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
| no. 42
Kapβ2, suggesting its nuclear import step can be regulated to
modulate its splicing output. Indeed, WNK3 induces cytoplasmic
retention of Fox-1 and its kinase activity is crucial for this phe-
nomenon. One hypothesis we considered, that WNK3 might in-
experimentally (Fig. S8 B and C). WNK3 may regulate steps of
nuclear import other than the recognition by Kapβ2, or Kapβ2
may not be a major importin for Fox-1.
Related to the involvement of WNK3 in neuronal activity, most
research has focused on the regulation of proteins at a post-
translational level. Our findings provide a unique mechanism for
WNK3 regulation of factors important for neuronal excitability
through control of the spliced forms that are expressed. This type
of regulation, like control of membrane proteins, can have a po-
tent impact on the pathogenesis of neurodevelopmental diseases.
GST Pull-Down Assays. GST-tagged proteins were prepared as described (25).
One microgram of GST-tagged proteins was immobilized to 20 μL of a 50%
(vol/vol) glutathione-Agarose slurry (Pierce) at 4 °C, overnight. After three
washes, 100 ng of recombinant FLAG-Fox-1 (F:Fox-1) was incubated with
immobilized GST-proteins at 4 °C for 1 h. Reactions were washed three times
and analyzed by immunoblotting.
RNA Preparation and RT-PCR for Splicing Assays. TotalRNAwasprepared from
SuperScript II reverse transcriptase (Invitrogen) using gene-specific reverse
Primer sequences are as follows: for endogenous genes (FMNL3-ex25-F GAA-
CACCGGCCTGTTTATGAG, FMNL3-ex26-R AAGTGCTTCTGCCTCCGAGAG) and
for minigenes (DUP-ex1-F AAGGTGAACGTGGATGAAGTTGGT, DUP-ex3-R AC-
AGATCCCCAAAGGACTCAAAGAAC). To quantify the relative ratio of specific
exon included or excluded products,32P end-labeled primers were added to
PCR reactions with few cycles. PCR products were resolved on denaturing gels
and exposed using PhosphorImager screens. Radioactivity in bands was quan-
was determined as ([cpm of exon included product/(cpm of exon included
product and exon skipped product)] × 100%) and the skipping ratio was de-
termined as ([cpm of exon skipped product/(cpm of exon included product
and exon skipped product)] × 100%).
In Vitro Kinase Assays. FLAG-tagged Fox proteins (F:Fox) were expressed and
immunoprecipitated with M2-agarose beads from HEK293 cells for kinase
assays. The beads were washed three times with detergent buffer [0.25 M Tris
(pH 7.4), 1 M NaCl, 0.1% Triton X-100, 0.1% sodium deoxycholate] and once
with 10 mM Hepes (pH 7.6), and then further incubated with indicated
proteins in 50 μL of 20 mM Hepes (pH 7.6), 5 μM ATP (5 μCi of [γ-32P]ATP),
10 mM MgCl2, 10 mM glycerol phosphate at 30 °C for 1 h. Reactions were
stopped by adding 15 μL of 5× sample buffer followed by boiling for 2 min.
The reactions were analyzed by polyacrylamide gel electrophoresis in SDS
RNA Electrophorectic Mobility-Shift Assay. For RNA EMSA, phosphorylated
andnonphosphorylated F:Fox-1 were preparedas follows.M2-immobilized F:
Fox-1 was incubated in the kinase reaction solution as above with or without
purified WNK3 kinase domain at 30 °C for 2 h. After washing, proteins were
released from beads with elution buffer [20 mM Tris·Cl (pH 7.9), 20% glyc-
erol, 0.2 mM EDTA, 100 mM KCl, 0.03% Nonidet P-40, and 0.2 mg/mL FLAG
peptide] and the indicated amount of protein was used for the assay.
The RNA sequence template (AAACCAGCAUGAACGAUUUACCAAG) was
selected as reported (1) and a biotinylated RNA was obtained from Dhar-
macon. Biotinylated RNA (20 nM) was incubated with binding buffer [10 mM
Hepes (pH 7.3), 20 mM KCl, 1 mM MgCl2, 1 mM DTT, 2 μg tRNA] in the
absence or presence of the proteins as indicated. After 30 min at room
temperature, the reactions were analyzed in 6% polyacrylamide gels and
then transferred to nylon membrane in 0.5× Tris·borate-EDTA at 400 mA for
30 min. The membrane was crosslinked with a hand-held UV lamp with
a 254-nm bulb for 3 min. Biotinylated RNA on the membrane was visualized
using Chemiluminescent Nucleic Acid Detection Module (Pierce) according
to the manufacturer’s protocol.
Subcellular Fractionation. Subcellular fractionation was performed as de-
scribed (27) with some modifications. Briefly, transfected HEK293 cells were
lysed with 0.3 mL of cytoplasmic lysis buffer and centrifuged at 3,500 × g,
4 °C, for 15 min. The pellet was washed with 1 mL of cytoplasmic lysis buffer
without Nonidet P-40 and lysed with 0.3 mL of nuclear lysis buffer and
centrifuged at 15,000 × g, 4 °C, for 20 min to separate a nucleoplasmic
fraction. Insoluble material was further incubated with 0.3 mL of nuclease
incubation buffer with 25 U/μL Benzonase at room temperature for 30 min.
The reaction was centrifuged at 20,000 × g, 4 °C, for 20 min. The supernatant
was collected and mixed with the nucleoplasmic fraction as the nuclear
fraction and each fraction was analyzed by immunoblotting.
Other procedures are described in SI Text.
ACKNOWLEDGMENTS. We thank Kristen Lynch and Lynch laboratory mem-
Glo1 construct, human genomic DNA, andtheir help setting up splicing assays.
This work was supported by National Institutes of Health Grant GM53032 and
Robert A. Welch Foundation Grant I1243.
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