites are often referred to as hydrophobic motif and turn motif sites. From structural studies of these kinases, hydrophobic motif phosphorylation promotes activation by docking a C-terminal extension onto a regulatory site on the catalytic core (40). Phosphorylation of the turn motif enhances stability of the enzyme by protecting it from degradation directly or indirectly and perhaps by providing additional ligands for interaction between the C-terminal extension and the kinase core (28, 41).

OSR1 is phosphorylated on the activation loop and an additional site in the PF1 region C-terminal to the core kinase domain by WNK1. Phosphorylation of S325 or its mutation to glutamate modestly increased OSR1 activity (6). However, most dramatic activity increase comes from phosphorylation of the activation loop by WNK1. Additional phosphorylations have been reported to further increase activity including phosphorylation of S339 (37). Additionally, OSR1 S339 and the comparable site in SPAK (as well as several additional sites in the PF1 region) have been identified by MS in several proteomic studies (www.phosphositeplus.com) (35, 42). Here we identify a major S339 kinase as mTORC2, and we confirm that mutation of S339 to alanine reduces OSR1 activity. Phosphorylation by mTORC2 on the OSR1 PF1 segment may facilitate the interaction of PF1 with the kinase core to enhance the active state of the enzyme. The structural impact of dual PF1 phosphorylation by WNK1 and mTORC2 may resemble the impact of Akt hydrophobic and turn motif phosphorylation by mTORC2, which remains to be determined.

Calcium binding protein 39 (CAB39; originally known as MO25) together with the Ste20-related adaptor STRAD, an OSR1/SPAK pseudo kinase homolog, activate the protein kinase LKB1 (43, 44). OSR1 and SPAK have also been shown to bind and be activated by CAB39. CAB39 is thought to bind the region of OSR1 and SPAK that contains the mTORC2 phosphorylation site and increase their activity independently of WNK1. Modification of this site may recruit CAB39 or other regulators to OSR1 and SPAK. It might also be involved in stabilizing the protein. Another Ste20 kinase was recently shown to autophosphorylate on a comparable C-terminal site (45). The phosphorylation itself was not thought to directly change activity, but was proposed to be involved in docking CAB39 (44). Thus, the PF1 is a regulatory nexus in these kinases.

The turn motif and hydrophobic motif sites of Akt are phosphorylated by mTORC2 with distinct kinetics. The turn motif is phosphorylated cotranslationally, whereas the hydrophobic motif is phosphorylated in response to insulin and growth factors (28, 46). The fact that KU0063794 rapidly interfered with activation of OSR1 suggests that phosphorylation of S339 by mTORC2 is not cotranslational but instead occurs acutely in response to the osmotic stimulus, and may underlie regulation of OSR1 by insulin (34). A second not mutually exclusive possibility is that this site is phosphorylated cotranslationally to stabilize OSR1 before association with partners, yet turns over rapidly as a consequence of its localization with NKCC. In addition to binding OSR1, the OSR1-activated cotransporter NKCC binds the phosphatase protein phosphatase 1, which contributes to rapid changes in NKCC activity (47, 48). This phosphatase may also dephosphorylate OSR1 and SPAK when complexed to NKCCs. Active OSR1 and SPAK associated with NKCC substrate may require rephosphorylation in situ to retain activity.

We found that OSR1 was less sensitive to mTOR inhibition with longer times of drug treatment. The reduced capacity of inhibitors to block OSR1 phosphorylation with longer times of treatment most likely accounts for the fact that this site was not attributed to mTOR previously (35). It seems likely that one or more other kinases can also phosphorylate the PF1 site on OSR1 following prolonged mTOR inhibition. Enzymes other than mTORC2 phosphorylate the Akt hydrophobic motif under certain circumstances; these include Akt itself, the DNA-dependent protein kinase (DNA-PK), and the Tank binding kinase (30, 49–51). DNA-PK and casein kinase II are predicted to phosphorylate OSR1 S339 (http://scansite.mit.edu). Akt, like the Ste20 kinase noted above, can autophosphorylate its own hydrophobic motif site; this mechanism is enhanced by mutation of the pleckstrin homology domain or prolonged inhibition of mTORC1 (30, 49, 52). Autophosphorylation is an intriguing possibility because OSR1 can exist as an activation loop-swapped dimer (53).

WNKs are interconnected with PI3K-dependent pathways. Akt phosphorylates WNK1 on a threonine residue, T60 (T58 in rat), preceding the kinase domain and the comparable residue in WNK4 (24, 36, 54, 55). Phosphorylation of this site has little effect on WNK1 kinase activity. However, mutation of T60 inhibits SGK activation by WNKs, suggesting that phosphorylation of T60 is a permissive event that exerts positive feedback on some WNK1 functions (24, 36). Phosphorylation of T60 is required for maximal activation of ENaC by WNK1, consistent with the effect on SGK1 activation (36); regulation of the renal outer medullary potassium channel by WNK1 also depends on phosphorylation of T60 (56). WNK1 activity is essential for phospholipase C beta PLCβ function downstream of Gq-coupled receptors, which may also involve PI3K (57). The PI3K pathway has recently been implicated in WNK-OSR1/SPAK-NCC pathway in animal studies (58). With the finding that activation of OSR1/SPAK by WNK1 is enhanced by mTORC2, we have demonstrated the mechanism underlying another WNK1-regulated cascade that requires a coincident signal from PI3K. We conclude that cell homeostasis requires the multilevel integration of WNK osmosensing and PI3K survival pathways.

Experimental Procedures

Materials.

Human Sin1 siRNA oligonucleotides were from Ambion (hSin1, sense, gauuagaacgacuccgaaaTT, antisense, uuucggagucguucuaaucTT). Rapamycin and wortmannin were from LC Laboratories, KU0063794 from Tocris biosciences, and ouabain (Na⁺/K⁺ inhibitor) and bumetanide (NKCC inhibitor) from Sigma. Antibodies were from the following: Sin1 (NovusB biological), mTOR (Santa Cruz Biotechnology, immunoblotting) and Cell Signaling (Santa Cruz Biotechnology, immunoprecipitation), and Akt and pS473 Akt (Cell Signaling). Anti-WNK1 (Q256), anti-OSR1 (U5567) and plasmids encoding WNKs and NKCC2 were as described (59). ⁸⁶Rb and [γ-³²P]ATP were from Perkin-Elmer. Recombinant proteins used for kinase assays were purified from Escherichia coli strain BL21 using standard protocols.

Cell Culture and Transfection.

HeLa cells were grown in DMEM supplemented with 10% (vol/vol) FBS (Sigma) and 2 mM glutamine. Cells were harvested in 50 mM Hepes, pH 7.7, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 10% (vol/vol) glycerol, 100 mM NaF, 0.2 mM NaVO₄, 50 mM β-glycerophosphate, 0.1% Triton X-100, 0.1 µM phenylmethyl sulfonyl fluoride, 10 mg/L each N-α-p-tosyl-l-lysine chloromethyl ester, N-α-p-tosyl-l-arginine methyl ester, N-α-p-tosyl-l-lysine chloromethyl ketone, leupeptin, and pepstatin A as described (60).

Immunoprecipitation and Immunoblotting.

Proteins were immunoprecipitated overnight at 4 °C from 750 µg of soluble lysate protein with 5 µL each antibody. Antibodies were collected with 30 µL of a 50% protein A-Sepharose (GE Healthcare) slurry following a 2-h incubation at 4 °C. Beads were washed 3× in lysis solution and proteins were eluted using 5X electrophoresis sample buffer. Proteins in cell lysates or immunoprecipitates were resolved by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose. Immunoblots were developed using a Li-COR Odyssey infrared imaging system.

siRNA.

HeLa cells were detached from 10-cm dishes with trypsin and immediately transfected with 20 nM Sin1 dsRNA oligonucleotides (hSin1), or 2.5 nM small interfering WNK1 dsRNA oligonucleotides [sense, cagacagugcaguauucacTT; antisense, gugaauacugcacugucugTT; (Ambion) using Lipofectamine RNAiMax (Invitrogen)] according to manufacturer’s protocols.

Kinase Assays.

For immune complex kinase assays, 20 µL immunoprecipitate on beads were incubated with 50 µM ATP (5,000–13,000 dpm/pmol [γ-³²P]), 10 mM MgCl₂, and 20 mM Hepes pH 7.4 for 30 min at room temperature. Proteins were resolved as above. Gels were dried and exposed to film for autoradiography. Incorporation of radioactivity was quantified by scintillation counting of the bands excised from the gel. To obtain a 0.1-mol/mol incorporation of phosphate into substrate, 2 µM OSR1 K46R were used as a substrate with 500 µM ATP and the reactions were incubated for 4 h at 30 °C.

NKCC Assay.

Knockdown proceeded 48 h following transfection and then cells were washed 2× in 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM Hepes pH 7.4, 10 mM glucose, 10 mM Na pyruvate, and 0.1% BSA. After 30 min, cells were incubated for 5 min with 10⁷ cpm/mL ⁸⁶Rb and 0.5 mM ouabain with or without 10 μM bumetanide. After washing 2× in cold 100 mM MgCl₂ and 10 mM Hepes pH 7.4 buffered with Tris, cells were lysed in 2% SDS. ⁸⁶Rb uptake was measured in 100 μL lysates by liquid scintillation counting. Lysate protein concentration was determined using Pierce MicroBCA Protein Assay Kit. Uptake was an average of triplicates. NKCC activity (bumetanide-sensitive uptake) was taken as the difference with and without bumetanide. Effects of treatments are expressed relative to the control.

Statistical Analysis.

Comparison between two groups was made using the two-tailed unpaired t test. One-way ANOVA followed by Tukey’s multiple comparison test were performed as indicated. P values are indicated in figure legends.

Proteomics and MS.

Protein bands from polyacrylamide gels in SDS were excised and digested overnight with trypsin (Promega) after reduction and alkylation with DTT and iodoacetamide (Sigma-Aldrich). The resulting samples were analyzed by tandem MS using a Q Exactive mass-spectrometer (Thermo Electron) coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system (Dionex). Peptides were loaded onto a 180 μm inner diameter, 10 cm self-packed column containing 3 μm C18 resin (Dr. Maisch), and eluted with a gradient of 1–41% buffer B in 40 min. Buffer A contained 0.1% formic acid in water and buffer B, 0.1% formic acid in acetonitrile. The Q Exactive instrument acquired up to 10 high-energy collision-induced dissociation fragment spectra for each full spectrum acquired.

Raw MS data were converted to peak list format using ProteoWizard msconvert (version 3.0.3535) (61). The resulting files were analyzed using the Central Proteomics Facilities Pipeline (CPFP) (62). Peptide identification was performed using the X!Tandem (63) and Open MS search algorithm (64) search engines against the UniProtKB human whole proteome sequence database (release 2012_02) (65), with reversed decoy sequences appended (66). Fragment and precursor tolerances of 20 ppm and 0.5 Da were specified and two missed cleavages were allowed. Carbamidomethylation of cysteine was specified as a fixed modification. Oxidation of methionine and phosphorylation (Ser/Thr/Tyr) were specified as variable modifications. Validation and combination of results was performed within CPFP using Trans-Proteomic Pipeline tools (67). Identifications were filtered to <1% false discovery rate. Initial assessment of localization ambiguity for phosphorylation assigned by the search engines was performed using the modification localization score tool within CPFP, based on the post translational modification score method (68, 69).