Protein kinase WNK3 regulates the neuronal splicing
A-Young Leea,1, Wei Chena,1,2, Steve Stippeca, Jon Selfa,3, Fan Yangb, Xiaojun Dingb, She Chenb, Yu-Chi Juanga,4,
and Melanie H. Cobba,5
aDepartment of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390; andbNational Institute of Biological Sciences, Beijing
Contributed by Melanie H. Cobb, September 5, 2012 (sent for review July 25, 2012)
We report an action of the protein kinase WNK3 on the neuronal
mRNA splicing factor Fox-1. Fox-1 splices mRNAs encoding proteins
important in synaptic transmission and membrane excitation.
WNK3, implicated in the control of neuronal excitability through
actions on ion transport, binds Fox-1 and inhibits its splicing activity
in a kinase activity-dependent manner. Phosphorylation of Fox-1 by
WNK3 does not change its RNA binding capacity; instead, WNK3
increases the cytoplasmic localization of Fox-1, thereby suppressing
Fox-1–dependent splicing. These findings demonstrate a role of
WNK3 in RNA processing. Considering the implication of WNK3
and Fox-1 in disorders of neuronal development such as autism,
WNK3 may offer a target for treatment of Fox-1–induced disease.
required for their mRNA splicing activity (1, 2). Depending on the
position of the (U)GCAUG stretch relative to the target exon,
Fox proteins promote either exon inclusion or skipping (3). Fox-1
splices pre-mRNAs in a tissue-specific manner. In brain it has been
7). Comparative profiling of splicing in autism spectrum disorder
(ASD) and normal brain demonstrated differential Fox-1–mediated
in autistic brain (8). Analysis of Fox-1−/−brains revealed splicing
With No Lysine (K) (WNK) protein kinases are large enzymes,
notable for a uniquely positioned catalytic lysine, that regulate ion
ubiquitous, WNK3 is highly expressed in brain, kidney, and some
other tissues (12, 13). WNK3 modulates neuronal excitability
of the genomic location encompassing the WNK3 gene has been
found in individuals with neurodevelopmental disorders including
ASD and schizophrenia (14–16). Nonsynonymous mutations in
WNK3 identified in ASD patients may be disease-relevant (17).
WNK proteins are pleiotropic, suggesting versatile signal trans-
the present study, we found that WNK3 is associated with RNA
binding proteins, including Fox-1, and we show that WNK3 mod-
ulates Fox-mediated mRNA splicing by causing its subcellular
ammalian Fox family members recognize a (U)GCAUG el-
ement through a conserved RNA recognition motif (RRM)
WNK3 Binds mRNA Processing Factors. To explore unique biological
roles of WNK3, yeast two-hybrid screens were performed with
three WNK3 baits and a neonatal mouse brain cDNA library.
More than 100 putative WNK3 partners were identified; several
are involved in mRNA splicing and translation (Fig. 1A), sug-
gesting a function of WNK3 in these processes. Several of these
interactions were confirmed by coimmunoprecipitation (Fig. 1 B–
G). Consistent with yeast two-hybrid results, the WNK3 C ter-
minus bound three splicing factors, Fox-1, Fox-2, and Celf4 (Fig.
1 B–D). WNK3 also bound the general translation elongation
factor, EEF1A1, located not only at the ribosome but also in
RNA processing complexes (18), and another translation elon-
gation factor, EEF2 (Fig. 1 E–G).
Mapping Interacting Regions on WNK3 and Fox-1. To evaluate the
significance of these interactions we focused on the convergence of
tissue specificity and physiological and pathological involvement of
found that the coiled-coil domain in the C terminus of WNK3 was
important to its interaction with Fox-1; from coimmunoprecipita-
tion, its binding was equivalent to the full-length protein (Fig. 2A
and Fig. S1A and D). Because that coiled coil is conserved among
WNKs, it seemed likely that Fox-1 would bind other WNKs. Two-
hybrid tests indicated no interaction of Fox-1 with the comparable
region of WNK2 or WNK4 (Fig. S1B). Models of the surface-
regions that could confer specificity (Fig. S1C). We tested binding
of Fox-1 to WNK1 directly and found no coimmunoprecipitation,
suggesting that Fox-1 binds WNK3 with selectivity (Fig. S1A).
WNK3 isoforms include one nonneuronal form (isoform 1, Fig.
2A) and three brain-specific forms (isoforms 2–4) produced by al-
ternative splicing of exons 18b and 22; these exons are absent from
the more broadly expressed WNK3 isoform (13, 19). Fragments
representing all possible spliced products were generated as GST
fusion proteins for in vitro pull-down assays. Fox-1 bound not only
fragments containing the WNK3 two-hybrid bait, but also a brain-
specific fragment, residues 991–1412, indicating the importance of
exon 18b residues (Fig. 2A). This suggests that Fox-1 differentially
binds neuronal relative to nonneuronal forms of WNK3.
Basedona series ofoverlappingFox-1 fragments,Fox-1 residues
291–326 accounted for its ability to bind WNK3 (Fig. 2 B and C).
Brain and muscle forms of Fox-1 are well conserved except in two
regions (Fig. S2A), one at the N terminus and a second in the seg-
ment that binds WNK3. The RRM and the WNK3-binding region,
residues 291–326, are most similar between Fox-1 and Fox-2; thus,
this region of Fox-2 is also likely recognized by WNK3 (Fig. S2B).
WNK3 Regulates Splicing by Fox-1 and Fox-2. To test the functional
relationship between WNK3 and Fox-1, we first established an
assay for alternative splicing mediated by Fox-1. The Fox-1 target
gene formin-like protein 3 (FMNL3) contains four UGCAUG
Author contributions: A.-Y.L. and W.C. designed research; A.-Y.L., W.C., S.S., J.S., F.Y.,
X.D., and S.C. performed research; A.-Y.L., W.C., and Y.-C.J. analyzed data; and A.-Y.L.
and M.H.C. wrote the paper.
The authors declare no conflict of interest.
1A.-Y.L. and W.C. contributed equally to this work.
2Present address: Mary Kay Global Research and Development, Dallas, TX 75379.
3Present address: Wenderoth Patent Law, Washington, DC 20005.
4Present address: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto,
ON, Canada M5G 1X5.
5To whom correspondence should be addressed. E-mail: melanie.cobb@utsouthwestern.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| October 16, 2012
| vol. 109
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elements downstream of exon 25a (20). Inclusion of the exon
introduces an early stop codon that produces a shorter isoform,
FMNL3A (Fig. 3A, Upper) (20, 21). An FMNL3 minigene splicing
template was generated by inserting the FMNL3 sequence sur-
rounding exon 25a in between beta-globin constitutive exons 1 and
2 (Fig. 3A). When exogenously expressed with the minigene, Fox-1
and Fox-2 increased the inclusion ratio of exon 25a up to 12-fold
(Fig. 3B). F125 and F159 in the Fox-1 RRM domain are crucial for
RNAsequence recognition (1,22, 23). As expected, Fox-1(F125A)
and (F159A) mutants failed to induce exon inclusion, supporting
type Fox proteins (Fig. 3B). To verify that splicing depended on
UGCAUG elements in the minigene, mutations were introduced
in two of the four contiguous elements that the Fox-1 RRM rec-
ognizes (M1 and M2 of four), individually or in combination (Fig.
3A). The expression of single-element mutants (M1 or M2) re-
duced the inclusion ratio compared with the wild-type template,
and activity was further reduced if two elements were mutated [M
(1+2)], consistent with the importance of UGCAUG element
recognition in Fox-mediated alternative splicing (Fig. 3C).
The minigene was used to test effects of WNK3 on Fox splicing
activity. As shown in Fig. 3D, Fox-1–mediated exon inclusion was
significantly down-regulated by coexpression with wild-type
WNK3, whereas expression of the K-Cl cotransporter (KCC3b),
a probable target of WNK3, had no effect. WNK3 kinase activity
was required for exon skipping, because inactive WNK3 showed
on inclusion. We conclude that WNK3 specifically inhibits Fox-1–
induced exon inclusion in a kinase activity-dependent manner.
The role of endogenous WNK3 in a neuronal system was tested
in stable cell lines expressing control shRNA or shRNA against
WNK3 (Fig. S3A). Under conditions promoting neuronal dif-
ferentiation, knockdown of WNK3 down-regulated the skipping
ratio of the endogenous FMLN3 25a exon, further supporting the
idea that WNK3 inhibits Fox-mediated exon inclusion (Fig. S3B).
To explore WNK3 regulation of splicing mediated by endoge-
nous Fox proteins, the assay was validated in HEK293 cells, which
express a detectable amount of Fox-2 endogenously. As shown in
Fig. 3E, about 95% of endogenous FMNL3 exists in the exon 25a-
dependence on Fox-2 of FMNL3 exon 25a splicing. Up-regulated
exon skipping by siFox-2 was partially rescued by Fox-1 over-
CUGBP, Elav-like family member 4
Feminizing gene on X
mitochondrial ribosomal protein L24
mitochondrial ribosomal protein L14
ribosomal protein L41
ribosomal phosphoprotein P0
eukaryotic translation initiation factor 4H
eukaryotic translation initiation factor 4E nuclear import factor 1
eukaryotic translation elongation factor 2
eukaryotic translation elongation factor 1 alpha 1
t i aB3KNWEMANLLUF SYMBOL
(A) List of mRNA processing factors identified as
WNK3-binding partners from yeast two-hybrid
screens. (B) FLAG-tagged Fox-1 (F:Fox-1) was expressed
in HEK293 cells with or without Myc-tagged proteins
as indicated. The protein complex was coimmuno-
precipitated with anti-Myc antibody and analyzed
by immunoblotting. (C) Myc-tagged proteins were
expressed in HEK293 cells and anti-Myc antibody
was used for coimmunoprecipitation to detect bound
endogenous Fox-2. In B and C, the arrow marks the
position of F:Fox-1 or endogenous Fox-2, respectively.
In both, the asterisk represents the position of IgG.
(D) Transfection and coimmunoprecipitation were as
described in B and proteins were immunoblotted. (E
and F) Proteins were expressed in HEK293 cells as
indicated and then immunoprecipitated with M2
(anti-FLAG antibody) resin followed by immuno-
blotting. Alpha-tubulin was used as a loading marker.
(G) HEK293 cells were lysed and immunoprecipitated
with anti-WNK3 antibody. Associated proteins were
detected by immunoblotting with the indicated
WNK3 binds to mRNA processing factors.
| www.pnas.org/cgi/doi/10.1073/pnas.1215406109 Lee et al.
collection owing to their identical RRM domains. Expression of
a WNK3 brain-specific isoform (isoform 4, Fig. 2A) in HEK293
cells dramatically increased the exon 25a skipping ratio, consistent
with the minigene results (Fig. 3F). A catalytically inactive WNK3
mutant was less effective than wild-type WNK3.
Because results from the minigene system and analysis of en-
dogenous Fox-2 function revealed the importance of WNK3 ki-
nase activity in regulating splicing, we asked whether the WNK3
kinase domain was sufficient to regulate splicing. Neither wild-
type nor inactive WNK3 kinase domains had any effect on Fox-
mediated splicing, suggesting that not only its kinase activity but
also the region of WNK3 that binds Fox proteins is necessary for
proper splicing regulation (Fig. 3F). Confirming the WNK se-
lectivity, WNK1 did not change the exon skipping ratio.
WNK3 Influences the Subcellular Localization of Fox-1 in a Kinase
Activity-Dependent Manner. Because the kinase activity of WNK3
is important for the regulation of Fox-mediated splicing, we tested
WNK3 kinase activity toward Fox proteins. The WNK3 kinase
domain phosphorylated both Fox-1 and Fox-2, whereas neither
kinase-dead WNK3 nor wild-type WNK1 did so (Fig. 4A and Fig.
S4A). Full-length WNK3 phosphorylated Fox proteins in vitro to
an extent comparable to the kinase domain, consistent with the
suggestion that the differential effect of WNK3 (1–411) shown in
dependence on UGCAUG element binding of Fox proteins for
for Fox negative regulation, we asked whether WNK3 phosphor-
ylation of Fox-1 reduced its RNA recognition. Bacterially purified
Fox-1 wild-type and RNA binding mutants (F125A, F159A) were
assay was validated by confirming that only wild-type Fox-1 shifted
in mobility as a function of increasing protein concentration (Fig.
S5B). However, WNK3-mediated phosphorylation of Fox-1 did
recognized the UGCAUG template with a marginal increase in
binding (Fig. 4B). Instead, wild-type WNK3 bound to Fox-1 more
contain the C-terminal Fox-1 binding site (Figs. 1B and 4C). More
S4C). This could be due to an effect of phosphorylation on the
of its C-terminal region for interaction with Fox proteins.
As previously suggested, Fox-1 carries a hydrophobic PY nuclear
localization consensus sequence recognized by Karyopherin beta 2
(Kapβ2) (4, 24). The sequence lies near the C terminus, which di-
verges betweencytoplasmic and nuclear Fox-1 isoforms (2). Indeed,
nuclear Fox-1 directly bound Kapβ2 and the interaction was greatly
reduced by addition of GTP-Ran, displaying the appropriate speci-
changes the localization of Fox-1, thereby affecting its splicing out-
expression of wild-type, but not kinase-dead, WNK3 increased the
amount of Fox-1 in the cytoplasm (Fig. 4 D and E). The kinase
domain (1–411) in the absence of C-terminal sequences was in-
sufficient to promote cytoplasmic retention of Fox-1. This result
Fox-mediated splicing is due to the increased cytoplasmic and thus
Consistent with the fractionation assay, exogenously expressed
to the cytoplasm (Fig. S6). Kinase-dead mutant of WNK3 did not
inducecytoplasmic Fox-1, confirming the requirementof its kinase
of Fox-1, thereby regulating Fox-mediated splicing output.
The identification of mRNA processing factors as WNK3 binding
partners implies an unexpected role of WNK3 in mRNA splicing
and translation. Besides its distinct involvement in each event,
WNK3 may regulate the complex consisting of the factors impli-
cated in these stages of mRNA processing; this seems more plau-
evident. This notion has been supported by the recent report
showing that Fox-2 and EEF1A, both WNK3 binding proteins, are
found in a pre-mRNA 3′ processing complex (18). Perhaps WNK3
affects formationofthecomplex orcoordinates a separate complex
including other proteins found as binding partners.
CC Kinase AI
indicate protein domains including Kinase, AI (auto inhibitory), and CC (coiled-coil) domains. Outside these regions, WNKs are poorly conserved. Numbers are
the amino acid residues of full-length protein or the boundaries of small fragments linked to GST. GST pull-down assays with bacterially purified F:Fox-1 are
presented (Right). (B) Schematic view of mouse Fox-1. RRM = RNA recognition motif. Fragments were generated for WNK3 interaction assays and binding
results are summarized on the right. (C) Coimmunoprecipitation of Myc-WNK3 (1474–1743) with FLAG-tagged Fox-1 fragments from HEK293 cells.
Mapping the interacting regions on WNK3 and Fox-1. (A) Schematic representation of protein domains in human WNK3 (Left). Shaded regions
Lee et al.PNAS
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1 2 3 4
t n t n
41 115 58
Inclusion (%) 0.6 6.9 4.7 0.5 0.95
Ratio (Fox-1/ )7.9
WT M1M2 M(1+2)
Inclusion (%) 1.6 12.70.7
3.6 0.1 0.3
WT M1 M2M(1+2)
41 115 58
25 25a 26
Skipped (%) 5 93 63
25 25a 26
Skipping (%)899 23 1510 10
including exons 25, 25a, and 26 and introns (Upper). FMNL3 minigene content (Lower). The region marked with dotted lines was inserted between β-globin
exons 1 and 2 in the pcAT7-Glo1 plasmid. (B) FMNL3 minigene and FLAG-tagged Fox proteins were expressed in HEK293 cells as indicated and then subjected
to the splicing assay described in Experimental Procedures. Alternative splicing products are depicted on the left of the gel. Error bars indicate SD (Right). (C)
Minigene splicing assay with wild-type or UGCAUG element-mutated templates in the absence or presence of F:Fox-1 expression (mean ± SD). (D) Minigene
splicing assay with F:Fox-1 and Myc-tagged proteins as indicated. Error bars show the SD in three experiments. (E) Endogenous FMNL3 splicing assay. Fox-2
down-regulation by siRNA against Fox-2 (SiFox-2) and F:Fox-1 expression were monitored by immunoblotting. (F) Effect of Myc-tagged WNK proteins on
endogenous Fox-2–mediated FMNL3 splicing (mean ± SD).
WNK3 regulates splicing activity of Fox-1 and Fox-2. (A) Genomic organization and alternative splicing patterns of an endogenous FMNL3 region
| www.pnas.org/cgi/doi/10.1073/pnas.1215406109Lee et al.
Among the newly identified WNK3-binding partners, knowledge
of Fox-1 and WNK3 has revealed several intersections. These fac-
links to control of neuronal excitability, and both have been impli-
cated in neurological diseases such as ASD and schizophrenia.
Current ideas concerning a connection between WNK3 and ASD
the WNK3 gene in autistic brain and its modulation of neuronal
has been shown to affect transcripts encoding many genes impli-
cated in ASD and is responsible for generating proper alternative
splicing variants required for normal neuronal excitability and syn-
aptic transmission (8, 9). Our finding that WNK3 regulates Fox-
that implicates WNK3 in neurological diseases through reprog-
ramming splicing patterns.
Fox regulation by WNK3 is dependent on its kinase activity,
which also affects the interaction between the two factors in the
cellular environment. It is likely that both of the physical and
kinetic relationships are required for proper regulation. There are
atleastthreepossibilities abouthow this physio-kineticinteraction
might occur. The first and most expected is that phosphorylation
may induce conformational changes in Fox-1 favorable for the
interaction with WNK3. This possibility was investigated by map-
ping WNK3 phosphorylation sites on Fox-2. The sites, S174 and
T337, conserved in Fox-1 and Fox-2, were tested through point
mutations and kinase assays (Figs. S2B and S7A). However, the
mutants functioned equivalently to and were localized like wild-
type Fox-2 (Fig. S7 B–D). It is conceivable that additional Fox
phosphorylation sites that were not detected could mediate this
effect. WNK3 may also phosphorylate other factors that enhance
the interaction between WNK3 and Fox-1. Finally, it is also pos-
sible that WNK3 catalytic activity induces conformational changes
in itself that enable the interaction with Fox-1. Given the major
function of WNK proteins as scaffolds, this possibility must be
We report here Kapβ2 as a putative importin for Fox-1. Con-
sistent with its sequence information, Fox-1 is recognized by
1 2 - 1 2 - 1 2
Myc:WNK3B WT KD
+ ++ +
C N C N C N C N C N
Cytoplasmic Retention (%)
+ Myc:WNK3B (WT)
+ Myc:WNK3B (KD)
Cytoplasmic Retention (%)
domains and F:Fox-1 and F:Fox-2 (Upper). Following these reactions, Fox proteins contained ∼2 mol phosphate/mol protein. Coomassie blue staining of
proteins used for the assay (Lower). Asterisks indicate positions of Fox proteins. (B) RNA electrophoretic mobility-shift assay with the RNA template including
a UGCAUG element and nonphosphorylated or WNK3-phosphorylated F:Fox-1. Triangles represent increasing amounts (0.1, 0.3, and 1 μg) of proteins used.
(C) Myc-tagged WNK3 proteins and F:Fox-1 were expressed in HEK293T cells for coimmunoprecipitation. (D) F:Fox-1 was coexpressed with Myc-tagged WNK3
proteins in HEK293T cells and then subjected to subcellular fractionation as described in Experimental Procedures. C and N represent cytoplasmic and nuclear
fractions, respectively. Alpha-tubulin and histone 3 (H3) were detected as markers of cytoplasmic and nuclear fractions, respectively. To calculate the per-
centage of cytoplasmic retention, the cytoplasmic F:Fox-1 intensity was divided by the total intensity of both fractions and multiplied by 100 (mean ± SD). (E)
Immunostaining of F:Fox-1 (green), Myc:WNK3 (red), and DAPI (blue). HeLa cells were transfected for 60 h as indicated. Cytoplasmic and nuclear intensities
were quantitated individually through ImageJ and calculated as described in D.
WNK3 modulates the subcellular localization of Fox-1 in a kinase activity-dependent manner. (A) In vitro kinase assay with purified WNK kinase
Lee et al.PNAS
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