Supplementary Materials and Methods
Materials. Sequencing-grade trypsin (Promega); Protease-inhibitor cocktail tablets
(Roche); Protein G–Sepharose and glutathione–Sepharose (Amersham Biosciences);
[γ32P]ATP and 86Rb (PerkinElmer); Anti-FLAG M2-agarose, Tween-20, fish skin
gelatin, Triton X-100, phenformin hydrochloride, ouabain, bumetanide, tetracycline
and compound C (Sigma-Aldrich); Streptavidin Sepharose (GE Healthcare); Colloidal
blue staining kit, precast SDS polyacrylamide Bis-Tris gels, blasticidin and
hygromycin (Invitrogen); Nonidet P40 (Fluka); hydromount (BDH). Peptides were
synthesised by Graham Bloomberg (university of Bristol School of Medical Sciences,
UK).
General methods. Restriction enzyme digests, DNA ligations, other recombinant
DNA procedures, electrophoresis, cell culture, and transfections were performed
using standard protocols. All mutagenesis was performed using the QuikChange sitedirected mutagenesis method (Stratagene) with KOD polymerase (Novagen). DNA
constructs used for transfection were purified from Escherichia coli DH5α using
QIAGEN kits according to the manufacturer’s protocol. All DNA constructs were
verified by DNA sequencing, which was performed by the Sequencing Service
(School of Life Sciences, University of Dundee, U.K.), using DYEnamic ET
terminator chemistry (GE Healthcare) on Applied Biosystems automated DNA
sequencers.
Antibodies. The following antibodies were raised in sheep and affinity purified on the
appropriate antigen: NKCC2 total antibody (residues 1-174 of human NKCC2,
S838B), NKCC2 phospho-Ser91 antibody (residues 86-97 of human NKCC2
phosphorylated at Ser91, FHAYDpSHTNTYY, S451C), NKCC2 phospho-Thr105
antibody (residues 99-110 of human NKCC2 phosphorylated at Thr105,
QTFGHNpTMDAVP, S378C), NKCC2 phospho-Ser130 antibody (residues 123-137
of human NKCC2 phosphorylated at Ser130, GPKVNRPpSLLEIHEQ, S432C), NCC
total antibody (residues 907-926 of human NCC, HTKRFEDMIAPFRLNDGFKD,
S962B), NCC phospho-Thr55 antibody (residues 41-60 of human NCC
phosphorylated at Thr55, HPSHLTHSSTFCMRpTFGYNT, S908B), NCC phosphoThr60 antibody (residues 54-66 of human NCC phosphorylated at Thr60,
RTFGYNpTIDVVPT, S995B), NKCC1 total antibody (residues 1-260 of shark
NKCC1, S841B), NKCC1 phospho-Thr203/Thr207/Thr212 antibody (residues198217 of human NKCC1 phosphorylated at Thr203, Thr207 and Thr212,
HYYYDpTHTNpTYYLRpTFGHNT, S763B), WNK1-total antibody (residues 23602382 of human WNK1, QNFNISNLQKSISNPPGSNLRTT, S062B), WNK1
phospho-Ser382 antibody (residues 377-387 of human WNK1 phosphorylated at
Ser382, ASFAKpSVIGTP, S099B), SPAK-total antibody (full-length human SPAK
protein, S637B), OSR1 total antibody (full-length human OSR1 protein, S636B),
SPAK/OSR1 (T-loop) phospho-Thr233/Thr185 antibody (226–238 of human SPAK
or residues 178–190 of human OSR1, TRNKVRKpTFVGTP, S204C), SPAK/OSR1
(S-motif) phospho-Ser373/Ser325 antibody (367–379 of human SPAK,
RRVPGSpSGHLHKT, which is highly similar to residues 319–331 of human OSR1
in which the sequence is RRVPGSpSGRLHKT, S670B), ERK1 total antibody (fulllength human ERK1 protein, S221B), GST-total antibody (raised against the
glutathione S-transferase protein, S902A). The AMPK total antibody (#2532),
AMPK phospho-Thr172 antibody (#2535S), and the Raptor phospho-Ser792
antibody were from Cell Signaling Technology. The total AMPK1 antibody
employed for immunoprecipitation kinase assays was generated and donated by D.
Graham Hardie (University of Dundee). The Insulin Receptor  antibody (sc-711)
was purchased from Santa Cruz Biotechnology, the anti-FLAG antibody (F 1804) was
purchased from Sigma-Aldrich, and the anti-GAPDH antibody (ab8245) was
purchased from Abcam. Secondary antibodies coupled to horseradish peroxidase used
for immunoblotting were obtained from Pierce. Preimmune IgG used in control
immunoprecipitation experiments were affinity purified from preimmune serum using
protein G-Sepharose. Alexa Fluor secondary antibodies used for
immunocytochemistry were obtained from Invitrogen.
Buffers. Lysis buffer was 50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 50
mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1%
(w/v) NP-40, 0.27 M sucrose, 0.1% (v/v) 2-mercaptoethanol, and protease inhibitors
(1 tablet per 50 ml). Buffer A was 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, and
0.1% (v/v) 2-mercaptoethanol. TBS-Tween buffer (TTBS) was Tris/HCl, pH 7.5, 0.15
M NaCl, and 0.2% (v/v) Tween-20. SDS sample buffer was 1× NuPAGE LDS sample
buffer (Invitrogen), containing 1% (v/v) 2-mercaptoethanol. Basic buffer was 135
mM NaCl, 5 mM KCl, 0.5mM CaCl2, 0.5mM MgCl2, 0.5 mM Na2HPO4, 0.5mM
Na2SO4, and 15mM HEPES (pH 7.4) (Moriguchi et al., 2005). Hypotonic lowchloride buffer was 67.5 mM Na gluconate, 2.5 mM K gluconate, 0.25mM CaCl2,
0.25mM MgCl2, 0.5 mM Na2HPO4, 0.5mM Na2SO4, and 7.5mM HEPES (pH 7.4)
(Moriguchi et al., 2005). Isotonic uptake buffer was 40 mM NaCl, 46 mM N-MethylD-glucamine, 10 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5.0 mM HEPES (pH 7.4)
(Ponce-Coria et al., 2008). Hypotonic uptake buffer was 40 mM NaCl, 36 mM NMethyl-D-glucamine, 10 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5.0 mM HEPES
(pH 7.4) (Ponce-Coria et al., 2008).
Plasmids. Cloning of human SPAK, human OSR1 (Vitari et al., 2005) and human
NCC (Richardson et al., 2008) have been described previously. In order to obtain fulllength cDNA for human NKCC2, PCR products from EST DKFZp686014110
(covering amino acids 210-1099) and from kidney RNA (amino acids 1-209) were
combined. The resulting cDNA corresponding to NKCC2 variant F (NCBI acc.
EAW77346) was sequenced and subcloned into bacterial and mammalian expression
vectors. This clone was used as a template for the generation of NKCC2 variant A
(NCBI acc. ABU69043) and NKCC2 variant B (NCBI acc. CAA06478) employing
PCR methods to modify exon 4. Required amino acid mutations were introduced
using site-directed mutagenesis by Quickchange method (Stratagene).
Expression and purification of FLAG-NKCC2 in HEK-293 cells. Typically 5-10
10-cm diameter dishes of HEK-293 cells were cultured and each dish was transfected
with 5-10 g of the pCMV5 construct encoding wild type or different mutant forms of
FLAG-NKCC2 using the polyethylenimine method (Durocher et al., 2002). The cells
were cultured for a further 36 h and lysed in 0.3 ml of ice-cold lysis buffer. The
pooled lysate was clarified by centrifugation at 4C for 10 min at 26,000 g. The
FLAG-fusion proteins were affinity-purified on FLAG-agarose and eluted in either
Buffer A with 0.1 mg/ml FLAGtide peptide (DYKDDDD) or Buffer A containing
LDS sample buffer.
Expression and purification of GST-tagged SPAK, OSR1 and NKCC2[1-174] in
E.coli. All pGEX-6P-1 constructs were transformed into BL21 E. coli cells and 1-litre
cultures were grown at 37 °C in Luria Broth containing 100 g/ml ampicillin until the
absorbance at 600 nm was 0.8. Isopropyl b-D-thiogalactopyranoside (30 M) was
then added and the cells were cultured for a further 16 h at 26°C. Cells were isolated
by centrifugation, resuspended in 40 ml of ice-cold Lysis Buffer and lysed in one
round of freeze/thawing, followed by sonication (Branson Digital Sonifier; ten 15 s
pulses with a setting of 45% amplitude) to fragment DNA. Lysates were centrifuged
at 4°C for 15 min at 26 000 g. The GST-fusion proteins were affinity-purified on 0.5
ml glutathione-Sepharose and eluted in Buffer A containing 0.27 M sucrose and 20
mM glutathione.
Immunoblotting. Cell lysates (20–40 mg), purified proteins, or immunoprecipitates
in SDS sample buffer were subjected to electrophoresis on a polyacrylamide gel and
transferred to nitrocellulose membranes. The membranes were incubated for 30 min
with TTBS containing 10% (w/v) skimmed milk. The membranes were then
immunoblotted in 5% (w/v) skimmed milk in TTBS with the indicated primary
antibodies overnight at 4C. Sheep antibodies were used at a concentration of 1–2
μg/ml, whereas commercial antibodies were diluted 1,000-5,000 fold. The incubation
with phosphospecific sheep antibodies was performed with the addition of 10 μg/ml
of the dephosphopeptide antigen used to raise the antibody. The blots were then
washed six times with TTBS and incubated for 1 h at room temperature with
secondary HRP-conjugated antibodies diluted 5,000 fold in 5% (w/v) skimmed milk
in TTBS. After repeating the washing steps, the signal was detected with the
enhanced chemiluminescence reagent. Immunoblots were developed using a film
automatic processor (SRX-101; Konica Minolta Medical), and films were scanned
with a 300-dpi resolution on a scanner (PowerLook 1000; UMAX). Quantitative
immunoblotting was performed employing the LI-COR Odyssey imaging system and
Figures were generated using Photoshop/Illustrator (Adobe).
Immunoprecipitation and assay of AMPK1. 50 g of clarified HEK-293 cell
lysate was incubated with 1 mg of the AMPK1 antibody conjugated to 5 ml of
protein G–Sepharose and incubated for 1 h at 4°C with gentle agitation. The
immunoprecipitates were washed twice with 1 ml of lysis buffer containing 0.5 M
NaCl and twice with 1 ml of buffer A. The AMPK1 immunoprecipitates were
assayed with the AMARA peptide substrate (AMARAASAAALARRR) (Dale et al.,
1995; Sakamoto et al., 2004). Assays were set up in a total volume of 50 ml in buffer
A containing 10 mM MgCl2, 0.1 mM [γ32P]ATP and 200 M AMARA peptide. After
incubation for 20 min at 30°C, the reaction mixture was applied onto P81
phosphocellulose paper, the papers were washed in phosphoric acid, and incorporation
of 32P-radioactivity in AMARA peptide was quantified by Cerenkov counting.
Assay of NKCC2 phosphorylation by SPAK and OSR1 and phosphorylation site
mapping. GST-NKCC2[1-174] and GST-NKCC2[1-174] mutant proteins (5-7.5 g)
purified from E.coli were incubated with 1.5-5 g of the following also purified from
E.coli (all GST tagged): kinase-inactive SPAK - SPAK[D212A], active SPAK SPAK[T233E], kinase-inactive OSR1 – OSR1[D164A], or active OSR1 –
OSR1[T185E]. Reactions were for 0-80 min at 30C in a 25 l reaction volume in
Buffer A containing 10 mM MgCl2, 0.1 mM [γ32P]ATP (between 300 cpm/pmol and
11,000 cpm/pmol) and terminated by addition of LDS Sample Buffer. For
phosphorylation site mapping, Dithiothreitol (DTT) was added to a final
concentration of 10 mM, the samples heated at 95°C for 4 min and cooled for 20 min
at room temperature, iodoacetamide was then added to a final concentration of 50
mM, and the samples left in the dark for 30 min at room temperature to alkylate
cysteine residues. The samples were subjected to electrophoresis on a Bis-Tris 10%
polyacrylamide gel, which was stained with Colloidal Blue and then
autoradiographed. The phosphorylated NKCC2[1-174] bands were excised, cut into
smaller pieces and incorporation of 32P-radioactivity quantified by Cerenkov
counting. Excised gel pieces were then washed sequentially for 10 min on a vibrating
platform with 1 ml of the following: water; 1:1 (v/v) mixture of water and
acetonitrile; 0.1 M ammonium bicarbonate, 1:1 mixture of 0.1 M ammonium
bicarbonate and acetonitrile, and finally acetonitrile. The gel pieces were dried, and
incubated for 16 h at 30C in 25 mM triethylammonium bicarbonate containing 5
g/ml trypsin as described previously (Woods et al., 2001). Following tryptic
digestion, >95% of the 32P radioactivity incorporated in the gel bands was recovered
and the samples were chromatographed on a Vydac 218TP5215 C18 column
(Separations Group, Hesperia, CA, U.S.A.) equilibrated in 0.1% trifluoroacetic acid in
water. The column was developed with a linear acetonitrile gradient (diagonal line in
Supplementary Figs 1C & 2) at a flow rate of 0.2 ml/min and fractions of 0.1 ml were
collected. Some phosphopeptide fractions were further purified by immobilized
metal-chelate affinity chromatography on Phos-Select resin (Sigma) as described
previously (Beullens et al., 2005).
Identification of phosphorylation sites on FLAG-NKCC2 expressed in HEK-293
cells. HEK-293 cells were transfected with wild type FLAG-NKCC2 and 36 h posttransfection, cells were treated with either control basic or hypotonic low-chloride
medium for 30 min. The treated cells were lysed with 0.3 ml of ice-cold lysis
buffer/dish and the lysates clarified by centrifugation at 4C for 10 min at 26,000 g.
FLAG-NKCC2 was purified from basic or hypotonic low-chloride treated samples by
incubating 30 l of FLAG-agarose beads with 7 mg of the clarified lysate for 2 h at
4C. The immunoprecipitates were washed four times with 1 ml lysis buffer
containing 0.5 M NaCl and twice with 1 ml of buffer A. Proteins were eluted from the
FLAG beads by the addition of 25 l 1× NuPAGE LDS sample buffer to the beads.
Eluted proteins were reduced by the addition of 10 mM DTT followed by heating at
95C for 4 min. The samples were then alkylated by the addition of 50 mM
Iodoacetamide followed by incubation in the dark for 30 min at room temperature.
Samples were then electrophoresed on a 4–12% polyacrylamide gel, which was
stained with Colloidal blue. The NKCC2 bands were excised from the gel, cut into
smaller pieces and washed as described above. For the identification of
phosphorylation sites, the tryptic digests were analyzed by LC-MS on an Applied
Biosystems 4000 QTRAP system with precursor ion scanning as described previously
(Williamson et al., 2006). The resultant data were searched using the Mascot search
algorithm (www.matrixscience.com) run on a local server. Tryptic phosphopeptides
were also analysed by LC-MS-MS using a linear ion trap-orbitrap hybrid mass
spectrometer (LTQ-Orbitrap, Thermo Fischer Scientific) equipped with a
nanoelectrospray ion source (Thermo) and coupled to a Dionex Ultimate 3000-nanoHPLC system. The 5 most intense ions, above a specified minimum signal threshold
(20,000) were fragmented by collision-induced dissociation and recorded in the linear
ion trap. Multi-Stage-Activation was used to provide an MS3 scan of any parent ions
showing a neutral loss of 48.9885, 32.6570 or 24.4942, from 2+, 3+ and 4+ ions
respectively. The resulting MS3 scan was automatically combined with the relevant
MS2 scan prior to searching peptide spectra against an in house database, containing
the NKCC2 sequence, using the Mascot search algorithm.
Phosphopeptide sequence analysis. Isolated phosphopeptides were analysed by
MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight)-MS on an
Applied Biosystems 4700 Proteomics Analyser using 5 mg/ml a-cyano-4hydroxycinnamic acid containing 2 mM ammonium phosphate as the matrix. Spectra
were acquired in reflector mode and the phosphopeptides were analysed further by
performing MALDI–TOF–TOF on selected masses. The characteristic loss of
phosphoric acid (M-98 Da) from the parent phosphopeptide was seen. The site of
phosphorylation of all the 32P-labelled peptides was determined by solid-phase Edman
degradation on an Applied Biosystems 494C sequencer of the peptide coupled to
Sequelon-AA membrane (Milligen) as described previously (Campbell and Morrice,
2002).
Supplementary References
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of protein kinase MELK. J Biol Chem 280, 40003-11.
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phosphorylation sites by a combination of mass spectrometry and solid phase Edman
sequencing. J Biomol Tech 13, 119-30.
Dale, S., Wilson, W. A., Edelman, A. M. and Hardie, D. G. (1995). Similar
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Natsume, T., Matsumoto, K. and Shibuya, H. (2005). WNK1 regulates
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Supplementary Figure 1 – Identification of residues on NKCC2 that are
phosphorylated by SPAK in vitro. (A) E. coli expressed GST-NKCC2[1-174] was
incubated with the indicated active and kinase-inactive forms of GST-OSR1 or GSTSPAK in the presence of Mg-[32P]ATP for 40 min. Phosphorylation of NKCC2[1174] was determined following electrophoresis and subsequent autoradiography of the
Colloidal blue-stained bands corresponding to NKCC2[1-174]. Similar results were
obtained in three separate experiments. Abbreviations KI-SPAK, kinase-inactive
SPAK[D212A]; Act-SPAK, activated-SPAK[T233E]; KI-OSR1, kinase-inactiveOSR1[D164A]; Act-OSR1; activated OSR1[T185E] (B) E. coli expressed GST
NKCC2[1-100] was incubated with active GST-SPAK in the presence of Mg[32P]ATP for the indicated times and phosphorylation of NKCC2[1-174] was
determined as in (A). (C) GST-NKCC2[1-174] was phosphorylated with active GSTSPAK in the presence of Mg-[32P]ATP for 60 min. Phosphorylated NKCC2[1-174]
was digested with trypsin and chromatographed on a C18 column. The peak fractions
containing the major 32P-labelled peptides are labelled P1, P2a, P2b and P3. (D)
Summary of the mass spectrometry and solid phase Edman sequencing data obtained
following analysis of the indicated phosphopeptides. The deduced amino acid
sequence of each peptide is shown and the phosphorylated residue is indicated by (p).
Abbreviation: (m) in peptide sequence indicates methionine sulphoxide. (E) The
indicated forms of GST-NKCC2[1-174] were phosphorylated with active GST-SPAK
and phosphorylation was analysed as in (C). (F) The indicated forms of GSTNKCC2[1-174] were phosphorylated with kinase inactive or active GST-SPAK or
GST-OSR1. Phosphorylation was analysed as in (A) as well as by quantification of
32
P-radioactivity associated with Colloidal blue-stained bands corresponding to
NKCC2[1-100]. Results of the latter are presented as a percentage of phosphorylation
compared to wild type NKCC2[1-174] by active SPAK/OSR1. Results of duplicate
samples are shown and similar results were obtained in two separate experiments.
Supplementary Figure 2 – OSR1 phosphorylation of NKCC2[1-174] in vitro. As
described in supplementary Figure 1C except that active OSR1 was used in place of
active SPAK.
Supplementary Figure 3 – Analysis of NKCC2 Ser130 phosphorylation
employing an AMPK inhibitor termed compound C. HEK-293 T-Rex stable cells
were induced to express wild type human FLAG NKCC2 (isoform F). 24 h postinduction, cells were either untreated or pretreated with an AMPK inhibitor termed
compund C for a period of 30 min. Cells were subsequently treated with basic buffer
for 30 min, hypotonic low-chloride buffer for 30 min, or basic buffer containing 2
mM phenformin (AMPK activator) for 60 min and lysed. Total cell extracts were
immunoblotted with the indicated antibodies and NKCC2 Ser130 phosphorylation
was monitored by immunoblotting following immunoprecipitation of FLAG NKCC2.
Supplementary Figure 4 – Investigation of the bumetanide sensitivity of
endogenous NKCC1 and overexpressed NKCC2 in HEK-293 cells. (A) HEK-293
cells were transfected with pCMV5 empty vector or a construct encoding wild type
human NKCC2 isoform B. 36 h post-transfection, 86Rb uptake was assessed in control
basic or hypotonic low-chloride treated cells in the absence or presence of 0.1 mM
bumetanide in the uptake medium as described in the materials and methods. For each
transfection construct used, bumetanide-sensitive 86Rb uptake (i.e. bumetanideinsensitive counts subtracted) is plotted for both basic and hypotonic low-chloride
conditions. The results are presented as the mean 86Rb uptake ± SD for triplicate
samples. Control immunoblotting with the indicated antibodies was performed in
parallel. (B) Bumetanide dose-response curves of endogenous NKCC1 (hypotonic
low-chloride treated) and overexpressed NKCC2 isoform B (basic treated) in HEK293 cells. Assays were performed as described in (A) in the absence or presence of
the indicated concentrations of bumetanide and the calculated IC50 values for NKCC1
and NKCC2 are indicated.
Supplementary Figure 5 – NKCC2 phosphorylated at Thr105 localises to the
plasma membrane under both basic and hypotonic low-chloride treatment
conditions. (A) HEK-293 cells were transfected with constructs encoding the
indicated wild type and mutant form of human NKCC2 isoform B. 36 h posttransfection, cells were treated with either basic (B) or hypotonic low-chloride buffer
(H) for 30 min and lysed. Total cell extracts were immunoblotted with the indicated
antibodies. Abbreviation: 5A corresponds to a quintuple NKCC2 mutant in which
Ser91, Thr95, Thr100, Thr105 and Ser130 are changed to Ala. (B) As described in
(A), except that following buffer treatment cells were fixed in 4% paraformaldehyde.
The cells were subsequently permeabilised and immunostained with either anti-FLAG
(Total NKCC2) followed by Alexa Fluor 594-labelled anti-mouse antibody or antiNKCC2 phospho-Thr105 antibody followed by Alexa Fluor 594-labelled anti-sheep
antibody. Fluorescence imaging was performed on a confocal microscope and the
cells shown are representative images.