Phosphopeptide Screen Uncovers Novel Phosphorylation
Sites of Nedd4-2 That Potentiate Its Inhibition of the
Kenneth R. Hallows‡1, Vivek Bhalla§1,2, Nicholas M. Oyster‡, Marjolein A. Wijngaarden¶, Jeffrey K. Lee‡, Hui Li‡,
Sindhu Chandran§, Xiaoyu Xia§, Zhirong Huang?, Robert J. Chalkley**, Alma L. Burlingame**, and David Pearce?
From the‡Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine,
FranciscoSchoolofPharmacy,San Francisco, California 94158
proteins, including the epithelial Na?channel (ENaC). Nedd4-2
decreases apical membrane expression and activity of ENaC.
Although it is subject to tight hormonal control, the mechanistic
basis of Nedd4-2 regulation remains poorly understood. To char-
acterize regulatory inputs to Nedd4-2 function, we screened for
novel sites of Nedd4-2 phosphorylation using tandem mass spec-
trometry. Three of seven identified Xenopus Nedd4-2 Ser/Thr
matched the consensus for a MAPK target sequence. Further in
vitro and in vivo phosphorylation experiments revealed that
Nedd4-2 serves as a target of JNK1, but not of p38 MAPK or
ERK1/2. Additional rounds of tandem mass spectrometry identi-
fied two other phosphorylated residues within Nedd4-2, includ-
ing Thr-899, which is present within the catalytic domain.
Nedd4-2 with mutations at these sites had markedly inhibited
activity, and significantly reduced ubiquitin ligase activity.
These data identify phosphorylatable residues that activate
Nedd4-2 and may work together with residues targeted by
inhibitory kinases (e.g. SGK1 and protein kinase A) to govern
Nedd4-2 regulation of epithelial ion transport.
Nedd4-2, a member of the HECT (homology to the E6-asso-
ciated protein C terminus) family of E3 ubiquitin ligases (1, 2),
is a physiologically important regulator of the epithelial Na?
channel (ENaC)3(1, 3). ENaC likely exists as an ???-hetero-
trimer at the apical membrane in a variety of epithelial tis-
sues, including the kidney collecting duct, where it controls
renal Na?reabsorption (4). Nedd4-2 decreases ENaC cell-
surface expression, and inactivation of Nedd4-2 in cultured
cells increases surface expression and Na?current (5),
whereas genetic deletion of Nedd4-2 in mice causes salt-
sensitive hypertension (6). In humans, the clinical manifes-
tations of Liddle syndrome underscore the physiological
tension results from ENaC mutations that abolish interac-
tion with Nedd4-2 and cause renal Na?retention (7, 8).
Potential effects of Nedd4-2-mediated ENaC ubiquitination
include increased endocytosis from the plasma membrane
and enhanced proteasomal and/or lysosomal degradation (9,
10). The effects of Nedd4-2 on ENaC trafficking are medi-
ated in part through direct ubiquitination of ENaC and pos-
sibly through ubiquitination of components of the traffick-
ing machinery. Furthermore, Nedd4-2 may decrease ENaC
activity by limiting its cell-surface residence time and thus
cleavage of ?- and ?-ENaC subunits by activating proteases
Phosphorylation is an important mechanism for the regula-
tion of Nedd4-2 and other E3 ligases. For example, Nedd4-2
phosphorylation by SGK1 (serum- and glucocorticoid-regu-
three residues (12–14). SGK1 expression and activity are stim-
Therefore, Nedd4-2 is a convergence point for hormonal regu-
lation of epithelial Na?transport in the distal nephron. The
inhibitory effects of SGK1 and PKA on Nedd4-2 are mediated
by Nedd4-2 phosphorylation-induced interaction with 14-3-3
scaffolding proteins, which prevent interaction with and ubiq-
uitination of ENaC (15–17). Mutation of SGK1 target sites
inhibits Nedd4-2 and leads to enhanced ENaC cell-surface
Grants P30 DK079307 (to the Pittsburgh Kidney Research Center), R01
DK075048(toK. R. H.),K08DK071648(toV. B.),andR01DK056695(toD. P.).
This work was also supported by a grant from the Dutch Kidney Founda-
tion(toM. A. W.).MassspectrometryanalysiswasprovidedbytheUniver-
sity of California San Francisco Mass Spectrometry Facility (A. L. B. and
R. J. C.),supportedbytheBiomedicalResearchTechnologyProgramofthe
National Center for Research Resources (NCRR) and National Institutes of
Health NCRR Grants RR01614 and RR015804.
supplemental “Experimental Procedures” and Figs. 1–5.
1Both authors contributed equally to this work.
2To whom correspondence should be addressed: Div. of Nephrology, Stan-
1443; E-mail: email@example.com.
3The abbreviations used are: ENaC, epithelial Na?channel; PKA, protein
kinase A; GST, glutathione S-transferase; xNedd4-2, Xenopus Nedd4-2;
MAPK, mitogen-activated protein kinase; GFP, green fluorescent protein;
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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
JULY 9, 2010•VOLUME 285•NUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 21671
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been suggested, including Akt, which is related to SGK1 (18).
Furthermore, we have shown recently that I?B kinase-?
stimulates ENaC through phosphorylation of Nedd4-2 (19),
whereas AMP-activated protein kinase negatively regulates
ENaC through a stimulatory phosphorylation of Nedd4-2,
which enhances the interaction of ?-ENaC with Nedd4-2
Other E3 ligases are regulated by phosphorylation as well.
HECT E3 ligase, is phosphorylated at three residues by JNK1
(c-Jun N-terminal kinase 1), which disrupts an intramolecular
interaction between the catalytic HECT domain and a proline-
rich N-terminal domain of the ligase. This conformational
change activates the ligase and increases auto-ubiquitination
and ubiquitination of Itch targets (22). Phosphorylation by
SGK3/cytokine-independent survival kinase (a close relative of
SGK1) and Fyn negatively regulate Itch/AIP4 (23, 24). Thus,
phosphorylation has been described as a mechanism for mod-
With these observations in mind, we sought to identify novel
phosphorylation-dependent regulatory inputs to Nedd4-2
using tandem mass spectrometry coupled with in vitro phos-
phorylation assays and site-directed mutagenesis. We then
confirmed the effects on Nedd4-2 and ENaC function through
biochemical and electrophysiological assays. Using this ap-
proach, we have identified several novel sites of phosphoryla-
tion on Nedd4-2 and quantified SGK1-mediated phosphoryla-
phosphorylation and support the idea that specific kinases
(possibly JNK1) may activate Nedd4-2 and inhibit ENaC.
Cell Culture—Human embryonic kidney (HEK293) cells and
mouse polarized kidney cortical collecting duct (mpkCCDc14)
Generation of Recombinant Nedd4-2—Wild-type or mutant
N-terminal glutathione S-transferase (GST)-tagged Xenopus
Nedd4-2 (xNedd4-2; homologous to human Nedd4-2) sub-
cloned in pGEX4T1 (gift of Dr. Olivier Staub) was expressed in
Escherichia coli Rosetta 2 BL21 cells (EMD Biosciences). Indi-
vidual colonies were grown overnight at 30 °C to A600? 0.6,
induced with 40 ?M isopropyl ?-D-thiogalactopyranoside at
room temperature overnight, and isolated using glutathione-
Sepharose 4B beads (Amersham Biosciences) following the
manufacturer’s protocol. Uniform expression was confirmed
by SDS-PAGE and Coomassie Blue staining and quantified by
the Bradford assay.
In Vitro Phosphorylation Assays—Assays of Nedd4-2 in vitro
phosphorylation were performed with either recombinant
Nedd4-2 or FLAG-xNedd4-2 (20) transfected and immunopu-
rified from HEK293 cells. In vitro phosphorylation assays were
performed using 0.8, 1.6, 0.8, and 0.2 ng of purified active
JNK1?1, p38 MAPK?, ERK1 (extracellular signal-regulated
kinase 1), and SGK1, respectively (Upstate) or control buffer
and with 10 ?Ci of [?-32P]ATP following the manufacturer’s
instructions. For recombinant Nedd4-2, phosphorylated sam-
ples were blotted onto phosphocellulose P81 squares (What-
man), washed, and counted in a scintillation counter. The
respective positive control peptides supplied by the manufac-
cell-expressed Nedd4-2, the reaction product was analyzed as
described previously (20).
JNK1 Phosphorylation of Nedd4-2 in Cultured Cells—
and either green fluorescent protein (GFP; pGreenLantern,
experimentation. In vivo [32P]orthophosphate labeling assays of
Xenopus Oocyte Coexpression Assay and Two-electrode Volt-
age Clamp—Maintenance of Xenopus laevis frogs, surgical
extraction of ovaries, and collagenase treatment of oocytes
were carried out as described (25). cRNAs for all proteins
(mouse ?-, ?-, and ?-ENaC subunits and wild-type (WT) and
mutant Nedd4-2) were synthesized using the mMESSAGE
mMACHINE kit (Ambion) and were injected into Stage V-VI
oocytes. Two-electrode voltage clamp (TEV) measurements of
ENaC currents in oocytes were performed as described previ-
ously (21). Lysis and immunoblotting of oocytes for expression
of FLAG-tagged Nedd4-2 were performed using SDS-PAGE,
anti-FLAG antibody M2 (Sigma), and anti-?-actin antibody
(Sigma) as described previously (26).
ENaC Ubiquitin Assays—Ubiquitin assays were performed
as described previously (15), except that HEK293 cells grown
on 6-cm2dishes were transfected using the FuGENE HD
(Roche Applied Science) technique with 1.5 ?g of plasmid
ENaC subunits (hemagglutinin (HA)-tagged ?-ENaC, V5-
tagged ?-ENaC, and Myc-tagged ?-ENaC) and 2 ?g of FLAG-
tagged WT or mutant Nedd4-2. Samples were analyzed by
SDS-PAGE and immunoblotted with anti-ubiquitin antibody
(Covance) or anti-FLAG- or anti-HA-horseradish peroxidase
antibody (Roche Applied Science). Densitometry was mea-
sured using NIH ImageJ software, and values for ubiquitinated
ENaC were normalized to vector or WT Nedd4-2 as indicated.
Auto-ubiquitin Assay of Nedd4-2—Auto-ubiquitination of
FLAG-Nedd4-2 was performed as described previously (27).
HEK293 cells were transfected with GFP or FLAG-tagged WT
or mutant Nedd4-2. Immunoprecipitated FLAG-Nedd4-2 was
then incubated with yeast E1, ATP, and HA-tagged ubiquitin
with or without rabbit E2 (Boston Biochem) for 1 h at room
temperature. Samples were analyzed by SDS-PAGE.
Transient Transfection and Electrophysiological Mea-
surements in mpkCCDc14Cells—Approximately 2 ? 106
mpkCCDc14cells at 80–90% confluency were trypsinized
and then resuspended in 200 ?l of Nucleofector T solution
(Amaxa Biosystems) containing 4 ?g of pGreenLantern
(GFP), pcDNA3-JNK1?1-WT, pcDNA3-JNK1?1-T183E/
Y185E (active mutant), or pcDNA3-JNK1?1-K55M (domi-
nant-negative mutant) plasmid cDNA/transfection. Cells
were electroporated with the plasmid DNA in the Nucleo-
fector electroporation device according to the manufactur-
er’s instructions using Amaxa Program K-29. An equal vol-
ume of warm culture medium was then added, and the cell
suspension was plated onto a plastic 6-well plate containing
2 ml of culture medium. The following day, the cells were
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trypsinized and plated onto 0.33-cm2Transwells (Corning
Costar 3413) at superconfluency. The cells were used for
electrophysiological study 2–4 days later when the cell
monolayers reached transepithelial resistances ?1000 ohm-
cm2. Transepithelial resistance and potential difference
across monolayers were measured using an epithelial volt-
ohm meter as described previously (21). Equivalent short-
circuit currents were calculated by Ohm’s law (28).
Statistical Analysis—TEV data generated from different
oocyte batches were pooled and analyzed using an analysis of
ity in amiloride-sensitive ENaC currents. For other experi-
ments, statistics were performed using paired Student’s t tests.
In all cases, p values ?0.05 were considered significant. Error
bars indicate S.E.
Initial Identification of Phosphorylated Residues in Nedd4-2—
trophoresis, enzymatically digested, and then analyzed by tan-
dem mass spectrometry. This initial screen revealed seven
phosphorylation sites in Nedd4-2, all of which are conserved
from Xenopus to human Nedd4-2 but interestingly are not
present in the related isoform Nedd4-1 (Fig. 1A). Through effi-
cient elution of Nedd4-2 and the use of multiple enzymes for
peptide digestion, 90% sequence coverage (873 of 971 amino
acids) was obtained for analysis (Fig. 1B), although in some
cases, the exact phosphorylated residue could not be unambig-
uously identified (supplemental Fig. 1).
cells with a phosphatidylinositol 3-kinase (PI3K) inhibitor
(LY294002), which inhibits PI3K-dependent activation of
SGK1 (29), or by cotransfecting with constitutively active
SGK1. We then compared the ratio of peak intensities of phos-
mass spectrometry under these conditions. Three residues
showed a higher ratio of phosphorylated to non-phosphory-
lated peptide in samples from cells cotransfected with active
SGK1 versus those treated with LY294002: Ser-338, Thr-363,
previously by mutation analysis as a site of SGK1/PKA-medi-
ated phosphorylation (12, 14). The SGK1-dependent increase
in phosphorylation of Ser-338 and Thr-363 was quite robust
(?4.5-fold), whereas the increase at Ser-444 was only modest
(?1.5-fold) and showed higher basal (PI3K-independent)
Ser-293 was one of four novel phosphorylated residues iden-
tified by mass spectrometry (supplemental Fig. 1). Ser-293 is
embedded within a canonical target motif for the proline-di-
ulate ENaC (31). The related E3 ligase Itch/AIP4 is also acti-
vated by MAPK (JNK1) phosphorylation in a similar proline-
rich domain (22). These observations prompted us to further
characterize the role of Ser-293 in regulation of Nedd4-2.
JNK1 Phosphorylates Nedd4-2 in Vitro and in Cultured Cells—
sought to identify kinase(s) that could phosphorylate this resi-
phorylate Nedd4-2 at Ser-293 by incubating purified GST-
MAPKs: JNK1, p38 MAPK?, and ERK1. Purified constitutively
active SGK1 served as a positive control for Nedd4-2 phosphor-
ylation. Both SGK1 and JNK1, but not p38 MAPK? or ERK1,
significantly enhanced phosphorylation of GST-Nedd4-2 in
vitro (Fig. 2A). JNK1-dependent phosphorylation of the GST-
tagged S293A Nedd4-2 mutant was also significantly reduced
by ?30% (Fig. 2B, left panel), but SGK1-mediated phosphory-
lation was comparable between WT and S293A Nedd4-2. Sim-
ilar amounts of Nedd4-2 fusion proteins were used in these
assays (Fig. 2B, right panel).
Relative to expression of GFP (control), expression of wild-
type JNK1 along with Nedd4-2 in HEK293 cells increased
[32P]orthophosphate in vivo labeling of Nedd4-2 by ?150%
(Fig. 2, C and D). JNK1 coexpression enhanced phosphoryla-
tion of S293A Nedd4-2 by only ?40% (Fig. 2D). These results
indicate that Ser-293 serves as a site for JNK1-mediated
FIGURE 1. Phosphorylation of Nedd4-2. A, schematic of Nedd4-2 domains
residues. Ser-338, Thr-363, and Ser-444 are previously described targets of
SGK1 and PKA. B, sequence of Nedd4-2. Phosphorylated residues identified
non-phosphorylated peptide (%) for each of the three known SGK1/PKA-
ditions. White bars indicate treatment of HEK293 cells with 25 ?M LY294002
constitutively active SGK1 (S422D) and Nedd4-2.
JULY 9, 2010•VOLUME 285•NUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 21673
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Nedd4-2 phosphorylation in vitro and in cells. However, these
results suggest that additional JNK1 phosphorylation site(s)
exist within Nedd4-2.
Ser-293 Potentiates Inhibition of ENaC by Nedd4-2—Using a
Xenopus oocyte coexpression assay and the TEV technique to
measure whole-cell currents, we found that the S293A muta-
tion in Nedd4-2 modestly but significantly reduced the ability
pared with oocytes not expressing exogenous Nedd4-2, there
was an ?90% ENaC current inhibition with coexpression of
WT Nedd4-2. However, there was a modest but significant
reduction in the inhibition of ENaC current (by only ?80%)
with S293A Nedd4-2 coexpression (supplemental Fig. 2).
However, this mutation did not affect regulation of Nedd4-2
through previously known mechanisms, including interac-
kinase, interaction with ENaC channels, and ubiquitination
of ENaC (supplemental Fig. 3).
JNK1 phosphorylates Nedd4-2 partially at Ser-293, and the
Therefore, we sought to identify the additional JNK1 phosphor-
ylation site(s) within Nedd4-2.
Mass Spectrometry Identifies Additional JNK1 Phosphoryla-
tion Sites within Nedd4-2—We performed in vitro phosphory-
lation of Nedd4-2 in the presence or absence of recombinant
JNK1. Using phosphopeptide enrichment followed by tandem
mass spectrometry, two new putative phosphorylation sites
were identified. On the basis of this screen, we quantitated
JNK1-mediated phosphorylation of Nedd4-2 with or without
and T899A mutants were phosphorylated significantly less than
WT. The T408A mutation alone or with S293A did not further
decrease phosphorylation, but the triple mutant (S293A/T408A/
cantly attenuated compared with S293A alone (Fig. 3). SGK1 was
still able to phosphorylate the triple mutant with efficacy compa-
Mutations of Additional Sites Modulate ENaC Current and
Disrupt ENaC Ubiquitination—We next tested whether the
phosphorylation-deficient triple mutant of Nedd4-2 altered
FIGURE 2. JNK1 phosphorylates Nedd4-2 at Ser-293. A, in vitro kinase assay with recombinant kinase incubated with recombinant GST-WT Nedd4-2 (N42)
B, left panel, in vitro kinase assay comparing targets GST, GST-WT Nedd4-2, and GST-S293A Nedd4-2. *, p ? 0.0001, versus GST; #, p ? 0.002, versus WT (in
triplicate). Right panel, Coomassie Blue-stained gel of recombinant proteins. Lane M, molecular mass markers. C, in vivo phosphorylation of Nedd4-2 by JNK1.
Upper panel, representative one-dimensional PAGE analysis showing phosphopeptide screen image of immunoprecipitated FLAG-tagged WT versus S293A
Nedd4-2 cotransfected with JNK1 versus GFP and incubated with [32P]orthophosphate. Lower panel, immunoblot of the same membrane with anti-FLAG
versus S293A; #, p ? 0.001, JNK1 versus GFP (n ? 3).
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of the Nedd4-2 triple mutant abrogated the inhibition of ENaC
currents that occurred with expression of WT Nedd4-2 to the
extent that the currents were similar to those measured with
mutant Nedd4-2 protein expression levels in the oocytes were
confirmed for these experiments (Fig. 4B). Taken together, our
results suggest that these three residues can be phosphorylated
by JNK1 and are important for the functional regulation of
the mechanism more clearly.
We first tested whether the triple mutant affected the
strength of interaction between Nedd4-2 and any of the ENaC
subunits (supplemental Fig. 5). As measured by reciprocal co-
immunoprecipitation assays in HEK293 cells cotransfected to
express ENaC subunits and Nedd4-2, we did not observe any
significant differences in the apparent binding affinity of triple
mutant versus WT Nedd4-2 for the ?-, ?-, and ?-ENaC sub-
units with coexpression of WT Nedd4-2 relative to the triple
mutant (supplemental Fig. 5A). Together, these results suggest
bition of cellular ENaC abundance found with expression of
ENaC subunits. We next performed ubiquitination assays in
HEK293 cells to test whether these residues affect the ability of
Nedd4-2 to ubiquitinate ?-ENaC (Fig. 5). Expression of the
triple mutant significantly decreased ?-ENaC ubiquitination.
Interestingly, stepwise inclusion of each of the mutations at
these phosphorylatable residues progressively decreased ubiq-
uitinated ENaC versus vector control, but mutation of Thr-899
alone was sufficient to demonstrate the effect. Ubiquitination
of ?-ENaC was attenuated by 57% with coexpression of triple
mutant Nedd4-2 compared with WT.
Mutation of Thr-899 Is Sufficient to Disrupt Ubiquitin Ligase
Activity of Nedd4-2—Transfection of Nedd4-2 or mutants in
HEK293 cells was followed by immunoprecipitation and an in
and HA-ubiquitin (Fig. 6). WT Nedd4-2 exhibits potent auto-
ubiquitination activity, but the Nedd4-2 triple mutant did not
significantly ubiquitinate itself compared with the vector con-
trol (GFP). Abolition of Thr-899 was again sufficient to repro-
duce this effect.
JNK1 Inhibits ENaC-mediated Na?Transport—To probe
the effect of JNK1 on ENaC currents in epithelial cells that
endogenously express Nedd4-2 and ENaC (13), we overex-
pressed WT, constitutively active (T183E/Y185E), or kinase-
ative to WT JNK1, expression of kinase-dead JNK1 increased
FIGURE 3. JNK1 phosphorylates Nedd4-2 at three sites. A, FLAG-Nedd4-2
was transfected and immunoprecipitated from HEK293 cells, followed by an
sentative phospho-screen image (upper panel) and immunoblot (IB; lower
S293A (n ? 4–11 experiments).
?-, and ?-ENaC subunits were coexpressed either alone (white bar) or with
Xenopus oocytes (2 ng of each cRNA). A, using the TEV technique, amiloride-
sensitive ENaC currents with WT Nedd4-2 were significantly decreased, but
alone. *, p ? 0.002, versus ENaC alone (N ? four batches, n ? 39 eggs).
B, equivalent expression levels of FLAG-Nedd4-2 in oocytes were verified by
Western blotting, and currents shown were normalized for protein expres-
sion as quantitated by densitometry of the bands relative to ?-actin. IB,
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ENaC-dependent equivalent short-circuit currents by 31 ?
15%, whereas constitutively active JNK1 decreased currents by
38 ? 11% (Fig. 7). These results suggest that JNK1 (a candidate
kinase for these novel residues) modulates ENaC currents in
polarized kidney epithelial cells.
Nedd4-2 regulates several ion transport proteins in addition
to ENaC (32–35), and recent animal studies have confirmed
its importance in the pathogenesis of hypertension (6). The
only mode of ENaC regulation by Nedd4-2 described to
date involves modulation of the Nedd4-2/ENaC interaction.
Our identification of additional phosphorylated residues in
Nedd4-2 led us to hypothesize that other kinase networks may
modulate Nedd4-2 function and thus ion transport. We have
presented an unbiased approach toward elucidating these reg-
anism of Nedd4-2 and Na?transport. Phosphorylation-defi-
cient Nedd4-2 mutants are unable to either ubiquitinate ENaC
or inhibit ENaC currents effectively in cells. These mutants
directly alter ubiquitin ligase activity, which implies a broader
role for these sites in the scope of Nedd4-2 actions.
Our initial mass spectrometry phosphopeptide screen re-
vealed Ser-293 and at least six other residues as phosphoryla-
conserved across mammalian species. Although three of these
residues had been implicated previously in SGK1- and PKA-
mediated stimulation of ENaC (12, 14), Ser-338 and Thr-363
had not been definitively shown to be modified by SGK1 or
other PI3K-dependent kinases prior to this study. Nedd4-2
tagged Nedd4-2 constructs (WT, phosphorylation-deficient mutants, and
ligase-dead C938S) were cotransfected with differentially tagged ???-ENaC.
A, cell dissociation followed by immunoprecipitation (IP) of the HA-tagged
?-ENaC subunit and immunoblotting (IB) with anti-ubiquitin (upper panel)
and anti-HA (middle panel) antibodies. Also shown is an immunoblot of the
whole-cell lysate (WCL) with anti-FLAG antibody (lower panel). B, densitome-
try comparing the signals of ubiquitinated adducts of ?-ENaC. *, p ? 0.02,
versus WT Nedd4-2 (n ? 3).
FIGURE 6. Auto-ubiquitination of Nedd4-2. FLAG-tagged Nedd4-2 con-
structs (WT, triple mutant, and T899A) were transfected into HEK293 cells.
Following immunoprecipitation (IP) with anti-FLAG beads, samples were
incubated with the E1 and E2 enzymes, ATP, and HA-tagged recombinant
ubiquitin in vitro and analyzed by SDS-PAGE, followed by immunoblotting
with anti-HA (upper panel) and anti-FLAG (lower panel) antibodies succes-
sively. As a negative control, GFP was substituted for FLAG-Nedd4-2 (upper
panel, first lane), or E2 was not included (fifth lane). A representative experi-
ment (n ? 3) is shown.
FIGURE 7. JNK1 inhibits endogenous ENaC-mediated sodium currents.
were grown to high resistance on permeable supports. Amiloride-sensitive
(DN) or constitutively active (CA) JNK1 relative to WT JNK1. *, p ? 0.02, versus
WT; #, p ? 0.002, versus dominant-negative JNK1 (n ? 3).
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phosphorylation at these sites was dramatically changed by
dependent phosphorylation at these sites in vivo (Fig. 1C).
estly increased, similar to aldosterone-treated epithelial cells
(13). The higher basal phosphorylation may represent SGK1-
independent pathways such as Akt, PKA, and I?B kinase-?
(14, 18, 19).
Mutation of the previously unrecognized phosphorylation
site Ser-293 to Ala modestly reduced the ability of Nedd4-2 to
inhibit ENaC. Mutation of two additional putative JNK1 phos-
ENaC inhibition (Fig. 4) by reducing catalytic activity (Fig. 6)
without disrupting other known properties of Nedd4-2 (e.g.
phosphorylation by SGK1 and interaction with ENaC). Muta-
tion of Thr-899 in Nedd4-2 appears to be dominant over Ser-
293 or Thr-408, but abolition of phosphorylation at Ser-293
ther Thr-408 nor Thr-899 was detected in the initial phos-
phopeptide screen. For the second mass spectrometry experi-
ment, we phosphorylated Nedd4-2 with JNK1 in vitro and
performed phosphopeptide enrichment to identify these addi-
tional JNK1 phosphorylation sites. The need for these addi-
tional approaches to detect the other two sites might imply a
difference in the relative stoichiometry of phosphorylation at
these residues. Thr-899 resides within the HECT domain of
Nedd4-2 and is conserved from Xenopus to human and across
the Nedd4 family. To our knowledge, this is the first identifica-
tion of a phosphorylation site within a HECT domain. It may
thus play an important general role in the regulatory mecha-
nism of HECT E3 ligases.
Although our data indicate that JNK1 both regulates ENaC
currents in mpkCCDc14cells (Fig. 7) and phosphorylates
Nedd4-2 in vitro and in vivo (Figs. 2 and 3), we were unable to
confirm in additional experiments that JNK1-dependent regu-
lation of ENaC requires the phosphorylation of Nedd4-2 at
these sites (data not shown). We thus speculate that additional
kinase(s), independent of JNK1, also play an important role in
phosphorylating these sites (especially Thr-899) that are criti-
cal in maintaining Nedd4-2 catalytic function in cells. It is also
possible that the lack of measurable effect of JNK1 on Nedd4-2
ubiquitin ligase activity could reflect a limitation of the experi-
mental method. Future experiments will be needed to charac-
may phosphorylate these residues, and their relative in vivo
Phosphorylation of Nedd4-2, and E3 ligases in general,
represents a powerful regulatory mechanism to alter the fate
of ion channels and other targets of ubiquitination. We have
identified novel sites of phosphorylation of Nedd4-2 using
mass spectrometry and have characterized the functional
importance of phosphorylation at some of these sites on
Nedd4-2. The MAPK JNK1 phosphorylates Nedd4-2 and
negatively regulates ENaC-mediated Na?transport. Through
these studies, we have described a novel mode of Nedd4-2 reg-
ulation via phosphorylation of the HECT domain and have
introduced JNK1 as a potential mediator in the regulation of
Acknowledgments—We thank Thomas Kleyman, Alan Pao, and
Rama Soundararajan for suggestions.
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21678 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 285•NUMBER 28•JULY 9, 2010
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Burlingame and David Pearce
2010, 285:21671-21678. J. Biol. Chem.
Zhirong Huang, Robert J. Chalkley, Alma L.
K. Lee, Hui Li, Sindhu Chandran, Xiaoyu Xia,
M. Oyster, Marjolein A. Wijngaarden, Jeffrey
Kenneth R. Hallows, Vivek Bhalla, Nicholas
Potentiate Its Inhibition of the Epithelial
Phosphorylation Sites of Nedd4-2 That
Phosphopeptide Screen Uncovers Novel
doi: 10.1074/jbc.M109.084731 originally published online May 13, 2010
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