MOLECULAR AND CELLULAR BIOLOGY, Jan. 2006, p. 689–698
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 26, No. 2
Characterization of SPAK and OSR1, Regulatory Kinases of the
Kenneth B. E. Gagnon, Roger England, and Eric Delpire*
Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
Received 3 September 2005/Returned for modification 21 October 2005/Accepted 29 October 2005
Our recent studies demonstrate that SPAK (Ste20p-related Proline Alanine-rich Kinase), in combination
with WNK4 [With No lysine (K) kinase], phosphorylates and stimulates the Na-K-2Cl cotransporter (NKCC1),
whereas catalytically inactive SPAK (K104R) fails to activate the cotransporter. The catalytic domain of SPAK
contains an activation loop between the well-conserved DFG and APE motifs. We speculated that four
threonine residues (T231, T236, T243, and T247) in the activation loop might be sites of phosphorylation and
kinase activation; therefore, we mutated each residue into an alanine. In this report, we demonstrate that
coexpression of SPAK (T243A) or SPAK (T247A) with WNK4 not only prevented, but robustly inhibited,
cotransporter activity in NKCC1-injected Xenopus laevis oocytes. These activation loop mutations produced an
effect similar to that of the SPAK (K104R) mutant. In vitro phosphorylation experiments demonstrate that
both intramolecular autophosphorylation of SPAK and phosphorylation of NKCC1 are significantly stronger
in the presence of Mn2?rather than Mg2?. We also show that SPAK activity is markedly inhibited by
staurosporine and K252a, partially inhibited by N-ethylmaleimide and diamide, and unaffected by arsenite.
OSR1, a kinase closely related to SPAK, exhibited similar kinase properties and similar functional activation
of NKCC1 when coexpressed with WNK4.
Cation-chloride cotransporters (e.g., Na-K-2Cl and K-Cl co-
transporters) serve multiple fundamental functions in a wide
variety of tissues and organs. These include influencing ion and
fluid movements in secreting or reabsorbing epithelia, control
of CNS excitability, and cell volume regulation, proliferation,
and survival (for a review, see reference 19). On the basis of
the observations that the cotransporters are phosphoproteins
and that phosphatase inhibitors affect cotransport activity, it is
generally agreed that cotransporter regulation is mainly medi-
ated through phosphorylation-dephosphorylation mechanisms.
Activity of NKCC1, for instance, correlates directly with the
phosphorylation state of the protein (33). For the past 20 years,
the identity of the kinase in question has remained elusive. We
have recently shown direct interaction between cation-chloride
cotransporter and two Ste20p-related serine/threonine kinases,
SPAK and OSR1 (45).
There are some 30 protein kinases identified in mammals
related to the budding Saccharomyces cerevisiae sterile 20 pro-
tein kinase (Ste20p). In 1991, Dan et al. divided the mamma-
lian Ste20p-like kinases into two subgroups: the p21-activated
kinases (PAKs; two subfamilies), which are characterized by a
carboxyl-terminal catalytic domain, and the germinal center
kinases (GCKs; eight subfamilies), which have their catalytic
domain located at the amino terminus (10). Most Ste20p-like
kinases stimulate mitogen-activated protein kinase (MAPK)
cascades and thus participate in the modulation of cell motility
(7), cell growth (31), and apoptosis (23).
The GCK6 subfamily comprises two kinases: SPAK/PASK
(Ste20-related proline alanine-rich kinase) (26, 52) and OSR1
(oxidative stress response 1) (50). These two closely related
kinases share 66% identity overall at the amino acid level, are
structurally reminiscent of MST kinases, and possess a well-
conserved, short, and unique C-terminal region. This unique
region was demonstrated to be a critical domain of the kinase,
interacting with RFxV motifs in membrane transport systems
such as cation-chloride cotransporters (45) and chloride chan-
nels (12) but also associating with other proteins such as heat
shock protein 105, WNK4, apoptosis-associated tyrosine ki-
nase, gelsolin (44), the actin cytoskeleton (51), and p38 MAPK
Functionally, SPAK increases the activity of the Na-K-2Cl
cotransporter (13, 18), reduces the activity of the neuron-spe-
cific K-Cl cotransporter, KCC2 (18), and inactivates the cell
cycle-dependent CLC anion channel in Caenorhabditis elegans
(12). Consistent with their role in ion transport, SPAK and
OSR1 expression increases in killifish opercular epithelium as
the fish adapts from seawater to fresh water (35). Thus, mod-
ulation of ion transport seems to be a major function of the
kinase and, not surprisingly, the highest levels of kinase ex-
pression are seen in the secretory cell in C. elegans (12) and in
mouse secreting and absorbing epithelia (36, 45, 52). However,
OSR1 and SPAK have also been shown to carry out additional
functions, such as acting as intermediates in cell signaling. For
example, increasing concentrations of sorbitol activate OSR1,
which phosphorylates the N-terminal regulatory domain of
PAK1, desensitizing the kinase to activation by small G pro-
teins (8). In 2004, Li et al. (30) found that SPAK serves as an
intermediate in a T-cell-receptor-induced signaling pathway.
Activation of the transcription factor AP-1 requires protein
kinase C-? (PKC?) phosphorylation of two specific SPAK
serine residues: S311 and S325. We recently demonstrated that
SPAK activation of NKCC1 and deactivation of KCC2 in Xe-
nopus laevis oocytes requires a functional interaction with an-
* Corresponding author. Mailing address: Department of Anesthe-
siology, Vanderbilt University Medical Center, T-4202 Medical Center
North, 1161 21st Avenue South, Nashville, TN 37232. Phone: (615)
343-7409. Fax: (615) 343-3916. E-mail: firstname.lastname@example.org.
other upstream kinase: WNK4 (18). Vitari and coworkers con-
firmed this interaction between SPAK, WNK1, and WNK4
(54). They also showed that the two WNK kinases phosphor-
ylate SPAK at residue T243 in the activation loop and at
residue S383 in the regulatory subdomain (mouse sequence).
Thus, SPAK is a target for at least three upstream kinases:
PKC?, WNK4, and WNK1. Furthermore, there seems to be
some specificity to the residues phosphorylated by these up-
Through in vitro phosphorylation studies, we examined the
modalities of mouse SPAK and OSR1 autophosphorylation
and transphosphorylation of the N-terminal tail of NKCC1.
We show that the in vitro activity of both SPAK and OSR1 is
stronger in the presence of Mn2?rather than Mg2?and that
both have high affinities for ATP and are inhibited by high Cl?
concentrations. We demonstrate that autophosphorylation oc-
curs through intramolecular rather than intermolecular reac-
tions and that two of the four threonine residues located in the
activation loop of the kinase (T243 and T247) are critical for
SPAK activity in vitro as well as in vivo activation of NKCC1 in
Xenopus laevis oocytes.
MATERIALS AND METHODS
Mutagenesis of SPAK. A 800-bp AflII-BglII fragment from mouse SPAK
cDNA containing the “activation loop” between the DFG and APE motifs was
PCR amplified and subcloned into pGEM-T Easy vector (Invitrogen, Carlsbad,
CA). Complementary sense and antisense oligonucleotides containing the codon
GCN (Ala) instead of ACN (Thr) were used to individually mutate the four
threonine residues into alanines (QuikChange; Stratagene, La Jolla, CA). The
parental DNA was digested with DpnI to cleave methylated GATC sequences,
and a 1-?l aliquot of the PCR was transformed into Escherichia coli. Several
clones were isolated to verify proper sequence and mutation. The AflII-BglII
fragment was then reinserted into the original SPAK clone in pGEX. Each of the
four Thr3Ala mutant SPAK clones were also moved to the Xenopus expression
vector pBF with EcoRI-XhoI. Two PCRs using nested sense and antisense
oligonucleotides were used to excise the proline- and alanine-rich region (PAPA
box) located upstream of the catalytic domain of SPAK. The sequence of the
final PCR product was verified, and the EcoRI-EcoRV fragment was reinserted
into the original SPAK clone in pGEX and pBF vectors. A SacI fragment from
the full-length catalytically inactive SPAK was then moved into the shortened
SPAK (-PAPA box) clone to create a smaller, catalytically inactive SPAK.
Cloning of mouse OSR1. We obtained IMAGE clone 5341146 (mouse OSR1)
from ATCC, subcloned it into pBluescript vector (pBSK?), and sequenced the
entire insert. A SacI-BamHI fragment was PCR amplified from mouse brain,
sequenced, and then substituted into the original clone to correct for an inser-
tion. Adaptors made of complementary oligonucleotides (KpnI-XhoI at the 5?
end and BstXI-NotI at the 3? end) were used to remove both 5? and 3? untrans-
lated regions. The full-length mouse OSR1 clone was then moved into pBF and
GST fusion protein production. Wild-type and mutant SPAK, OSR1, and
NKCC1 constructs in pGEX were transformed into protease-deficient E. coli
cells and incubated at 30°C for 3 to 4 h until reaching an optical density at 600
nm ? 0.5. Fusion protein production was then induced by the addition of 24
mg/liter IPTG (isopropyl-?-D-thiogalactopyranoside) for an additional 4 h, and
then the cells were collected by centrifugation at 4°C, resuspended in column
buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4)
containing protease inhibitors, and frozen at ?20°C. E. coli cells were lysed by
sonication, incubated with Triton X-100 (1% final) for 30 min on ice, and spun
down at 10,000 ? g for 10 min at 4°C. The supernatant was passed through a
column containing glutathione-Sepharose beads, and the fusion protein-bead
complex was washed overnight with phosphate-buffered saline at 4°C. Bacterial
chaperone proteins attached to misfolded glutathione S-transferase (GST) fu-
sion proteins were removed using a 10 mM Mg-ATP wash (according to the
Pharmacia manual instructions). Fusion proteins were eluted using 10 mM glu-
tathione in 50 mM HEPES and concentrated using a 30-kDa cutoff Amicon ultra
In vitro kinase assays. Kinase reactions with each of the GST fusion proteins
were carried out using a 30-?l volume containing 20 mM HEPES (pH 7.4), 2 mM
MnCl2, 5 mM dithiothreitol (DTT), 2 ?M cold ATP, and 8 ?Ci [?-32P]ATP at
37°C for 45 min. Reactions were stopped by adding 30 ?l of 2? sodium dodecyl
sulfate (SDS) sample buffer followed by denaturing at 70°C for 15 min. Reaction
products were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on
a 10% polyacrylamide gel. Gels were washed three times for 10 min each in a
buffer containing 1% sodium pyrophosphate and 5% trichloroacetic acid and
dried at 60°C for 2 h, and phosphorylated products were visualized by autora-
Western blot analysis. GST-SPAK fusion proteins (?5 ?g) were resolved by
SDS–10% PAGE and electroblotted onto polyvinylidene difluoride membranes.
Membranes were blocked for 2 h at room temperature with 5% nonfat dry milk
in TBST (150 mM NaCl, 10 mM Tris-HCl, 0.5% Tween 20 [polyoxyethylene-
sorbitan monolaurate]) and then incubated overnight at 4°C with either a poly-
clonal N-terminal or C-terminal anti-SPAK antibody (1:1,000) in TBST–5%
nonfat dry milk. Membranes were washed extensively in TBST, and protein
bands were visualized by enhanced chemiluminescence (ECL Plus; Amersham
Biosciences, Piscataway, NJ).
cRNA synthesis. All cDNA clones in pBF were linearized with MluI and
transcribed into cRNA by use of Ambion’s mMESSAGE mMACHINE SP6
transcription system (Ambion, Austin, TX). RNA quality was verified by gel
electrophoresis (1% agarose–0.693% formaldehyde), and RNA was quantitated
by measurement of absorbance at 260 nm.
Isolation of Xenopus laevis oocytes. Stage V to VI Xenopus laevis oocytes were
isolated from eight different frogs as previously described (44, 49) and main-
tained at 16°C in modified L15 medium (Leibovitz’s L15 solution diluted with
water to a final osmolarity of 195 to 200 mosM and supplemented with 10 mM
HEPES and 44 ?g gentamicin sulfate). Oocytes were injected on day 2 with 50
nl water containing 15 ng NKCC1 cRNA and on day 3 with 50 nl water contain-
ing 10 ng of each kinase cRNA. Control oocytes were injected with 50 nl water.
86Rb uptake determinations were performed on day 5 postisolation.
K?uptakes in Xenopus laevis oocytes. Groups of 20 oocytes in a 35-mm dish
were washed once with 3 ml isosmotic saline solution (96 mM NaCl, 4 mM KCl,
2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES buffered to pH 7.4) and preincubated
for 15 min in 1 ml of the same isosmotic saline solution containing 1 mM
ouabain. The solution was then aspirated and replaced with 1 ml isosmotic flux
solution containing 5 ?Ci86Rb. Two 5 ?l aliquots of flux solution were sampled
at the beginning of each86Rb uptake period and used as standards. After 1 h
uptake, the radioactive solution was aspirated and the oocytes were washed four
times with 3 ml ice-cold isosmotic solution. Single oocytes were transferred into
glass vials, lysed for 1 h with 200 ?l 0.25N NaOH, and neutralized with 100 ?l
glacial acetic acid, and
counting. NKCC1 flux is expressed in nanomoles K?/oocyte/hour.
Yeast two-hybrid analysis. Full-length SPAK and portions of PCR-amplified
NKCC1 were inserted in the yeast vector pGBDUc2 and transformed into
competent PJ69-4A yeast cells. Yeast cells containing SPAK or NKCC1 were
then transformed with either the regulatory domain of SPAK or full-length
WNK4 inserted in pACTII. The transformed yeast cells were plated on double
dropout (uracil and leucine dropout) plates for measuring transformation effi-
ciency and triple dropout (uracil, leucine, and histidine dropout) plates for
determining protein-protein interaction. Yeast survival was assessed after 2 to 4
days at 30°C.
Statistical analyses. Differences between86Rb uptake groups were tested by
one-way analysis of variance followed by multiple comparisons using Student-
Newman-Keuls, Bonferroni, and Tukey posttests. P ? 0.001 was considered to be
86Rb tracer activity was measured by ?-scintillation
Schematic and structural representation of SPAK. The gen-
eral features of SPAK and amino acid residues mutated in this
study are indicated schematically in Fig. 1A. The kinase con-
tains a short N-terminal sequence rich in proline and alanine
residues (PAPA box), located upstream of the catalytic do-
main. Four threonine residues (T231, T236, T243, and T247)
identified in the activation loop are located upstream of a
region containing a quartet of highly conserved hydrophobic
residues (F244, M251, P253, and L278). The representation
also identifies serine residues S321 and S383, which are targets
690GAGNON ET AL.MOL. CELL. BIOL.
of PKC? and of WNK1 and WNK4, respectively. The C-ter-
minal regulatory domain contains a caspase cleavage site and a
relatively large region that interacts with R/KFxV/I motifs in
many proteins. A wire-frame representation of the kinase do-
main of SPAK was constructed by SWISS-MODEL, based on
the crystal structures of two Ste20 kinases, PAK1 and TAO2
(Fig. 1B). The model shows the typical propeller-like structure
created by ? strands I to VII, the two hydrophobic residues
valine and leucine that sandwich the adenosine ring of ATP,
four hydrophobic residues, and threonines 243 and 247 in the
vicinity of the P?1 pocket.
Kinase activity of SPAK. Wild-type and mutant SPAK pro-
teins were all expressed as GST fusion proteins and purified as
described in Materials and Methods. After incubation with
[?-32P]ATP, SDS-PAGE, and autoradiography, wild-type
SPAK was visible as two major bands at 87 kDa and 70 kDa
(Fig. 2A). Mass spectrometry and Western blot analyses (not
shown) confirmed that the 87 kDa band represents full-length
SPAK, whereas the smaller 70 kDa band represents a trun-
cated N-terminal portion of SPAK. Wild-type SPAK autophos-
phorylation is substantial in 2 mM MnCl2but negligible in
either 2 or 5 mM MgCl2(Fig. 2B), indicating a striking (in
vitro) divalent cation specificity. Incorporation of
duced substantially with excess cold ATP but not GTP, dem-
32P is re-
onstrating that GTP cannot be used as a donor source of
phosphate (Fig. 2C). We also show that dithiothreitol concen-
trations greater than 5 mM do not enhance SPAK autophos-
phorylation (Fig. 2D) and that kinase activity is sensitive to
chloride at physiologically relevant concentrations (Fig. 2E). In
vitro autophosphorylation of SPAK is time sensitive, with max-
imal activity observed between 60 and 90 min at 37°C (Fig. 2F),
and the Kmof the kinase for ATP is relatively low, ranging
from 5 to 10 ?M (Fig. 2G).
Mutation of threonine residues within the activation loop of
SPAK prevents autophosphorylation. First, we performed se-
quential mutation of each of the four threonine residues lo-
cated between the DFG and APE motifs of the activation loop
of SPAK. We identified T243 and T247 as the key residues
necessary for SPAK autophosphorylation and transphosphory-
lation of the N terminus of NKCC1 (Fig. 3A). Single-point
mutation of each threonine residue into an alanine verified
that neither T231A nor T236A interfered with the kinase ac-
tivity of SPAK. In contrast, we found that T243A significantly
reduces both autophosphorylation and transphosphorylation
of NKCC and that mutating T247A completely inhibits all32P
incorporation, in similarity to the results seen with the kinase-
inactive SPAK (K104R) mutant (Fig. 3A). To test whether the
C-terminal regulatory domain of SPAK is involved in auto-
FIG. 1. Schematic and structural representation of SPAK. (A) The catalytic domain of SPAK with the N-terminal PAPA box, activation loop,
and P?1 hydrophobic pocket are depicted. A caspase cleavage site and a large protein-protein interaction region within the regulatory domain are
also indicated. Specific residues mutated in this study are identified. (B) SWISS-PROTEIN model of the catalytic domain of SPAK highlighting
conserved hydrophobic residues (V89, L205) which sandwich the adenosine ring of ATP; the activation loop (red backbone) with residues T243
and T247; and four residues (F244, M251, P253, L278) lining a hydrophobic P?1 pocket for substrate interaction.
VOL. 26, 2006CHARACTERIZATION OF SPAK AND OSR1 691
phosphorylation and transphosphorylation of NKCC1, we in-
troduced stop codons at the end of the catalytic domain and
within the regulatory domain, before the region of divergence
between SPAK and OSR1. We show that removal of the ex-
treme carboxyl tail containing the cotransporter interaction
domain (region “f” in Fig. 3B) and the small region of low
homology between SPAK and OSR1 (region “e” in Fig. 3B)
does not affect SPAK autophosphorylation or SPAK phos-
phorylation of NKCC1. In contrast, although removal of the
entire regulatory domain resulted in the absence of NKCC1
phosphorylation, the kinase was still able to autophosphory-
Absence of SPAK-SPAK protein interaction. The Ste20p-
like kinase MST1 contains within its regulatory domain a re-
gion identified as the site of protein-protein dimerization (21).
To assess the presence of such a region in SPAK, we trans-
formed yeast cells with full-length SPAK in pGBDUc2 and the
entire regulatory domain of SPAK in pACTII. As indicated in
Fig. 4A, there was no yeast survival when full-length SPAK was
cotransformed with the regulatory domain of SPAK, whereas
there was yeast survival with our positive-control interactions
between NKCC1 and the regulatory domain of SPAK and
full-length SPAK with full-length WNK4. These data therefore
demonstrate the absence of SPAK-SPAK interaction and sug-
gest the absence of a dimerization domain.
Autophosphorylation of SPAK is intramolecular. To deter-
mine whether SPAK autophosphorylation occurs within one
SPAK molecule (intramolecular reaction) or between separate
SPAK molecules (intermolecular reactions), we examined the
effect of protein concentration on kinase autophosphorylation.
Indeed, if autophosphorylation occurs intermolecularly, as
molecules have to interact, incorporation of ?-32P should be
dilution dependent. Our data in Fig. 4B and C show a decrease
of ?-32P incorporation proportional to protein concentration.
Although these data support the idea of an intramolecular
autophosphorylation mechanism, the range of protein concen-
trations was limited by the sensitivity of the kinase assay, leav-
ing the possibility of a dilution effect at lower protein concen-
trations. Therefore, we used a more direct approach by
combining wild-type SPAK with catalytically inactive SPAK
(K104R). To clearly identify both proteins on polyacrylamide
gel, we shortened the catalytically inactive kinase by removing
the PAPA box. When full-length active SPAK was incubated
with the shorter inactive SPAK, the active kinase was unable to
phosphorylate the inactive kinase, demonstrating that auto-
phosphorylation of SPAK occurs intramolecularly (Fig. 4D).
We also shortened the catalytically active kinase to confirm
that absence of the PAPA box did not affect SPAK autophos-
phorylation or transphosphorylation of GST-NKCC1 (Fig. 4D,
fourth lane). Furthermore, coexpression of the shortened cat-
alytically active kinase in Xenopus laevis oocytes confirmed that
the absence of the PAPA box did not affect cotransporter
stimulation in the presence of WNK4 (see Fig. 6B).
FIG. 2. Optimization of kinase activity of SPAK. (A) Autoradiography of a 10% SDS-PAGE gel showing autophosphorylated SPAK at both
87 and 70 kDa. (B) Autophosphorylation of SPAK in the presence of MnCl2and MgCl2. (C) Effect of addition of cold Mg-ATP and Mg-GTP on
SPAK32P incorporation. (D to E) Effect of increasing DTT concentrations (1 to 20 mM) and chloride concentrations (4 to 40 mM) on SPAK
autophosphorylation. (F) Time course of SPAK autophosphorylation as measured at 37°C. (G)32P incorporation as a function of ATP concen-
trations. The Kmvalues for ATP were measured at 5 ?M and 8.3 ?M in two separate experiments.
692GAGNON ET AL.MOL. CELL. BIOL.
Kinase activity of OSR1. As previously mentioned, OSR1 is
the protein most closely related to SPAK, sharing 66% identity
at the amino acid level. To determine whether this kinase
possesses properties similar to those of SPAK, we performed
in vitro phosphorylation experiments using a GST-OSR1 fu-
sion protein and the reaction conditions already established for
SPAK (Fig. 5A). In similarity to SPAK results, OSR1 auto-
phosphorylation was substantial in 2 and 5 mM MnCl2and
negligible in either 2 or 5 mM MgCl2(Fig. 5B) and displayed
some sensitivity to high concentrations of chloride (Fig. 5C).
We determined the Kmfor ATP to be around 1 ?M (Fig. 5D).
Activation of NKCC1 by the SPAK-WNK4 complex requires
SPAK activation loop threonine phosphorylation. In a previ-
ous report, we demonstrated isotonic activation of NKCC1 by
coexpressing SPAK and WNK4 in NKCC1-injected Xenopus
laevis oocytes (18). In the next set of experiments, we tested
whether mutation of any of the threonine residues within the
activation loop (T231A, T236A, T243A, and T247A) affected
the capacity of SPAK to activate the cotransporter. We show
that coexpression of WNK4 with either SPAK (T231A) or
SPAK (T236A) does not significantly affect the stimulation of
cotransporter activity (Fig. 6). However, coexpression of
WNK4 with SPAK (T243A) or SPAK (T247A) in NKCC1-
injected Xenopus laevis oocytes completely inhibits NKCC1
activity, consistent with an absence of in vitro32P incorporation
Activation of NKCC1 by OSR1-WNK4. Consistent with the
in vitro phosphorylation of NKCC1 by OSR1, coexpression of
OSR1 together with WNK4 in Xenopus laevis oocytes results in
a significant stimulation of cotransporter activity (Fig. 6), rem-
iniscent of the stimulation observed with SPAK. Expression of
OSR1 alone in NKCC1-injected oocytes did not stimulate co-
transporter activity above basal levels (data not shown).
Inhibitors and activators of cation-chloride cotransporters.
Staurosporine and K252A have been shown to reduce NKCC1
activity and to prevent NKCC1 activation in a variety of cells
(17, 33, 37, 40, 42). To determine whether these inhibitors
exert their influence on the cotransporter by modulating SPAK
transphosphorylation of the N-terminal tail of NKCC1 in the
presence of 0.1 ?M to 100 ?M staurosporine and K252a. As
shown in Fig. 7,32P incorporation decreased with increasing
concentrations of both kinase inhibitors. Note that the concen-
tration required for half activity ranges from 0.1 to 1 ?M for
staurosporine and 1 to 10 ?M for K252a. N-Ethylmaleimide
(NEM) markedly activates the activity of K-Cl cotransport (29)
while inactivating the Na-K-2Cl cotransporter (37). Although
NEM is a rather nonspecific sufhydral reagent, its effect on
cation-chloride cotransporter activity likely results from pro-
tein kinase inhibition (9, 34). We tested the effect of NEM, and
the sulfhydral oxidant diamide, on SPAK activity also using in
vitro phosphorylation experiments. As shown in Fig. 7, the two
thiol-reacting compounds had minimal impact on SPAK auto-
phosphorylation. However, at concentrations higher than 100
?M, NEM and diamide decreased SPAK phosphorylation of
NKCC1. In ferret red cells, arsenite has been shown to cause
marked stimulation of Na-K-2Cl cotransport (17), potentially
linking cotransporter activation to the involvement of stress-
activated protein kinases (27). We examined the role of arsen-
ite on SPAK autophosphorylation and SPAK phosphorylation
of NKCC1 and found no effect in the range of 1 to 1,000 ?M.
Finally, we show that H2O2, a strongly oxidizing reagent, re-
duces SPAK autophosphorylation and SPAK phosphorylation
of the N-terminal tail of NKCC1 in the mM range (0.03%
[wt/wt] or ?9 mM).
We have recently shown that SPAK and OSR1, two Ste20p-
like serine-threonine kinases, interact with cation-chloride co-
transporters, including the Na-K-2Cl cotransporter, NKCC1
(45). We later showed that WNK4, a kinase involved in
pseudohypoaldosteronism (55), also interacts with SPAK
through a RFQV binding motif (44) and that this interaction
between the two kinases results in significant activation of
NKCC1 under isosmotic conditions (18). Because the catalytic
activity of both kinases is required for NKCC1 activation and
because, in contrast to SPAK, WNK4 failed to interact directly
with the cotransporter, we proposed a hierarchy of events in
which WNK4 activates SPAK, which in turn phosphorylates
NKCC1 (18). Consistent with this idea, Vitari and coworkers
have recently presented direct evidence that WNK1 and
WNK4 phosphorylate and activate SPAK and OSR1 (54).
FIG. 3. In vitro phosphorylation studies of SPAK with activation
loop and deletion mutants. (A) Wild-type SPAK along with nine mu-
tants was combined with the N-terminal region of NKCC1. To evi-
dence equal protein loading, semiquantitative determination of the 87
kDa SPAK band was obtained using Western blot analysis with a
specific N-terminal SPAK antibody. (B) Schematic representation of
wild-type and two truncated forms of SPAK. Segment “b” represents
the proline-and alanine-rich region (PAPA box), “c” the catalytic do-
main, “d” the first segment of the C-terminal domain with high ho-
mology to OSR1, “e” the region with low homology to OSR1, and “f”
the cation-chloride cotransporter interaction domain. The amino acid
length of each construct is indicated. The results of an in vitro kinase
assay showing autophosphorylation of truncated SPAK mutants and
the absence of NKCC1 phosphorylation when segment “d” is missing
VOL. 26, 2006CHARACTERIZATION OF SPAK AND OSR1 693
Activation of many protein kinases requires phosphorylation
of the activation segment or loop between two highly con-
served tripeptide motifs (DFG. . . .APE) (38). Crystallographic
models of phosphorylated and nonphosphorylated protein ki-
nases have demonstrated large movements in their activation
loop, allowing better substrate access to the active site, sug-
gesting that the activation loop may function as a “door” to the
substrate pocket (25). On the basis of the high degree of
conservation between SPAK and this particular group of pro-
tein kinases, we speculated that phosphorylation of threonine
residues located in the activation loop might be critical in the
activation of the kinase. Our experiments show that mutating
FIG. 4. Autophosphorylation of SPAK is intramolecular. (A) Yeast two-hybrid analysis of SPAK-SPAK interaction showing no yeast survival.
Positive controls are SPAK-NKCC1 and SPAK-WNK4 interactions. (B) Level of32P incorporation of SPAK decreases (upper panel) with the
amount of protein present, as determined by Western blot analysis (lower panel). (C) Linear relationship between32P incorporation and protein
concentration as determined by a Bradford assay. (D) In vitro phosphorylation experiment mixing active and inactive forms of SPAK. Western blot
analysis using a C-terminal SPAK antibody confirmed the presence of each of the SPAK proteins. Active (wild-type) or inactive (nonfunctional
K104R mutant) forms of SPAK are indicated by the letters “a” and “i,” respectively.
FIG. 5. Characterization of OSR1 kinase activity. (A) Presence of
autophosphorylated OSR1 at 77 and 60 kDa. (B) OSR1 autophosphor-
ylation in the presence 2 and 5 mM MnCl2and MgCl2. (C) Effect of
increasing Cl?concentrations (4 to 40 mM) on32P incorporation by
the kinase. (D) OSR1 autophosphorylation as a function of ATP
concentration. The Kmwas measured at ?1 ?M.
FIG. 6. NKCC1 function requires SPAK activation via loop threo-
nine phosphorylation. (A) Functional analysis of SPAK mutants
through86Rb uptake in Xenopus laevis oocytes. NKCC1 RNA (15 ng)
was injected on day 2, and WNK4 and SPAK wild type and mutants (10
ng) were injected on day 3.86Rb uptake was measured on day 5 in an
isosmotic solution containing 1 mM ouabain. (B) Expression of WNK4
with full-length SPAK, shortened SPAK (-PAPA box), and OSR1 in
NKCC1-injected Xenopus laevis oocytes. Bars represent means ? stan-
dard errors of the means (n ? 20 oocytes). The experiment was
repeated twice with identical results.
694GAGNON ET AL.MOL. CELL. BIOL.
residues T243 or T247 into alanines greatly impacted kinase
activity, as determined through autophosphorylation and
transphosphorylation of the N-terminal tail of NKCC1,
whereas alanine substitution of T231 and T236 remained silent
(Fig. 3). In parallel to these in vitro phosphorylation experi-
ments, we performed functional experiments with the threo-
nine mutants in Xenopus laevis oocytes and confirmed the
requirement of T243 and T247 for NKCC1 activation (Fig. 6).
Substrate recognition of many protein kinases depends, at
least partly, on residues flanking the specific site of substrate
phosphorylation (or “P site”) with residues N terminal to the P
site numbered P?1, P?2, and P?3 and residues C terminal to
the P sites numbered P?1, P?2, P?3, etc. (1). In the case of
the Ste20p-like protein kinase TAO2, it was shown that its
physiological substrates MEK3 and MEK6 both possess hydro-
phobic residues immediately following the P site (P?1) in both
of their two phosphorylation sites (58). For NKCC1, one of the
physiological substrates of SPAK, Darman and Forbush have
identified the principal P site of shark NKCC1 as T189 (11).
Consistent with observations of MEK3 and MEK6, this site is
directly followed by a hydrophobic methionine residue. Amino
acid sequence alignment of the catalytic domain of mammalian
Ste20p-like kinases reveals conservation of a quartet of hydro-
phobic residues forming a pocket that interacts with the P?1
site. As seen in Fig. 1A, the hydrophobic residues lining the
P?1 pocket in SPAK are F244, M251, P253, and L278. Further
analysis of the activation loop of SPAK reveals that residues
T243 and T247, newly defined as critical for SPAK activity, are
themselves directly followed by hydrophobic residues, phenyl-
alanine following T243 and a proline residue coming after
T247, whereas T231 and T236 are followed by nonhydrophobic
glycine and arginine residues, respectively. This observation
suggests that T243 and T247 might be phosphorylated in a
manner similar to the phosphorylation of Ste20p substrates.
Interestingly, Vitari et al. identified T243 as a target of WNK1
and WNK4 (54), and both kinases possess the conserved quar-
tet of hydrophobic residues: V383, M390, P392, and M415 for
mouse WNK1 and V333, M340, P342, and P365 for mouse
WNK4. However, as our in vitro phosphorylation experiments
showed that SPAK itself can incorporate phosphates on T243
and T247 (autophosphorylation), whether or not the P?1
pocket is involved is currently unknown. On the basis of the
presence of a dimerization region in the regulatory domain of
MST1, a Ste20p-like kinase involved in apoptosis, Glantschnig
et al. (21) argued that dimerization promotes phosphorylation
between individual MST1 molecules. They indeed demon-
strated intermolecular phosphorylation by using kinase-active
and -inactive forms of MST1. Our yeast two-hybrid data indi-
cate the absence of SPAK-SPAK interaction, making it there-
fore unlikely that SPAK possesses a dimerization domain. This
observation is in agreement with the absence of any multim-
erization products on Western blots (44). Because of the ab-
sence of SPAK-SPAK interaction, we thought it was important
to ask whether, in the absence of dimerization, one could see
intermolecular phosphorylation. Our experiments clearly indi-
cate that autophosphorylation occurs intramolecularly, as the
ability of SPAK to autophosphorylate appears dilution inde-
pendent (Fig. 4B and C) and as active SPAK is unable to
phosphorylate catalytically inactive SPAK (Fig. 4D).
An interesting observation was that Mn2?, but not Mg2?,
was very effective as a cofactor in our in vitro phosphorylation
experiments. This particular divalent metal ion specificity has
been reported for a few kinases (2, 39, 43, 57) and nucleotide
binding proteins (56). Whether or not the same specificity
exists in vivo remains to be determined. Slightly different con-
formational changes of the SPAK active site might be gener-
ated depending upon the nature of the divalent metal coordi-
nated to the polyphosphate region of the nucleotide. Indeed,
X-ray crystallography of CheA showed that the active site of
this histidine kinase is greatly influenced by the divalent metal
ion bound to the nucleotide, with Mg2?enabling a more ex-
tensive conformational change than Mn2?(3). Stronger auto-
phosphorylation with Mn2?in our in vitro experiments might
indicate a more open or relaxed conformation of SPAK. In
some cases, Mn2?has also been shown to facilitate the use of
GTP as a phosphoryl group donor (22). This is clearly not the
case with SPAK, where GTP cannot substitute for ATP (Fig.
2). Since Mg2?is more physiologically relevant, it is possible
that in vivo, autophosphorylation of the threonines in the ac-
tivation loop might be minimal, allowing for phosphorylation
by other upstream kinases such as WNK1 and/or WNK4.
Experiments performed using internally dialyzed squid giant
axons in the late 1970s revealed that Na-K-2Cl cotransport is
inhibited by high intracellular Cl?concentrations. This effect,
reproduced in a variety of other cell types, is not related to the
ion driving force, since increased intracellular Cl?concentra-
FIG. 7. Pharmacological inhibition of SPAK autophosphorylation.
All in vitro phosphorylation reaction mixtures contained identical
amounts of SPAK, NKCC1, MnCl2, ATP, [?-32P]ATP, DTT, and Na-
HEPES but increasing concentrations of pharmacological agents. Con-
centrations of staurosporine and K252a ranged from 0.1 to 100 ?M.
Concentrations of NEM, diamide, and arsenite ranged from 1 ?M to
1 mM. Hydrogen peroxide was obtained as a 3% solution. This corre-
sponds to a concentration of ?900 mM H2O2. Reactions were loaded
on 10% acrylamide gels. Gels were washed, dried, and exposed to
autoradiography. The panels are representative of two to three inde-
VOL. 26, 2006CHARACTERIZATION OF SPAK AND OSR1695
tions also inhibit Na-K-2Cl cotransport-mediated efflux. Of
interest is the demonstration that NKCC1 phosphorylation
increases with decreases in Cl?concentrations (for a review,
see reference 46). We examined the Cl sensitivity of both
SPAK and OSR1 phosphorylation and found an inhibitory
effect in the physiological range (4 to 40 mM). Whether or not
the effect of Cl?concentrations on SPAK autophosphorylation
is large enough to account for the exquisite sensitivity of
NKCC1 to internal Cl?concentrations in vivo remains to be
Yeast two-hybrid experiments have previously demonstrated
that OSR1, a kinase closely related to SPAK, interacts with the
K-Cl cotransporter (KCC3) (44). Immunofluorescence studies
with polyclonal antibodies have also demonstrated colocaliza-
tion of OSR1 and NKCC1 (35). It is therefore not surprising
that OSR1, like SPAK, when coexpressed with WNK4 acti-
vates NKCC1 in Xenopus laevis oocytes. Consistent with these
results, our in vitro phosphorylation experiments indicate that
OSR1 exhibits many of the same kinetic properties as SPAK.
Previous in vivo studies examining OSR1 phosphorylation have
shown an increase in kinase activity when the cells were incu-
bated with sorbitol (8). This hyperosmotic activation of OSR1
is consistent with an increase in cotransporter activity under
similar conditions, a process we showed to be at least partially
related to SPAK (18). Taken together, these results indicate
that the shared homology between OSR1 and SPAK is suffi-
cient for either kinase to serve as a modulator of NKCC1
Over the past 25 years, a variety of inhibitors and activators
of both cotransporters have been identified. These pharmaco-
logical interventions generally produce opposite effects and
therefore are thought to act on the kinases and/or phospha-
tases which regulate cotransporter activity. Here, we had the
possibility of testing the direct effect of several of these agents
on both SPAK autophosphorylation and substrate phosphory-
lation of NKCC1. Staurosporine is a potent inhibitor of the
Na-K-2Cl cotransporter (17, 24, 37, 53) and an activator of the
K-Cl cotransporter (5, 16, 48). We report here that staurospor-
ine inhibits SPAK activity by ?50% at drug concentrations
between 0.1 and 1 ?M, consistent with a measured 50% inhib-
itory concentration of 0.7 ?M in avian erythrocytes (33).
K252a, another protein kinase inhibitor preventing Na-K-2Cl
cotransport stimulation in a variety of cells (40, 42), also in-
hibited SPAK activity, although requiring slightly higher con-
centrations than staurosporine. In contrast, arsenite, which
stimulates stress-activated protein kinases (27) and MAPKs
(32) and markedly activates Na-K-2Cl cotransport in ferret red
cells (17), had little effect on SPAK autophosphorylation. This
seems to indicate that the arsenite effect in ferret erythrocytes
is not directly associated with the kinase that phosphorylates
We also demonstrated a direct inhibitory effect of hydrogen
peroxide on SPAK autophosphorylation as well as transphos-
phorylation of NKCC1. Hydrogen peroxide (H2O2) has been
shown to stimulate K-Cl cotransport (4, 6, 41), although the
oxidant was believed to act through a phosphatase rather than
a volume-sensitive kinase, as calyculin substantially inhibited
the H2O2activation (4). Although there are no reports of a
H2O2effect on Na-K-2Cl cotransport, tert-butyl hydroperoxide
has been shown to have an inhibitory effect on the “regulatory”
kinase (15, 47). Thus, our data showing SPAK inhibition by
H2O2are consistent with oxidative reagents inhibiting K-Cl
cotransport and activating Na-K-2Cl cotransport. K-Cl co-
transport was first defined as a mechanism promoting volume-
induced (14) and NEM-induced (29) Cl?-dependent K?flux.
First, it was proposed that the alkylating reagent reacts with
thiol groups located on the transport molecule (for a review,
see reference 28). However, in light of the fact that NEM also
inhibits Na-K-2Cl cotransport, consensus has moved toward
the idea that NEM acts as a protein kinase inhibitor, antago-
nizing the effect of calyculin A (e.g., see reference 37). The
concentration of NEM (1 mM) required to affect K-Cl cotrans-
port in native red cells (29), rabbit KCC1 heterologously ex-
pressed in HEK293 cells (20), and Na-K-2Cl cotransport in
native red cells (37) is relatively high. We showed that 1 mM
NEM minimally affects SPAK autophosphorylation but signif-
icantly decreases the level of NKCC1 phosphorylation. This
observation suggests that despite SPAK autophosphorylation
of the activation loop threonine residues, NEM still prevents
kinase-substrate interaction, cotransporter phosphorylation,
and, ultimately, functional activation. Interestingly, the sulfhy-
dryl oxidant, diamide, produced similar effects on SPAK auto-
phosphorylation and NKCC1 transphosphorylation, further
evidence that the effector sites of these agents lie between
SPAK and the cotransporters.
The effect of NEM is intriguing, since it clearly delineates
two separate events: SPAK autophosphorylation and kinase
phosphorylation of the cotransporter. Data obtained with our
deletion mutants (Fig. 3B) also demonstrate that kinase auto-
phosphorylation by itself is not sufficient but that a portion of
the regulatory domain proximal to the catalytic domain is nec-
essary for substrate phosphorylation. The fact that WNK1 and
WNK4 phosphorylate SPAK on residue S383 (located within
this proximal region of the regulatory domain) indicates that
access of the substrate to the kinase domain might depend on
some conformation changes triggered by phosphorylation of
the C terminus.
In summary, our results demonstrate that SPAK and OSR1
are kinases regulated through activation segment phosphory-
lation. We found that two of the four threonine residues (T243
and T247) within the activation loop were critical for SPAK
autophosphorylation, and subsequent substrate phosphoryla-
tion and activation of NKCC1. Through mutagenesis studies,
we determined that in vitro phosphorylation of these key thre-
onine residues occurs by intramolecular autophosphorylation.
We also found that although truncation of the regulatory do-
main allowed SPAK autophosphorylation, a proximal portion
of the regulatory domain (?70 amino acids) was necessary for
NKCC1 phosphorylation. Finally, we demonstrate that phar-
macological inhibitors of NKCC1, i.e., staurosporine and
K252a, directly affect SPAK autophosphorylation and sub-
strate phosphorylation of the cotransporter. Taken together,
these results clearly indicate that SPAK (and OSR1) likely
represent two of the kinases which phosphorylate and activate
This work was supported by National Institutes of Health grant
NS36758 and by a grant-in-aid from the American Heart Association
696GAGNON ET AL.MOL. CELL. BIOL.
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