Glycogen synthase kinase 3ß functions as a positive effector in the WNK signaling pathway
Glycogen synthase kinase 3û functions as a positive effector in the WNK signaling pathway
Atsushi Sato 0 1 2
Hiroshi Shibuya 0 1 2
0 Department of Molecular Cell Biology and Joint Usage/Research Center for Intractable Diseases, Medical Research Institute, Tokyo Medical and Dental University (TMDU) , Bunkyo-ku, Tokyo , Japan
1 Aid for Scientific Research from the Ministry of Education , Culture, Sports , Science and Technology of Japan , 26460386, to Dr. Atsushi Sato; and Nanken-Kyoten, TMDU, 17M03, to Prof. Hiroshi Shibuya
2 Editor: Renping Zhou, Rutgers University , UNITED STATES
The with no lysine (WNK) protein kinase family is conserved among many species. Some mutations in human WNK gene are associated with pseudohypoaldosteronism type II, a form of hypertension, and hereditary sensory and autonomic neuropathy type 2A. In kidney, WNK regulates the activity of STE20/SPS1-related, proline alanine-rich kinase and/or oxidative-stress responsive 1, which in turn regulate ion co-transporters. The misregulation of this pathway is involved in the pathogenesis of pseudohypoaldosteronism type II. In the neural system, WNK is involved in the specification of the cholinergic neuron, but the pathogenesis of hereditary sensory and autonomic neuropathy type 2A is still unknown. To better understand the WNK pathway, we isolated WNK-associated genes using Drosophila. We identified Glycogen synthase kinase 3û (GSK3û)/Shaggy (Sgg) as a candidate gene that was shown to interact with the WNK signaling pathway in both Drosophila and mammalian cells. Furthermore, GSK3û was involved in neural specification downstream of WNK. These results suggest that GSK3û/Sgg functions as a positive effector in the WNK signaling pathway.
Data Availability Statement; All relevant data are within the paper
The with no lysine (WNK) protein kinases are atypical members of the serine/threonine kinase
family, and are conserved among many species [1±3]. The mammalian WNK family has four
members: WNK1±4. WNK1 and WNK4 have been identified as causative genes of
pseudohypoaldosteronism type II (PHAII) [
], and WNK1 is also a causative gene of hereditary sensory
and autonomic neuropathy type 2A (HSAN2A) [
]. Several groups including ours have
attempted to identify the functions of the WNK family. In the kidney, WNK1 and WNK4
phosphorylate and activate STE20/SPS1-related, proline alanine-rich kinase (SPAK) and
oxidative-stress responsive 1 (OSR1) kinases, which in turn regulate various ion co-transporters
[6±9]. Because knock-in mice of Wnk4D561A (the mutation found in PHAII patients) display
similar phenotypes to PHAII, dysregulation of this WNK signaling pathway was thought to
cause hypertension in PHAII patients [
]. In the neural system, a neural-specific alternatively
spliced isoform of WNK1 is expressed, which includes the neural-specific exon HSN2. In
HSAN2A patients, mutations were found in this HSN2 exon [
], but HSN2 knock-out mice
have no discernable morphological phenotype [
]. Furthermore, in other familial
HSAN2A patients, mutations were found outside the HSN2 exon in WNK1 [
]. Thus, the
pathogenesis of HSAN2A remains unclear.
WNK kinases are required for EGF-mediated ERK5 activation, and WNK family members
are also involved in proliferation, migration, and differentiation [14±16]. Recently, we found
that WNK1 and WNK4 induced Lhx8 expression and were important for neural specification
]. Moreover, WNK was identified as a positive regulator of the Wnt signaling pathway;
however, the detailed mechanisms of this are unknown [
]. Although WNK has a range of
functions during many developmental processes, little is known about the components of the
WNK signaling pathway, except for the main molecules WNK1/4±SPAK/OSR1. In the kidney,
ASK3 inhibits WNK1 [
], and the PI3K/AKT signaling pathway activates the WNK±SPAK/
OSR1±NCC pathway [
]. Other upstream or downstream component(s) are still unknown.
Glycogen synthase kinase 3û (GSK3û) is a ubiquitously expressed serine/threonine kinase
that was originally identified as the regulatory kinase of glycogen synthase. Since then, GSK3û
has been shown to be involved in many biological processes [
]. GSK3û plays important roles
in several signaling pathways, especially PI3K/AKT and Wnt signaling pathways. In the PI3K/
AKT signaling pathway, AKT phosphorylates Ser9 of GSK3û which inhibits its activity, thus
phosphorylating cyclin D1 and regulating the cell cycle [
]. In the Wnt signaling pathway,
GSK3û is a major component of the destruction complex that phosphorylates û-catenin,
which in turn is degraded by proteasomes [
In this study, we attempted to identify a new component of the WNK signaling pathway
using Drosophila, and identified the shaggy gene (sgg) as a possible candidate. Sgg is a
Drosophila homolog of mammalian GSK3û. We found that GSK3û worked as a positive effector
downstream of WNK in both mammalian and Drosophila cells.
Materials and methods
All the animal experiments were performed under the ethical guidelines of Tokyo Medical and
Dental University, and animal protocols were reviewed and approved by the animal welfare
committee of the Tokyo Medical and Dental University.
Fly stocks and genetics
Fly strains used in this study were; Canton-S, y w, EY10165 (UAS-Wnk; Bloomington Stock
Center #16970), UAS-frayS347D [
], WnkEY18 FRT2A [
], Akt104226 (Bloomington Stock
Center #11627), sgg1 (Bloomington Stock Center #9095), sggM11 (Bloomington Stock Center
#31308), UAS-sgg (Bloomington Stock Center #5435), hh-Gal4 (Bloomington Stock Center
#67046), arm-Gal4 (Bloomington Stock Center #1560), hsGFP hsCD2(y+) M(3)i55 Tub>Gal80
FRT2A (provided by G. Struhl).
We made hh-Gal4 EY10165 recombinant flies for screening. We crossed these flies with the
fray mutant and confirmed the suppression as described previously [
]. For initial screening, we
crossed several mutants and isolated candidate suppressor genes (data not shown), including sgg.
Histology and staining
All wings were mounted in GMM [
]. Images were obtained using SteREO Discovery and
Axioscope microscopes (Carl Zeiss), and were processed using Axiovision with extended focus
(Carl Zeiss) and Photoshop (Adobe).
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Cultured cell lines
Neuro2A cells [
] were grown in DMEM with 20% FBS. Polyethylenimine (Polysciences) was
used to transfect plasmids, and the TransIT-X2 Dynamic Delivery System (Mirus Bio) was
used to transfect small interfering (si)RNA and co-transfect siRNA and plasmids. The
plasmids we used are: pRK5-Flag-hWNK1, pRK5-Flag-hWNK1D368A, pRK5-Flag-OSR1,
pRK5T7-OSR1, pRK5-Flag-OSR1K46M, pRK5-T7-OSR1K46M, pRK5-Flag-OSR1S325D,
pcDNA-FlagGSK3û, pRK5-Flag-GSK3û, pRK5-Flag-GSK3ûK85M. siRNA target sequences were described
], and were as follows: mouse Osr1, 50-GAUAUUCGAUUUGAAUUUA-30; and
mouse GSK3ß, 50-GAAAUGAACCCAAAUUAUA -30. To differentiate Neuro2A cells, they were
induced in serum-free DMEM with 10 μM retinoic acid for 24 h.
Neruo2A cells were transfected with indicated expression vectors. Then, lysates were prepared
from transfected cells, and immunoprecipitated with indicated antibodies and Protein A/G
PLUS-agarose (Santa Cruz). Immunoprecipitates were subjected to SDS-PAGE and western
blotting, and bands were detected by the LAS-4000 mini (GE) image analyzer. For sequential
immunoprecipitation, the anti-Flag antibody (M2, Sigma) was used and eluted with Flag
peptides (Sigma). Eluates were divided and immunoprecipitated with anti-T7 antibodies and
control mouse normal IgG.
Total RNA was isolated by TRIzol (Invitrogen). cDNA synthesis was carried out using
Moloney murine leukemia virus reverse transcriptase (Invitrogen). GAPDH was used for the
normalization of cDNA samples. Primer pairs were described previously [
], and were as
follows: mouse GSK3ß, 50-GCAGCAAGGTAACCACAGTAGTGGC-30 and 50-TGGTGCCCT
In vitro kinase assay
Neuro2A cells were transfected with Flag-WNK1, Flag-WNK1D368A, Flag-OSR1,
FlagOSR1K46M, or Flag-OSR1S325D expression plasmids. Lysates were prepared from transfected
cells and immunoprecipitated with an anti-Flag M2 antibody (Sigma) and Protein A/G
PLUSagarose (Santa Cruz). Immunoprecipitates were incubated with bacterially-expressed GST
fusion proteins (GST-GSK3û K85M) in kinase buffer containing 10 mM HEPES (pH 7.4), 1
mM DTT, 5 mM MgCl2, and 5 μCi [γ-32P]-ATP at 30ÊC. Phosphorylated substrates were
subjected to SDS-PAGE, and bands were detected by the FLA3000 image analyzer (Fujifilm).
Antibodies used in this report were: mouse anti-Flag M2 (Sigma; 1:400 for
immunoprecipitation), rabbit anti-Flag (Sigma; 1:1000 for western blotting), rat anti-HA (Roche; 1:1000 for
western blotting), mouse anti-T7 (Merck; 1:2000 for immunoprecipitation), rabbit anti-T7
(MBL; 1:1000 for western blotting), anti-rabbit HRP-conjugated (GE; 1:10000 for western
blotting), and anti-rat HRP-conjugated (GE; 1:10000 for western blotting).
Quantification and statistical analysis
Quantitative PCR was performed with an Applied Biosystems 7300 Real-Time PCR Cycler
(ABI) using THUNDERBIRD SYBR qPCR Mix (TOYOBO). Primer sequences for Lhx8, ChAT,
Gad1, and GAPDH were described previously [
]. GAPDH was used for the normalization of
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cDNA samples. Neurite lengths were measured using ImageJ software (NIH). Data were
computed using Microsoft Excel (Microsoft) and StatPlus (AnalystSoft). Values and error bars
represent the means and SDs, and are representative of at least three independent experiments.
Shaggy is a novel candidate effector of the WNK signaling pathway
Overexpression of Drosophila WNK using hh-Gal4 driver resulted in an ectopic vein around
vein 5 in the adult wing (Fig 1B compared with Fig 1A; [
]). As we showed previously, a
heterozygous mutation of fray, which encodes a Drosophila homolog of SPAK/OSR1 that is a
downstream effector of WNK, suppressed this phenotype [
]. Therefore, we performed
screening to identify a new effector of the WNK signaling pathway using this system. We
selected several mutants known to be components of other signaling pathways, such as the
Wnt pathway, Notch pathway, TGFû pathway, and the EGF pathway. We obtained several
candidate suppressor genes, including sgg, which encodes the Drosophila homolog of
mammalian GSK3û whereas some genes, including Akt1, did not show any interaction (Fig 1C and
data not shown). Two independent sgg mutants (sgg1 and sggM11) suppressed the wing
phenotypes by the overexpression of Wnk (Fig 1D and 1E), suggesting that sgg is a suppressor of the
WNK signaling pathway and that this suppression is not an effect of the genetic background.
We further tested the interaction between sgg and fray. Ectopic expression of frayS347D (the
constitutively active form of fray) resulted in a similar phenotype to that seen following the
ectopic expression of Wnk (Fig 1F; [
]). The sgg1 mutant also repressed these phenotypes (Fig
1G) suggesting that sgg interacts with the WNK signaling pathway in Drosophila.
We next confirmed this genetic interaction between Wnk and sgg. Because WnkEY18 mutant
clones led to abdominal developmental defects (Fig 1H; [
]), we attempted to rescue this
phenotype by the overexpression of sgg. Using a combination of the FLP/FRT mosaic system and
Gal80 suppression, we induced the local expression of sgg in WnkEY18 minute clones. As
shown in Fig 1I, sgg overexpression partially rescued the abdominal phenotype of the WnkEY18
minute clones. These results suggest that sgg is a novel effector of the WNK signaling pathway,
not only in wing development but also in abdominal development.
GSK3û functions as a positive effector downstream of WNK
Because GSK3ß and the WNK pathway are highly conserved among many species [
2, 3, 22
we next examined whether the interaction between WNK and GSK3û was also conserved in
mammalian cells. In Neuro2A cells, WNK1 expression induced the expression of Lhx8 ([
see also Fig 2B lane 2). As shown in Fig 2, the expression of GSK3û also induced the expression
of Lhx8 in Neuro2A cells (Fig 2A lane 2). However, the kinase dead form of GSK3û (GSK3û
K85M) could not activate the expression of Lhx8, suggesting that GSK3û kinase activity is
required for its expression (Fig 2A lane 3).
Next, we examined the epistatic interaction between WNK1 and GSK3û. The induction of
Lhx8 was suppressed by the knockdown of GSK3ß (Fig 2B lane 4). However, Lhx8 induction
by GSK3û was not suppressed by the knockdown of both Wnk1 and Wnk4 (Fig 2C lanes 5 and
6), even though this knockdown did suppress Lhx8 induction by retinoic acid (RA) stimulation
(Fig 2C lanes 3 and 4).
The expression of OSR1, a downstream molecule of WNK, and its constitutively active
form (OSR1S325D) induced the expression of Lhx8 (Fig 2D lanes 3 and 5; [
]). This activation
was also suppressed by the knockdown of GSK3ß (Fig 2D lanes 4 and 6). In contrast, the
induction of Lhx8 by GSK3û was not suppressed by the knockdown of Osr1 (Fig 2E lanes 5 and 6),
although the induction of Lhx8 by WNK1 was suppressed by the knockdown of Osr1 (Fig 2E
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Fig 1. sgg is downstream of Wnk in the Drosophila wing vein and abdominal patterning. (A) Wild-type wing. (B)
Wing from EY10165 (UAS-Wnk) fly driven by hh-Gal4. Additional veins around vein 5 (arrowhead) were observed.
(C) Wing from fly overexpressing Wnk driven by hh-Gal4 with the Akt104226 heterozygous mutant. (D) Wing from fly
overexpressing Wnk driven by hh-Gal4 with the sgg1 heterozygous mutant. (E) Wing from fly overexpressing Wnk
driven by hh-Gal4 with the sggM11 heterozygous mutant. (F) Wing from UAS-frayS347D fly driven by hh-Gal4.
Additional veins around vein 5 (arrowhead) were observed. (G) Wing from fly overexpressing frayS347D driven by
hhGal4 with the sgg1 heterozygous mutant. (H) Abdomen from adult fly with DWNKEY18 minute clones. Thin black lines
indicate the clone border. (I) Abdomen from adult fly with DWNKEY18 minute clones and sgg overexpression. sgg was
expressed only in DWNKEY18 minute clones using the Gal80 suppression technique. Thin black lines indicate the clone
border (also the sgg expression area). Black arrowheads show rescued abdominal bristles. The detailed genotype is y w
hsflp; arm-Gal4 / UAS-sgg; WnkEY18 FRT2A / hsGFP hsCD2(y+) M(3)i55 Tub>Gal80 FRT2A. The numbers of wings or
abdomina showing the phenotypes and of total observed wings or abdomina are indicated.
lanes 3 and 4). These data suggest that the WNK±OSR1±GSK3û pathway is conserved not
only in flies but also in mammals, and that GSK3û functions as a positive effector downstream
of the WNK signaling pathway.
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Fig 2. GSK3û is a positive effector downstream of WNK-OSR1. (A) Gene expression determined by RT-PCR or
quantitative RT-PCR analysis was examined in Neuro2A cells overexpressing GSK3û or GSK3û K85M. The value
obtained from each sample was normalized to that of GAPDH. The value of Lhx8 from overexpressing GSK3û (lane 2)
was set to 100. (B) Gene expression detemined by RT-PCR or quantitative RT-PCR analysis was examined in Neuro2A
cells overexpressing hWNK1 under GSK3ß knockdown using siRNA. The value obtained from each sample was
normalized to that GAPDH. The value of Lhx8 from overexpressing hWNK1 (lane 2) was set to 100. (C) Gene
expression determined by RT-PCR or quantitative RT-PCR analysis was examined in Neuro2A cells stimulated by
retinoic acid (RA) or overexpressing GSK3û under both Wnk1 and Wnk4 knockdown using siRNA. The value
obtained from each sample was normalized to that of GAPDH. The value of Lhx8 from overexpressing GSK3û (lane 3)
was set to 100. (D) Gene expression determined by RT-PCR or quantitative RT-PCR analysis was examined in
Neuro2A cells overexpressing OSR1 or OSR1S324D (constitutively active form of OSR1) under GSK3ß knockdown
using siRNA. The value obtained from each sample was normalized to that of GAPDH. The value of Lhx8 from
overexpressing OSR1 (lane 3) was set to 100. (E) Gene expression determined by RT-PCR or quantitative RT-PCR
analysis was examined in Neuro2A cells overexpressing hWNK1 or GSK3û under Osr1 knockdown using siRNA. The
value obtained from each sample was normalized to that of GAPDH. The value of Lhx8 from overexpressing hWNK1
(lane 3) was set to 100.
WNK1 and OSR1 form a complex with GSK3û
We next investigated the biochemical interaction between WNK1 and GSK3û. We transiently
expressed Flag-tagged GSK3û together with HA-tagged WNK1 or T7-tagged OSR1. When cell
extracts were subjected to immunoprecipitation with anti-Flag, anti-HA (for WNK1), or
anti6 / 11
Fig 3. GSK3û forms a complex with WNK1, but is not directly phosphorylated. (A±B) Interactions between WNK1
(A) or OSR1 (B), and GSK3û were examined in Neuro2A cells by co-immunoprecipitation. Immunoprecipitates (IP)
were subjected to western blotting (WB) with the indicated antibodies. +, present; -, absent. (C) Interaction among
WNK1, OSR1 and GSK3û were examined in Neuro2A cells by sequential immunoprecipitation. An anti-FLAG antibody
was used for the first immunoprecipitation, and immunoprecipitates were subjected to a second immunoprecipitation
with IgG or T7 antibodies. (D) Phosphorylation of GSK3û by WNK1 or OSR1. Upper panel shows the result of an in
vitro kinase assay. Black arrow in the left upper panel (32P-Flag-WNK1/DA) represents the auto-phosphorylation of
WNK1. Black arrow in the right upper panel (32P-Flag-OSR1/KM/SD) represents the auto-phosphorylation of OSR1.
Grey arrows in both left and right upper panels indicated the size of GSK3û (32P-GST-GSK3û K85M). Lower panels show
the total protein. Arrowheads in both left and right lower panels represent the indicated proteins.
T7 (for OSR1) antibodies followed by immunoblotting, we found that GSK3û interacted with
WNK1 (Fig 3A lane 3), but not with OSR1 (Fig 3B lane 3). Because WNK1 bound to OSR1 [
], we investigated whether the WNK1±OSR1 complex interacted with GSK3û. As shown in
Fig 3C, GSK3û was immunoprecipitated after sequential IP by anti-Flag and anti-T7
antibodies, suggesting that GSK3û forms a complex with WNK1 and OSR1.
GSK3û is positively and negatively regulated by phosphorylation [
]. Because WNK1 and
OSR1 are both Ser/Thr kinases [
3, 25, 26
], and WNK1±OSR1±GSK3û forms the complex shown
above, we examined whether WNK1 or OSR1 directly phosphorylated and regulated GSK3û. To
perform the in vitro kinase assay, we purified Flag-tagged WNK1 or OSR1 from cultured cell
extracts, and produced a GST-fusion protein of the kinase dead form of GSK3û (GST-GSK3û
K85M) in bacteria. We did not observe phosphorylation of GSK3û by WNK1 or OSR1 (Fig 3D
lanes 1±5). These results suggest that GSK3û forms a complex with WNK1 and OSR1, but that the
regulation of GSK3û by the WNK signaling pathway does not depend on direct phosphorylation.
GSK3û is involved in neural specification
As we showed previously [
], WNK plays an important role in neural specification through
the regulation of Lhx8 expression. We examined whether GSK3û was also involved in neural
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specification downstream of WNK. Knockdown of GSK3ß caused the shortening of neurites
after RA stimulation (Fig 4B compared with Fig 4A, quantified in Fig 4C). Knockdown of
GSK3ß also decreased the expression of Lhx8 and the choline acetyltransferase gene (ChAT; a
marker for cholinergic neuron) (Fig 4D lane 4). However, the gene expression of glutamic acid
decarboxylase 1 (Gad1; a marker for GABAergic neurons) increased (Fig 4D lane 4). These
Fig 4. The WNK-OSR-GSK3û pathway is involved in the neural development. (A±B) siRNA-treated differentiated
Neuro2A cells induced by RA for 24 h; (A) Control siRNA, (B) siGSK3ß. (C) The average length of neurites in
siRNAtreated differentiated Neuro2A cells induced by RA for 24 h, shown in A and B (Control siRNA (n = 93), siGSK3ß
(n = 91)). p<0.0005 calculated by the Student's t-test. (D) Gene expression determined by RT-PCR or quantitative
RT-PCR analysis was examined in Neuro2A cells. Cells treated with siRNA against GSK3ß (siGSK3ß); (lanes 1 and 2)
undifferentiated cells, (lanes 3 and 4) cells differentiated by RA for 24 h. The value obtained from each sample was
normalized to that of GAPDH. The value of Lhx8, ChAT or Gad1 from differentiated cells under control siRNA
treatment (lane 3) was set to 100. (E±H) Differentiated Neuro2A cells were transfected with various combinations of
siRNAs and expression plasmids. (E) Control siRNA and control vector. (F) Control siRNA and GSK3û. (G) siWnk1
and siWnk4, and control vector. (H) siWnk1 and siWnk4, and GSK3û. (I) The average length of neurites in
siRNAtreated differentiated Neuro2A cells induced by RA for 24 h, shown in E-H (Control siRNA and Control vector
(n = 81), Control siRNA and GSK3û (n = 87), siWnk1 and siWnk4, and Control vector (n = 103), siWnk1 and siWnk4,
and GSK3û (n = 77)). p<0.0005 calculated by the Bonferroni correction. ns indicated non-significance. (J) Gene
expression determined by RT-PCR or quantitative RT-PCR analysis was examined in Neuro2A cells. Cells were treated
with various combinations of siRNAs and expression plasmids; (lanes 1±4) undifferentiated cells, (lanes 5±8) cells
differentiated by RA for 24 h, (lanes 1±2 and 5±6) control siRNA, (lanes 3±4 and 7±8) siWnk1 and siWnk4, (lanes 1, 3,
5 and 7) control vector, (lanes 2, 4, 6 and 8) GSK3û. The value obtained from each sample was normalized to that of
GAPDH. The value of Lhx8, ChAT or Gad1 from differentiated cells under the treatment of control siRNA (lane 5) was
set to 100.
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results suggest that GSK3û is involved in neural specification, similar to that induced by the
knockdown of both Wnk1 and Wnk4 as shown previously ([
]; see also Fig 4G and 4J).
We next examined whether the expression of GSK3û suppressed the neural specification
phenotypes caused by the knockdown of Wnk. While the expression of GSK3ß did not affect
the elongation of neurites (Fig 4F compared with Fig 4E, quantified in Fig 4I), the expression
of GSK3ß induced Lhx8 and ChAT expression, and reduced Gad1 expression after RA
stimulation in Neuro2A cells (Fig 4J lanes 5 and 6). Under conditions of both Wnk1 and Wnk4
knockdown, the expression of GSK3ß partially rescued the elongation of neurites (Fig 4H compared
with Fig 4G, summarized in Fig 4I), and Lhx8 expression (Fig 4J lanes 7 and 8); this in turn
increased ChAT expression and decreased Gad1 expression (Fig 4J lanes 7 and 8). These results
suggest that GSK3û is involved in neural development and functions downstream of the WNK
The WNK signaling pathway is involved in many biological processes, but the details of its
components are unclear, except for in the kidney. Here, we screened candidate genes that
genetically interact with the WNK signaling pathway in Drosophila. Among these, we
identified shaggy, which encodes the Drosophila homolog of mammalian GSK3û (Fig 1). We showed
that GSK3û activated Lhx8 expression and that GSK3û functions downstream of the WNK±
OSR1 pathway by epistasis analysis (Fig 2). We also showed that GSK3û might form a tertiary
complex with WNK1 and OSR1 (Fig 3). Furthermore, GSK3û was found to be involved in
neural specification and neurite elongation (Fig 4), and GSK3û rescued the neural phenotypes
induced by the knockdown of both Wnk1 and Wnk4 (Fig 4). However, we did not observe
direct phosphorylation of GSK3û by WNK1 or OSR1 (Fig 3). This suggests that GSK3û
functions as a positive downstream effector in the WNK signaling pathway, although the regulation
of GSK3û activity by the signaling pathway remains unclear and requires further study to
elucidate how WNK±OSR1 transduces the signal to GSK3û.
GSK3û plays many roles in various signaling pathways. In the PI3K/AKT signaling
pathway, AKT phosphorylates Ser-9 of GSK3û and inhibits GSK3û activity for cell proliferation
]. Previous research has shown that the PI3K/AKT signaling pathway activates the WNK±
OSR1±NCC pathway to regulate blood pressure [
]. However, GSK3 was reported to be a
negative regulator for the destruction complex in the Wnt signaling pathway [
]. A recent
study showed that WNK is a positive regulator of the Wnt signaling pathway [
]. Here, we
found that GSK3û is a positive regulator downstream of the WNK±SPAK/OSR1 signaling
pathway. These contradictions with regard to the regulation and role of GSK3û clearly indicate
that the WNK±SPAK/OSR1±GSK3û signaling pathway for neural development is independent
of PI3K/AKT or Wnt signaling pathways. The exact interaction between WNK and other
signaling pathways remains to be determined and will require further analyses.
GSK3û is also known to be involved in the Notch signaling pathway in which the
intercellular domain (ICD) of Notch directly regulates the transcription of target genes with several
]. GSK3û was previously shown to bind and phosphorylate the Notch ICD which
increased the transcriptional activity of the Notch ICD complex [
]. However, the
mechanisms of GSK3û activation in the Notch signaling pathway are still unclear. Since our initial
screening of Drosophila Wnk-related genes showed that Wnk has a weak genetic interaction
with the Notch signaling pathway (data not shown), we hypothesize that the WNK pathway
positively regulates the Notch signaling pathway through GSK3û in neural development.
Further study will be required to prove this hypothesis, which is likely to be important for
understanding the pathogenesis of HSAN2A.
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We thank Gary Struhl and the Bloomington stock center for fly stocks, Toshiyasu Goto for
helpful discussion, and Yoko Mitsutomo for technical assistance. We thank Sarah Williams,
PhD, from Edanz Group (www.edanzediting.com) for editing a draft of this manuscript.
Conceptualization: Hiroshi Shibuya.
Data curation: Atsushi Sato, Hiroshi Shibuya.
Formal analysis: Atsushi Sato.
Supervision: Hiroshi Shibuya.
Validation: Atsushi Sato.
Visualization: Atsushi Sato.
Writing ± original draft: Atsushi Sato.
Writing ± review & editing: Hiroshi Shibuya.
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