|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 4, 2897-2907, January 25, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, October 17, 2001
Nedd4 is a HECT domain-containing ubiquitin
ligase that mediates ubiquitylation and proteasome degradation of
target proteins. The molecular basis for the interaction of Nedd4 with
substrates lies in its WW domains, which can bind proline-rich (PY)
domains in target proteins. Nedd4 is a developmentally expressed
protein and may have a fundamental role to play in embryonic processes. However, whether Nedd4 has such a function is currently unknown, in
part because few developmentally regulated ubiquitylation substrates have been identified or characterized. We have carried out a yeast two-hybrid screen and identified four proteins expressed in the mid-gestation embryo that are able to interact with Nedd4.
Characterization of their functional interaction with Nedd4 in
vitro and in vivo demonstrated that three of the four
are bona fide Nedd4 binding partners, and two have the
capacity to be ubiquitylation substrates. One of these is the first
identified nonviral substrate for Nedd4-mediated monoubiquitylation.
Interestingly, neither of these two ubiquitylated proteins interacts
with Nedd4 through PY-mediated mechanisms. For one of the three Nedd4
binding partners, there was no discernable evidence of ubiquitylation.
However, this protein clearly associates with Nedd4 through its
PY domains and can alter the location of Nedd4 in cells, suggesting a
role other than as a ubiquitylation substrate.
A large number of recent findings have highlighted the importance
of protein modification by the covalent addition of small polypeptides
(1, 2). The prototype for this is ubiquitin, a 76-amino acid
polypeptide that, when added in multiple units to form a chain
(polyubiquitylation), targets proteins for degradation by the 26 S
proteasome (3). In contrast, the addition of a single ubiquitin moiety
(monoubiquitylation) has been implicated in receptor endocytosis (4,
5), activation of gene expression through histone modification (6), and
targeting of proteins to specific subnuclear locations (7). In
addition, protein modification by the ubiquitin-like proteins SUMO and
Nedd8 has been found to have a wide range of functions, from control of subcellular localization to modulation of protein stability (8, 9).
Ubiquitylation occurs at lysine residues, either within target proteins
or on ubiquitin already attached to the target protein, and involves
three distinct enzymatic activities. Ubiquitin-activating enzyme
(E1)1 activates ubiquitin to
a high energy state in an ATP-dependent manner and then
transfers it to a ubiquitin-conjugating enzyme (E2). The E2 either can
transfer ubiquitin directly to the target substrate or interact with a
ubiquitin protein ligase (E3), which then mediates the transfer of
ubiquitin to substrate proteins (10). There are two major classes of E3
ubiquitin ligases. Those containing a RING finger motif act in concert
with E2 enzymes in the direct transfer of ubiquitin from the E2 to the
target protein (11). Members of the second class of E3 ubiquitin
ligases accept activated ubiquitin from the E2 and transfer it to
substrate proteins. The first identified member of this class is E6-AP, which mediates polyubiquitylation of p53 in conjunction with human papillomavirus E6 protein (12, 13). Other members of this family
include Nedd4 and related Nedd4-like proteins (14). These all have a
carboxyl-terminal region known as the HECT (for homologous to E6-AP carboxyl terminus) domain,
which provides the ubiquitin ligase enzymatic activity.
Unlike E6-AP, members of the Nedd4 family have two to four
tryptophan-based WW domains, which bind certain proteins containing proline-rich motifs (Fig. 1A). The WW domains of Nedd4 have
been categorized as group I, which bind preferentially to the consensus sequence PPXY (PY domain) (15). Group II WW domains
typically bind to the sequence PPLP (16), whereas a third class is able to bind other proline-rich consensus sequences (17). The fourth class
of WW domains binds in a proline-independent manner, depending instead
on phosphoserine and/or phosphothreonine residues (18). The
best-characterized Nedd4 substrate is the epithelial sodium channel, a
plasma membrane protein composed of several subunits, two of which are
ubiquitylated by Nedd4. The WW domains of Nedd4 bind to PY domains in
the cytoplasmic region of these proteins. Truncation of the PY domains
results in a reduced turnover rate of epithelial sodium channel and
leads to Liddle's syndrome, an inherited form of hypertension (19,
20).
Nedd4 was identified originally as a gene expressed at high levels in
neural precursor cells during development and subsequently down-regulated in the adult (21). Nedd4 also is expressed at high
levels in proliferating chondrocytes in mid-gestation embryos (22, 23).
These sites of expression suggest an important role for Nedd4 in
regulating developmentally important gene function at the level of
protein half-life. Indeed, the actions of two Nedd4-like proteins,
Suppressor of Deltex and Smurf1, are consistent with such a role during
development. Smurf1 mediates polyubiquitylation of Smad1, a mediator of
bone morphogenetic protein signaling (24), and Suppressor of Deltex
regulates Drosophila Notch signaling (25). The Nedd4 family
member, Itch, also can ubiquitylate Notch (26). However, the specific
functions of Nedd4 itself in developmental processes are unknown, in
part because of the fact that very few developmentally expressed
ubiquitylation substrates have been characterized (27, 28), although a
number of potential binding partners have been identified (23).
Therefore, we searched for proteins expressed in the mid-gestation
embryo that interact with Nedd4 in a yeast two-hybrid screen, and
further characterized these to determine their functional interaction
with Nedd4 both in vitro and in vivo. We have
identified four proteins, which we designated as
Nedd4 binding partners
1-4 (N4BP1-4). We demonstrate here that N4BP1 and N4BP2, both novel
proteins, have the capacity to be ubiquitylation substrates. N4BP1 is a
bona fide substrate for Nedd4 ubiquitin ligase activity
in vivo, and the first identified nonviral target for
Nedd4-mediated monoubiquitylation. N4BP3, also a novel protein, is not
a ubiquitylation substrate, but it can alter the subcellular location
of Nedd4, indicating a functional interaction.
Yeast Two-hybrid Screening--
A fragment of mouse Nedd4
cDNA encoding amino acids 199-777 cloned into the GAL4 binding domain
vector pGBT9, and an 11-day mouse embryo cDNA library cloned into the
GAL4 activation domain vector pGAD10 were sequentially used to
transform Hf7c yeast cells according to the MATCHMAKER two-hybrid
system protocol (CLONTECH, Palo Alto, CA). Transformants were plated on
selection media (lacking tryptophan, leucine, and histidine) containing
25 mM 3-aminotriazole. After incubating 10 days at
30 °C, clones expressing HIS3 and Plasmid Construction and in Vitro Mutagenesis--
A full-length
N4BP1 expression vector was made in pcDNA3 (Invitrogen) by
assembling the original clone with mouse EST AA444325 and a fragment
containing the 5' part of the coding region, produced by PCR using a
N4BP1 genomic cosmid clone as template. KIAA0341, a human full-length
cDNA homologous to N4BP3, was obtained from Kazusa DNA Research
Institute as a pBluescript clone. The insert was removed and subcloned
into pCDNA3.1. Site-directed mutagenesis of the proline-rich sites
in full-length mouse N4BP1 and KIAA0341 was done using QuikChange
(Stratagene, La Jolla, CA).
Recombinant Protein Expression--
All Nedd4 GST fusion
constructs used in this study were described previously (29). They were
generated using the pGEX GST Gene Fusion System (Amersham Biosciences,
Inc.) and expressed in Escherichia coli BL21 (Novagen,
Madison, WI). The E1 from wheat cloned in pET3a and human UbcH5B cloned
in pET15b were expressed in E. coli BL21 using the pET
expression system (Novagen) as described previously (29). In
vitro expression and radiolabeling of proteins was performed by
coupled transcription and translation using the TnT wheat germ extract
kit (Promega, Madison, WI), in the presence of
[35S]methionine (Amersham Biosciences, Inc.) according to
the manufacturer's protocol. To produce antisera against N4BP1
(anti-N4BP1), the insert was subcloned from pGAD10 into the
EcoRI site of pGEX-4T-1 (Amersham Biosciences, Inc.) and
expressed in BL21 cells. Recombinant fusion protein was purified by
glutathione-Sepharose, and ~100 µg was injected into rabbits at
3-week intervals.
In Vitro Binding--
Nedd4 GST fusion proteins (GST-Nedd4,
GST-Nedd4:N, and GST-Nedd4:C) were adsorbed to glutathione-Sepharose
beads and then combined with each N4BP protein or E6-AP, which had been
in vitro translated and 35S-labeled, in binding
buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 5 mM dithiothreitol, 0.5% Nonidet P-40) with 2 mg/ml BSA. Mixtures were incubated 20 h at 4 °C with agitation and then
washed four times with five volumes of binding buffer to remove unbound proteins. Bound protein was eluted by boiling in reducing sample buffer, and then analyzed by SDS-PAGE and autoradiography.
In Vitro Ubiquitylation--
N4BP proteins, in vitro
translated and radiolabeled as above, were incubated in the presence of
GST-Nedd4, BL21 bacterial extracts of wheat E1and human UbcH5b, and
ubiquitin, in 25 mM Tris-HCl, pH 7.6, 120 mM
NaCl, 3 mM dithiothreitol, 1 mM
MgCl2, 1 mM phosphocreatine, 100 units of
creatine phosphokinase, 0.6 units/ml inorganic pyrophosphatase, and 5 mM ATP Cell Culture, Transfection, Western Blotting, and
Immunoprecipitation--
HEK293 cells were cultured in Dulbecco's
modified minimal essential medium (Invitrogen) supplemented with 10%
fetal bovine serum. Transfections were made using the calcium phosphate
method. After 24-48 h, cells were lysed in buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM
EDTA, 0.5% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin,
and 1 mM phenylmethylsulfonyl fluoride. The insoluble
fraction was removed by centrifugation, and the cleared supernatant was
either analyzed directly by Western blotting or used for
immunoprecipitations. For N4BP1 immunoprecipitation, lysates were
incubated for 16 h at 4 °C with anti-N4BP1 polyclonal antisera.
For N4BP2 and N4BP3 immunoprecipitation, lysates were incubated with
anti-Xpress or anti-HisG monoclonal antibody (Invitrogen). Immunoprecipitates were collected by incubating with protein
A/G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for an
additional 1 h. After brief centrifugation, the complexes were
washed three times with ice-cold 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100.
Proteins were recovered by boiling in SDS sample buffer, fractionated
by SDS-PAGE, and transferred to polyvinylidene difluoride membranes
(Novex, San Diego, CA). Membranes were blocked in 5% milk in TBS and
incubated for 2 h with the different primary antibodies diluted in
TBS. Antibody dilutions used were: anti-N4BP1, 1:2000 dilution;
anti-Nedd4, 1:1000; anti-Xpress and anti-HisG, 1:2000; and anti-Myc
monoclonal antibody (Invitrogen), 1:1000. After incubation with primary
antibodies, membranes were washed with TBST and incubated for 1 h
with the appropriate peroxidase-coupled secondary antibodies (Pierce).
After washing with TBST, chemiluminescent detection was performed using
Supersignal (Pierce). For proteasome inhibition, stock solutions of
MG101 (calpain inhibitor I), lactacystin, MG132, and epoxomycin
(Calbiochem, San Diego, CA) were prepared in Me2SO and
diluted immediately before use in Dulbecco's modified Eagle's medium
to final concentrations of 100 µM for MG101, 5 µM for lactacystin, 10 µM for MG132, and 5 µM for epoxomycin.
Subcellular Localization--
HEK293 cells were plated on glass
coverslips, transiently transfected, and, 24 h later, fixed in 4%
paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS.
Cells were then washed with 0.1% BSA in PBS, incubated for 2 h
with primary antibodies diluted in 0.1% BSA in PBS, washed in PBS, and
further incubated with fluorescein isothiocyanate-conjugated
anti-rabbit antibody (Vector Laboratories, Burlingame, CA) or
rhodamine-conjugated anti-mouse antibody (Pierce) for 1 h. N4BP1
was detected with anti-N4BP1 antiserum used at 1:400 dilution.
His-tagged N4BP2 and N4BP3 were detected with anti-HisG monoclonal
antibody used at 1:5000 dilution. HA-tagged Nedd4 was detected with
either anti-HA monoclonal antibody (Covance Research Products, Denver,
PA) at 1:1000 dilution or anti-Nedd4 polyclonal antiserum at 1:1000
dilution. After incubation with secondary antibodies, samples were
mounted in ProLong (Molecular Probes, Eugene, OR) plus
4',6-diamidino-2-phenylindole, observed with a Zeiss Axioplan
fluorescence microscope, and imaged with a SPOT2 digital camera.
Isolation of Developmentally Expressed Nedd4 Interacting
Proteins--
To identify potential Nedd4 ubiquitylation substrates
that might play a role in embryo development, a fragment of Nedd4
containing WW domains 2 and 3 and the HECT domain (Fig.
1B) was used as bait in a
yeast two-hybrid screen of a mid-gestation mouse embryo cDNA library. From 7 × 106 clones screened, four
interacting clones were identified. These were designated N4BP1-4
(Fig. 2). N4BP4 encodes an
amino-terminally truncated form of PLIC-2 (Fig. 2D), a
protein recently characterized as a regulator of the interaction
between the proteasome and ubiquitin ligases (30). N4BP1-3 show no
similarity to proteins with known functions. Taking advantage of
overlapping ESTs and, in the case of N4BP1, a genomic clone that we
isolated covering the 5' region of the gene, the full-length open
reading frames encoded by N4BP1 and N4BP3 were deduced (Fig. 2,
A and C). For clarity these are referred to as
flN4BP1 and flN4BP3, respectively. For N4BP1-3, amino acid sequences
of full-length human orthologs were found or deduced. For flN4BP1, the
893-amino acid human protein KIAA0615 was found to have 89%
similarity. N4PB2 was found to be 92% similar to the carboxyl region
of a full-length open reading frame encoding 1770 amino acids assembled
from KIAA1413 and EST AK001542. flN4BP3 was found to be 95% similar to
KIAA0341.
N4BP1-3 Bind Nedd4 in Vitro--
To confirm the yeast two-hybrid
results, N4BP1-4 were in vitro translated and radiolabeled
with [35S]methionine. These were then evaluated for
binding to glutathione-Sepharose-immobilized bacterially expressed
GST-Nedd4 fusion proteins that included either both the WW and HECT
domains (GST-Nedd4; Fig. 1C), the WW domain-containing
region only (GST-Nedd4:N; Fig. 1D) or the HECT domain only
(GST-Nedd4:C; Fig. 1E). N4BP1 bound to both GST-Nedd4 (Fig.
3A, lane 2) and
GST-Nedd4:N (Fig. 3A, lane 3). There was little
detectable binding to GST-Nedd4:C (Fig. 3A, lane
4) or to a non-WW domain HECT E3, E6AP, which was used as an
additional negative control (Fig. 3A, lane 6).
Because regions within the amino-terminal half of Nedd4 were used in
the two-hybrid screen, in vitro binding to GST-Nedd4:N
serves to validate the interaction in yeast. Similar results were found
for N4BP2 (Fig. 3B) and for N4BP3 and its full-length human
counterpart KIAA0341 (Fig. 3C). However, no binding to N4BP4
(Plic-2 carboxyl terminus) was observed. Therefore, no further studies
were carried out with this potential binding partner.
Nedd4 can bind other proteins through the interaction of its WW domains
with proline-rich domains in target proteins. Thus, identification of
proline-rich regions in N4BP proteins may provide clues for their
interaction with Nedd4. In fact, N4BP1 contains a PY motif (PPEY) and
two PPLP motifs. Strikingly, however, none of these three regions are
conserved in the human ortholog (KIAA0615). N4BP3 also contains a
consensus PY motif (PPPY), and this sequence is conserved in the
corresponding region of KIAA0341. In the case of N4BP2, neither this
isolate nor the corresponding region of the human ortholog has an
identifiable WW interaction domain. N4BP4 (PLIC-2) also lacks
proline-rich regions. The lack of conservation of the proline-rich
regions between N4BP1 and its human counterpart raises the possibility
that these domains are not required for the interaction with Nedd4. To
address this, key residues in the PY domain and two PPLP domains (PPLP1
and PPLP2; Fig. 2A) of N4BP1 were mutated. As shown in Fig.
3E, these mutations had no discernable effect on N4BP1
binding to Nedd4. Thus the interaction between Nedd4 and N4BP1 is, in
fact, independent of these proline-rich regions. Conversely, mutation
of the evolutionarily conserved PY domain in the human ortholog of
N4BP3 (KIAA0341) dramatically decreased Nedd4 binding (Fig.
3F), establishing that the binding of this protein to Nedd4
is dependent on interactions between the Nedd4 WW domains and the
proline-rich region of N4BP3.
N4BP1 and N4BP2 Are in Vitro Ubiquitylated by Nedd4--
As
N4BP1-3 all bind Nedd4, we next addressed their capacity to serve as
in vitro substrates for this E3. In the presence of GST-Nedd4, ubiquitin, and E1 and E2 enzyme activities, N4BP1 was ubiquitylated as indicated by the ubiquitin and
E2-dependent appearance of discrete higher molecular weight
species (Fig. 4A, lane
2, bracketed). Under the same conditions N4BP2
underwent what appeared to be polyubiquitylation, as demonstrated by
the appearance of a smear of higher molecular weight bands that
extended up the gel accompanied by a significant decrease in the amount
of the non-ubiquitylated form of this protein (Fig. 4B,
lane 2, bracketed). Again, this was dependent on
the presence of E1, E2, and ubiquitin (Fig. 4, A and
B, lanes 1, 3, 4, and
5). In contrast, full-length human N4BP3 (KIAA0341) was not
ubiquitylated despite its clear capacity to bind Nedd4 (Fig.
4C, lane 2).
N4BP2 and N4BP3 Bind Nedd4 in Vivo--
To extend the in
vitro observations to cells, eukaryotic expression vectors
encoding flN4BP1, N4BP2, or the full-length human N4BP3 (KIAA0341) were
co-transfected with Nedd4 into HEK293 cells and evaluated for
co-immunoprecipitation with Nedd4. Immunoprecipitation of N4BP1 was
performed using antiserum derived against bacterially expressed protein
(anti-N4BP1). N4BP2 and KIAA0341 were immunoprecipitated using
monoclonal antibodies directed against their amino-terminal His and
Xpress epitope tags. No co-immunoprecipitation of Nedd4 with N4BP1 was
detected (Fig. 5A, lanes
1-3), despite clear evidence for expression of this protein (Fig.
5B, lanes 1-3). In contrast, in vivo
association of N4BP2 and KIAA0341 could be discerned readily (Fig.
5A, lanes 5 and 7). Although there was
less Nedd4 co-immunoprecipitated with N4BP2 than with KIAA0341, the
expression level achieved in the transfection was much lower for N4BP2
than for KIAA0341 (compare lanes 5 and 7 in Fig.
5B). Thus, the level of interaction with Nedd4 may be
similar for these two proteins. Consistent with the in vitro
binding data, mutation of the PY domain in KIAA0341 resulted in a
dramatic decrease of co-immunoprecipitated Nedd4 (Fig. 5A, lane 9), underscoring the importance of the PY domain for
interactions between this protein and Nedd4.
N4BP1 Is Monoubiquitylated in Vivo by Nedd4--
The failure to
see co-immunoprecipitation of Nedd4 and N4BP1 does not preclude a
functionally significant in vivo interaction. To evaluate
this, N4BP1 ubiquitylation was analyzed in HEK293 cells. When we
co-transfected flN4BP1 with a Myc epitope-tagged ubiquitin expression
vector (31), a single Myc-ubiquitin immunoreactive band could be
detected after immunoprecipitation with anti-N4BP1 (Fig.
6A, lane 3). Its
mobility was consistent with its being a monoubiquitylated form of
flN4BP1. Strikingly, co-transfection with a Nedd4 expression vector
resulted in a marked increase in monoubiquitylated flN4BP1 (Fig.
6A, lane 4), despite the fact that the total
N4BP1 protein was equivalent in all lanes, as assessed by
immunoblotting with anti-N4BP1 (Fig. 6B, lanes
1-4). Thus, these results indicate a functional interaction
between Nedd4 and N4BP1 in vivo.
N4BP2 Is Polyubiquitylated in Vivo--
To assess in
vivo ubiquitylation of N4BP2 and N4BP3, His- and Xpress-tagged
forms of N4BP2 and N4BP3 were co-expressed with Myc-tagged ubiquitin in
HEK293 cells. Following immunoprecipitation with anti-His antibody and
Western blotting with anti-Myc antibody, multiple high molecular weight
forms could easily be discerned for N4BP2 (Fig.
7A, lanes 1 and
2 showing duplicate samples). In contrast, no such forms
were detected for N4BP3 (data not shown). To assess whether Nedd4
mediates N4BP2 polyubiquitylation in vivo, HEK293 cells were
co-transfected with Nedd4, N4BP2, and Myc-tagged ubiquitin expression
vectors. No discernable change in N4BP2 polyubiquitylation was detected
(Fig. 7A, lanes 3 and 4 showing
duplicate samples) despite clear overexpression of Nedd4 (Fig.
7C, lanes 3 and 4) compared with
control transfections (Fig. 7C, lanes 1 and
2). Equal loading was verified by reblotting with
anti-Xpress antibody (Fig. 7B, lanes 1 and
4). Overexpression of Nedd4 also did not result in any
detectable ubiquitylation of N4BP3 (data not shown). These data
correlate with the in vitro ubiquitylation results, in which
N4BP2 was polyubiquitylated and N4BP3 was not ubiquitylated at all.
However, there is no evidence that Nedd4 mediates N4BP3 polyubiquitylation in vivo.
Proteasome Inhibition Increases the Level of N4BP2 but Not
N4BP1--
Because proteins targeted for proteasomal degradation are
generally modified with chains of four or more ubiquitins, we reasoned that N4BP2 but not N4BP1 might be degraded by the proteasome. To assess
this, we determined the effect of proteasome inhibitors on steady state
levels of N4BP1 and N4BP2 expressed in HEK293 cells (Fig.
8). The steady state level of N4BP1, as
determined by immunoblotting of whole cell lysates with anti-N4BP1
antiserum, was not affected by any treatment (Fig. 8A). In
contrast, following treatment with different proteasome inhibitors and
immunoprecipitation and immunoblotting with anti-Xpress, a significant
increase in N4BP2 steady state levels was found (Fig. 8B,
bands marked by asterisk). Notably, levels of co-transfected
Xpress-tagged N4BP1-3 Have Distinct Subcellular Locations--
The functional
interaction between N4BP1 and Nedd4 in vivo, despite the
lack of co-immunoprecipitation, prompted us to determine the
subcellular location of N4BP1 and Nedd4 in transfected HEK293 cells
using immunofluorescence. In all transfected cells, N4BP1 was organized
into discrete circular structures, which were mainly located in the
nucleus and varied in number and size from cell to cell (Fig.
9, A and B). In
cells with high levels of transfected protein, there was also diffuse
staining in both the nucleus and cytoplasm. In contrast, Nedd4 was
found mostly throughout the cytoplasm (Fig. 9, C and
D), as reported previously (29). Co-transfection of the
N4BP1 and Nedd4 expression vectors did not alter the subcellular localization of either protein (Fig. 9, E-G). This small
degree of overlap (Fig. 9H) may underlie the failure to
detect co-immunoprecipitation.
We also examined the subcellular location of transfected N4BP2 and
N4BP3 in HEK293 cells. N4BP2 was found in an even distribution throughout the cytoplasm, similar to Nedd4 (Fig. 9I).
However, in cells co-transfected with Nedd4, there was some
redistribution of N4BP2 and Nedd4 into aggregates (Fig. 9,
J-L). Transfected N4BP3 was also cytoplasmic, but was
localized in vesicles of a wide range of sizes distributed throughout
the cytoplasm (Fig. 9, M and N). In cells
co-transfected with Nedd4, the location of Nedd4 protein was completely
altered (Fig. 9O). Rather than a uniform distribution
throughout the cytoplasm, all Nedd4 protein colocalized with N4BP3 in
the vesicular structures (Fig. 9P). The PY mutant of N4BP3
also was found in vesicles (Fig. 9Q), but in cells
co-transfected with Nedd4 the redistribution of Nedd4 was reduced
significantly (Fig. 9, R-T), again demonstrating the importance of the PY domain for the interaction between N4BP3 and Nedd4.
This study is a first step toward gaining insight into the roles
played by Nedd4 in mouse embryonic development. Three novel proteins
that are bona fide Nedd4 binding partners have been
identified from a yeast two-hybrid screen of a mid-gestation embryo
cDNA library. Two of these, N4BP1 and N4BP2, also have the capacity to be ubiquitylation substrates. For the third, N4BP3, we found no
discernable evidence of ubiquitylation. However, this protein clearly
associates with Nedd4 in cells, as assessed by co-immunoprecipitation and by the immunofluorescent co-localization of these proteins in
vesicular structures. Two recent reports have described screens in
which developmentally expressed proteins capable of physically interacting with Nedd4 were isolated (23, 28). One of these also was
shown to be a likely ubiquitylation substrate (27). However, none of
these were found in our screen, perhaps because of the use of different
screening methodologies and/or the use of a cDNA library from a
different stage of development.
Somewhat surprisingly, we found that the interaction of both N4BP1 and
N4BP2 with Nedd4 does not depend on proline-rich domains interacting
with the WW domains of Nedd4. N4BP1 contains three proline-rich
regions, but disruption of these had no effect on the in
vitro binding to Nedd4. The N4BP2 fragment we analyzed lacks
proline-rich domains altogether. A recent report on Rsp5, the yeast
homologue of Nedd4, has shown that its WW domains can bind target
proteins in a phosphorylation-dependent manner (18). It
will be interesting to determine whether a similar mechanism underlies
the Nedd4 interactions we have found.
For both N4BP1 and N4BP2, there was a good correlation between their
ubiquitylation in vitro and in cells. The in
vitro ubiquitylation of N4BP1 involves either the addition of a
single ubiquitin at a few sites or a single very short ubiquitin chain.
In transfected cells, only monoubiquitylation of N4BP1 was found. This
occurred in a Nedd4-dependent fashion and required the
amino-terminal region of N4BP1; the amino-terminally truncated form of
N4BP1 was not efficiently ubiquitylated in vivo, and
co-expression with Nedd4 increased only flN4BP1 monoubiquitylation
significantly. Only a few examples of Nedd4-mediated monoubiquitylation
have been described, and all involve viral proteins (32-34). Thus,
N4BP1 represents the first cellular protein identified as a Nedd4
monoubiquitylation substrate. This finding indicates that
monoubiquitylation is a true cellular function of Nedd4 and not an
aberrant activity co-opted by the viral replication machinery. In
contrast to N4BP1, Nedd4-mediated in vitro ubiquitylation of
N4BP2 resulted in an extensive range of higher molecular weight forms,
suggesting polyubiquitylation. Similarly, a wide range of very high
molecular weight ubiquitylated forms of N4BP2 was found in transfected
cells. Efficient in vivo ubiquitylation of N4BP2 was seen
even in the absence of co-transfected Nedd4, suggesting that N4BP2 may
be a substrate for other ubiquitin ligases in vivo, either
in addition to or instead of Nedd4. Alternatively, perhaps only limited
amounts of transfected N4BP2 are available for ubiquitylation in
vivo and endogenous Nedd4 levels in these cells are sufficient.
The clear functional interaction found between Nedd4 and N4BP1 was
surprising given that transfected N4BP1 protein was found predominantly
in the nucleus, and Nedd4 in the cytoplasm. However, it was shown
recently that Nedd4 is capable of entering the nucleus but its Rev-like
nuclear export sequence leads to a predominantly cytoplasmic location
(35). The transient presence of Nedd4 in the nucleus may be sufficient
to allow it to interact with N4BP1, as it does with the nuclear protein
hPRTB, which Nedd4 regulates through ubiquitylation (35).
Interestingly, hPRTB is found in discrete subnuclear structures, as is
true for N4BP1. We are currently determining whether the
N4BP1-containing nuclear bodies are the same as the hPRTB-containing
nuclear speckles, which are implicated in transcription and RNA
processing, or are related to other previously characterized classes of
nuclear bodies (36, 37). Nedd4-mediated monoubiquitylation of N4BP1 may
regulate N4BP1 assembly into these subnuclear structures. A recent
report described the monoubiquitylation of the Fanconi anemia protein
FANCD2 and showed that this modification is required for the formation
of a specific class of subnuclear structures, the ionizing
radiation-inducible foci that contain Fanconi anemia protein complexes
and BRCA1 (7). Although we detected no increase in the amount of
transfected N4BP1 contained in nuclear bodies, or in their size or
number, when monoubiquitylation was enhanced by overexpression of
Nedd4, perhaps only a low level of monoubiquitylation is necessary to
target N4BP1 to these structures. Alternatively, monoubiquitylation of
N4BP1 may regulate its activity within these structures.
In contrast to N4BP1 and N4BP2, N4BP3 is apparently not a
ubiquitylation substrate. mGrb10 also has been shown to bind to Nedd4
but not be targeted for ubiquitylation (38). These proteins may serve a
similar function to E6, which must physically associate with E6-AP to
allow ubiquitylation of the specific target protein p53 but does not
become ubiquitylated itself (12, 13). Alternatively, as has been shown
for annexin XIIIb (39), the function might be to localize or enrich
Nedd4 in a specific cellular compartment. N4BP3, which is assembled
into vesicles of varying sizes that are distributed throughout the
cytoplasm, is capable of relocating Nedd4 to these structures when the
two proteins are co-expressed. Unlike annexin XIIIb, which interacts
with the C2 domain of Nedd4, this relocalization is at least partially
dependent on the interaction of the PY domain of N4BP3 with the WW
domains of Nedd4, because there is some reversion to a general
cytoplasmic location when Nedd4 is co-transfected with the N4BP3 PY
mutant. We are currently investigating the nature of the
N4BP3-containing vesicles. The presence of a consensus
microsome-targeting signal (40) in the carboxyl terminus of both mouse
and human N4BP3 suggests that these vesicles might be related to
peroxisomes or other single-membrane organelles. Alternatively, these
vesicles might be part of the endocytic system that regulates turnover
of activated membrane receptors. Rsp5 has been shown to be essential in
yeast for the ubiquitylation of plasma membrane proteins, leading to
their internalization and targeting to the lysosome/vacuole for
degradation (4, 41). A possible role for N4BP3 in mammalian cells may
be to bring Nedd4 to the endocytic compartment and perhaps, by analogy
to E6, target the activity of Nedd4 to an as yet undetermined substrate.
In summary, we have identified three novel developmentally expressed
proteins that are able to interact functionally with Nedd4. Our
analysis has indicated a function for Nedd4 beyond polyubiquitylation
and proteasome degradation, involving monoubiquitylation of a novel
nuclear protein (N4BP1). Additionally, the PY-dependent interaction and co-localization with a second novel protein restricted to cytoplasmic vesicles (N4BP3) suggests a new function for Nedd4 in
the cytoplasmic compartment as well. Future studies will address the
developmental functions of these proteins and the importance of their
interaction with Nedd4.
We thank Jane P. Jensen, Alessandra
Magnifico, Swati Tiwari, and Shengyun Fang for invaluable discussions
and technical help, and Alfred Singer for critical reading of the manuscript.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Current address: Dept. of Molecular and Cellular Biology,
Medical Institute of Bioregulation, Kyushu University, Fukuoka
812-8582, Japan.
Published, JBC Papers in Press, November 20, 2001, DOI 10.1074/jbc.M110047200
The abbreviations used are:
E1, ubiquitin-activating enzyme;
E2, ubiquitin carrier protein;
E3, ubiquitin-protein isopeptide ligase;
EST, expressed sequence tag;
TBS, Tris-buffered saline;
TBST, Tris-buffered saline plus Tween 20;
Identification of Developmentally Expressed Proteins That
Functionally Interact with Nedd4 Ubiquitin Ligase*
,,
Experimental Immunology Branch, NCI,
National Institutes of Health, Bethesda, Maryland 20892-1360 and the
§ Regulation of Protein Function Laboratory, NCI, National
Institutes of Health, Bethesda, Maryland 20892-1152
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (-gal) were
identified. Plasmids were recovered according to MATCHMAKER protocols,
and transformed into yeast containing either pGBT9-Nedd4 or the pGBT9
empty vector. Inserts from plasmids that activated GAL4 transcription
with pGBT9-Nedd4 were excised from pGAD10 and subcloned into
pcDNA3.1 (Invitrogen, Carlsbad, CA), in frame with the
Xpress and 6-histidine (His) epitope tags.
S (Roche Molecular Biochemicals) for 1 h at
30 °C. Reactions were terminated with the addition of SDS-containing
reducing sample buffer, resolved by SDS-PAGE, and visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
[in a new window]
Fig. 1.
Structure of Nedd4 and Nedd4 constructs used
in this study. A, full-length Nedd4 protein containing
the amino-terminal C2 domain, three WW domains, and the
carboxyl-terminal HECT domain. B, an amino-terminally
truncated form of Nedd4, extending from amino acid position 199 to the
carboxyl terminus, used for yeast two-hybrid screening. C,
an amino-terminally truncated form of Nedd4, extending from amino acid
position 52 to the carboxyl terminus, used as a nearly complete Nedd4
for in vitro binding (GST pull-down experiments).
D, the amino-terminal half of Nedd4 used for in
vitro binding, extended from amino acid position 52 to 422. E, the carboxyl-terminal half of Nedd4 used for in
vitro binding, extended from amino acid position 423 to the
carboxyl terminus.
View larger version (23K):
[in a new window]
Fig. 2.
N4BP proteins identified in this study.
The four N4BP proteins are shown aligned with ESTs or known gene
sequences with which they exhibit significant homology. A,
the original N4BP1 isolate contains the carboxyl-terminal 685 amino
acids of the 893-amino acid full-length mouse protein (flN4BP1), which
is homologous to human KIAA0615. flN4BP1 was constructed using EST
AA444325 and a genomic fragment. The proline-rich domains in N4BP1
(thick black lines) are not conserved
in KIAA0615. B, the 374-amino acid original isolate of N4BP2
is homologous to the carboxyl region of a 1770-amino acid human protein
(flhN4BP2), deduced from KIAA1413 and EST AK001542. C, the
original N4BP3 isolate contains the middle 220 amino acids of the
537-amino acid full-length mouse protein (flN4BP3), which is homologous
to human KIAA0341. The proline-rich domains in N4BP3 (thick
black lines) are conserved in KIAA0341.
D, the original isolate of N4BP4 contains the
carboxyl-terminal 189 amino acids of the 638-amino acid protein, Plic2.
The ability (+) or inability ( 
) of specific N4BP proteins to bind or
to be ubiquitylated by Nedd4, either in vitro or in
vivo, is indicated in the columns to the
right. nd indicates not determined.
View larger version (49K):
[in a new window]
Fig. 3.
In vitro binding of
N4BP proteins to Nedd4. N4BP proteins were in vitro
translated with [35S]methionine and incubated with
bacterially produced GST-Nedd4, GST-Nedd4:N, GST-Nedd4:C, GST, or
GST-E6AP proteins bound to glutathione-Sepharose. Protein complexes
were collected by centrifugation, washed, and analyzed by SDS-PAGE and
autoradiography. 20% of the amount of each in vitro
translated protein used for binding (labeled 20% IVT) was
loaded as a control. A-C, N4BP1 (A), N4BP2
(B), and N4BP3 and KIAA0341 (C) each bind to
GST-Nedd4 (lane 2 in each panel) and GST-Nedd4:N (lane
3 in each panel) equally well, but show minimal binding to
GST-Nedd4:C (lane 4 in each panel) and GST-E6AP (lane
6 in each panel) and no binding to GST (lane 5 in each
panel). D, N4BP4 shows no binding to Nedd4. E,
N4BP1 carrying mutations in the PY motif (lane 6), in both
PPLP motifs (lane 8), or in the PY and PPLP motifs
(lane 10) binds Nedd4 identically to wild-type N4BP1
(lane 3). F, binding to Nedd4 by KIAA0341
carrying a mutated PY motif (lane 4) is greatly reduced
compared with wild-type KIAA0341 (lane 2).
View larger version (42K):
[in a new window]
Fig. 4.
In vitro ubiquitylation
of N4BP1-3. N4BP proteins were in vitro translated
with [35S]methionine and incubated in a ubiquitylation
reaction mix containing E1 activity, and with or without GST-Nedd4, E2
(UbcH5), and ubiquitin as indicated, followed by SDS-PAGE and
autoradiography. A, in the presence of all ubiquitylation
components (lane 2), N4BP1 shows only three higher molecular
weight bands (bracketed), indicating limited ubiquitylation.
In the absence of any component (lanes 1 and
3-5), there is no ubiquitylation. B, in the
presence of all ubiquitylation components (lane 2), N4BP2
shows a smear of higher molecular weight bands suggesting
polyubiquitylation. No higher molecular weight species are seen in the
absence of critical ubiquitylation reaction components (lanes
1 and 3-5). C, human N4BP3 (KIAA0341) is
not in vitro ubiquitylated.
View larger version (44K):
[in a new window]
Fig. 5.
In vivo binding of
N4BP1-3 to Nedd4. Expression vectors for flN4BP1, N4BP2,
and flhN4BP3 (KIAA0341) were transfected into HEK293 cells, with or
without Nedd4 overexpression. A, cell lysates were
immunoprecipitated using anti-N4BP1 antiserum (for N4BP1) or anti-His
monoclonal (for N4BP2 and N4BP3), followed by immunoblotting with
anti-Nedd4 antiserum. The arrow indicates the position of
co-immunoprecipitated Nedd4 found for N4BP2 (lane 5) and
KIAA0341 (lane 7). No co-immunoprecipitated Nedd4 was found
for N4BP1 (lane 2). A greatly reduced amount of
co-immunoprecipitated Nedd4 was found for the PY mutated N4BP3
(lane 9). B, the amount of each
immunoprecipitated N4BP protein was determined by reblotting with
anti-N4BP1 antiserum for N4BP1 and anti-Xpress monoclonal antibody for
N4BP2 and N4BP3. Asterisks mark the position of the bands
corresponding to each immunoprecipitated protein. C, the
amount of Nedd4 expression in each sample was determined by
immunoblotting whole cell lysates with anti-Nedd4 antiserum.
View larger version (39K):
[in a new window]
Fig. 6.
In vivo
monoubiquitylation of N4BP1 by Nedd4. HEK293 cells were
transfected with expression vectors for either N4BP1 or flN4BP1, with
or without Nedd4 overexpression, in the presence of Myc-tagged
ubiquitin. N4BP1 and flN4BP1were immunoprecipitated from cell lysates
using anti-N4BP1 antiserum. A, immunoblotting with anti-Myc
monoclonal antibody reveals a single band corresponding to a
monoubiquitylated form of flN4BP1 (lane 3). The amount of
monoubiquitylated flN4BP1 is greatly enhanced by Nedd4 overexpression
(lane 4). The truncated form of N4BP1 is not efficiently
ubiquitylated (lanes 1 and 2). B, an
equivalent amount of N4BP1 or flN4BP1 protein was expressed
(lanes 1 and 2 or lanes 3 and 4) as determined by reblotting with anti-N4BP1
antiserum. C, significant Nedd4 overexpression was achieved
(lanes 2, 4, and 5), as determined by
analyzing whole cell lysates by immunoblotting with anti-Nedd4
antiserum.
View larger version (41K):
[in a new window]
Fig. 7.
In vivo
polyubiquitylation of N4BP2. HEK293 cells were transfected
with an expression vector for His and Xpress-tagged N4BP2, with or
without Nedd4 overexpression, in the presence of Myc-tagged ubiquitin.
N4BP2 protein was immunoprecipitated with anti-His monoclonal, and
duplicate samples were analyzed. A, immunoblotting with
anti-Myc monoclonal revealed many higher molecular weight species,
which may represent polyubiquitylated forms of N4BP2 (lanes
1 and 2). There was no apparent enhancement with Nedd4
overexpression (lanes 3 and 4). B,
equivalent expression of N4BP2 was found in each sample (lanes
1-4), as determined by reblotting with anti-Xpress monoclonal.
C, significant Nedd4 overexpression was achieved
(lanes 3-6), as determined by analyzing whole cell lysates
by immunoblotting with anti-Nedd4 antiserum.
-gal, a stable protein insensitive to proteasome
inhibition used as a control, were unaffected (Fig. 8B,
arrow). Thus N4BP2, which is modified by multiple ubiquitin
moieties both in cells and in vitro, is subject to
proteasome-mediated degradation, whereas N4BP1, which demonstrates a
low stoichiometry of ubiquitylation in vitro and
Nedd4-mediated monoubiquitylation in cells, is not proteasome-degraded.
View larger version (41K):
[in a new window]
Fig. 8.
Effects of proteasome inhibition on N4BP1 and
N4BP2 levels. HEK293 cells were transfected with an expression
vector for flN4BP1, or expression vectors for His and Xpress-tagged
N4BP2 and -gal. 40 h after transfection, cells were incubated
with the indicated proteasome inhibitors for 6 h. A,
for flN4BP1, whole cell lysates were analyzed by immunoblotting using
anti-N4BP1 antiserum. Steady state levels of flN4BP1 (band marked by
asterisk) were unchanged following proteasome inhibition.
B, for N4BP2 and -gal, proteins were immunoprecipitated
and immunoblotted with anti-Xpress monoclonal. The arrow
marks the band corresponding to -gal, and the asterisk
marks the band for N4BP2. There was a marked increase in N4BP2 steady
state levels following proteasome inhibition, but -gal levels were
unchanged.
View larger version (107K):
[in a new window]
Fig. 9.
Subcellular localization of transfected N4BP
proteins. HEK293 cells were transfected with expression
vectors for flN4BP1, and for His and Xpress-tagged N4BP2 and
full-length human N4BP3 (KIAA0341). Indirect immunofluorescence was
done for flN4BP1 using anti-N4BP1 antiserum, and for N4BP2 and N4BP3
using anti-His monoclonal. In some experiments HA-tagged Nedd4 was
co-transfected and detected using either anti-Nedd4 antiserum or
anti-HA monoclonal antibody. Cells were stained with
4',6-diamidino-2-phenylindole to visualize nuclei. A and
B, N4BP1 was found in the nucleus, mainly in discrete
circular structures. C and D, Nedd4 was found in
the cytoplasm. E-H, there was no apparent change in the
distribution of N4BP1 or Nedd4 when cells were co-transfected with both
expression vectors. As shown in H, very little overlap in
distribution was apparent. A few N4BP1 dots that seem to be located
outside of the nucleus were seen. I, transfected N4BP2
protein showed a cytoplasmic distribution when transfected alone.
J-L, when co-transfected with Nedd4, N4BP2 formed
cytoplasmic aggregates that co-localized with Nedd4. M,
transfected KIAA0341 protein localized to cytoplasmic vesicles of a
wide range of sizes when transfected alone. N-P, when
co-transfected with Nedd4, KIAA0341 retained its distribution in
vesicles. However, Nedd4 was relocalized from a general cytoplasmic
distribution to the cytoplasmic vesicles containing KIAA0341.
Q, transfected KIAA0341 carrying the PY domain mutation
still localized to cytoplasmic vesicles. R-T, when
co-transfected with Nedd4, the KIAA0341 PY mutant retained its
distribution in vesicles but the amount of Nedd4 relocalized to the
cytoplasmic vesicles was less than with the wild-type KIAA0341.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Bldg. 10/Rm.
4B-36, 10 Center Dr., Bethesda, MD 20892-1360. Tel.: 301-435-6476; Fax:
301-496-0887; E-mail: mkuehn@mail.nih.gov.
ABBREVIATIONS
-gal, -galactosidase;
GST, glutathione S-transferase;
BSA, bovine serum albumin;
ATPS, adenosine
5'-O-(thiotriphosphate);
PBS, phosphate-buffered saline;
HA, hemagglutinin.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Muller, S.,
Hoege, C.,
Pyrowolakis, G.,
and Jentsch, S.
(2001)
Nat. Rev. Mol. Cell. Biol.
2,
202-210
2.
Weissman, A. M.
(2001)
Nat. Rev. Mol. Cell. Biol.
2,
169-178
3.
Hershko, A.,
and Ciechanover, A.
(1998)
Annu. Rev. Biochem.
67,
425-479
4.
Hicke, L.
(1999)
Trends Cell Biol.
9,
107-112
5.
Shih, S. C.,
Sloper-Mould, K. E.,
and Hicke, L.
(2000)
EMBO J.
19,
187-198
6.
Pham, A. D.,
and Sauer, F.
(2000)
Science
289,
2357-2360
7.
Garcia-Higuera, I.,
Taniguchi, T.,
Ganesan, S.,
Meyn, M. S.,
Timmers, C.,
Hejna, J.,
Grompe, M.,
and D'Andrea, A. D.
(2001)
Mol. Cell.
7,
249-262
8.
Jentsch, S.,
and Pyrowolakis, G.
(2000)
Trends Cell. Biol.
10,
335-342
9.
Hochstrasser, M.
(2000)
Nat. Cell Biol.
2,
E153-E157
10.
Ciechanover, A.,
Orian, A.,
and Schwartz, A. L.
(2000)
Bioessays
22,
442-451
11.
Joazeiro, C. A.,
and Weissman, A. M.
(2000)
Cell
102,
549-552
12.
Scheffner, M.,
Huibregtse, J. M.,
Vierstra, R. D.,
and Howley, P. M.
(1993)
Cell
75,
495-505
13.
Scheffner, M.,
Nuber, U.,
and Huibregtse, J. M.
(1995)
Nature
373,
81-83
14.
Harvey, K. F.,
and Kumar, S.
(1999)
Trends Cell Biol.
9,
166-169
15.
Chen, H. I.,
and Sudol, M.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
7819-7823
16.
Espanel, X.,
and Sudol, M.
(1999)
J. Biol. Chem.
274,
17284-17289
17.
Bedford, M. T.,
Reed, R.,
and Leder, P.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
10602-10607
18.
Lu, P. J.,
Zhou, X. Z.,
Shen, M.,
and Lu, K. P.
(1999)
Science
283,
1325-1328
19.
Staub, O.,
Dho, S.,
Henry, P.,
Correa, J.,
Ishikawa, T.,
McGlade, J.,
and Rotin, D.
(1996)
EMBO J.
15,
2371-2380
20.
Staub, O.,
Gautschi, I.,
Ishikawa, T.,
Breitschopf, K.,
Ciechanover, A.,
Schild, L.,
and Rotin, D.
(1997)
EMBO J.
16,
6325-6336
21.
Kumar, S.,
Tomooka, Y.,
and Noda, M.
(1992)
Biochem. Biophys. Res. Commun.
185,
1155-1161
22.
Weston, A. D.,
and Underhill, T. M.
(2000)
Mech. Dev.
94,
247-250
23.
Jolliffe, C. N.,
Harvey, K. F.,
Haines, B. P.,
Parasivam, G.,
and Kumar, S.
(2000)
Biochem. J.
351,
557-565
24.
Zhu, H.,
Kavsak, P.,
Abdollah, S.,
Wrana, J. L.,
and Thomsen, G. H.
(1999)
Nature
400,
687-693
25.
Cornell, M.,
Evans, D. A.,
Mann, R.,
Fostier, M.,
Flasza, M.,
Monthatong, M.,
Artavanis-Tsakonas, S.,
and Baron, M.
(1999)
Genetics
152,
567-576
26.
Qiu, L.,
Joazeiro, C.,
Fang, N.,
Wang, H. Y.,
Elly, C.,
Altman, Y.,
Fang, D.,
Hunter, T.,
and Liu, Y. C.
(2000)
J. Biol. Chem.
275,
35734-35737
27.
Pham, N.,
and Rotin, D.
(2001)
J. Biol. Chem.
276,
46995-47003
28.
Pham, N.,
Cheglakov, I.,
Koch, C. A.,
de Hoog, C. L.,
Moran, M. F.,
and Rotin, D.
(2000)
Curr. Biol.
10,
555-558
29.
Hatakeyama, S.,
Jensen, J. P.,
and Weissman, A. M.
(1997)
J. Biol. Chem.
272,
15085-15092
30.
Kleijnen, M. F.,
Shih, A. H.,
Zhou, P.,
Kumar, S.,
Soccio, R. E.,
Kedersha, N. L.,
Gill, G.,
and Howley, P. M.
(2000)
Mol. Cell
6,
409-419
31.
Ellison, M. J.,
and Hochstrasser, M.
(1991)
J. Biol. Chem.
266,
21150-21157
32.
Patnaik, A.,
Chau, V.,
and Wills, J. W.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
13069-13074
33.
Schubert, U.,
Ott, D. E.,
Chertova, E. N.,
Welker, R.,
Tessmer, U.,
Princiotta, M. F.,
Bennink, J. R.,
Krausslich, H. G.,
and Yewdell, J. W.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
13057-13062
34.
Ott, D. E.,
Coren, L. V.,
Chertova, E. N.,
Gagliardi, T. D.,
and Schubert, U.
(2000)
Virology
278,
111-121
35.
Hamilton, M. H.,
Tcherepanova, I.,
Huibregtse, J. M.,
and McDonnell, D. P.
(2001)
J. Biol. Chem.
276,
26324-26331
36.
Matera, A. G.
(1999)
Trends Cell Biol.
9,
302-309
37.
Zhong, S.,
Salomoni, P.,
and Pandolfi, P. P.
(2000)
Nat. Cell Biol.
2,
E85-E90
38.
Morrione, A.,
Plant, P.,
Valentinis, B.,
Staub, O.,
Kumar, S.,
Rotin, D.,
and Baserga, R.
(1999)
J. Biol. Chem.
274,
24094-24099
39.
Plant, P. J.,
Lafont, F.,
Lecat, S.,
Verkade, P.,
Simons, K.,
and Rotin, D.
(2000)
J. Cell Biol.
149,
1473-1484
40.
Recalcati, S.,
Menotti, E.,
and Kuhn, L. C.
(2001)
J. Cell Sci.
114,
1625-1629
41.
Rotin, D.,
Staub, O.,
and Haguenauer-Tsapis, R.
(2000)
J. Membr. Biol.
176,
1-17
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Al Sorkhy, R. Craig, B. Market, R. Ard, and L. A. Porter The Cyclin-dependent Kinase Activator, Spy1A, Is Targeted for Degradation by the Ubiquitin Ligase NEDD4 J. Biol. Chem., January 30, 2009; 284(5): 2617 - 2627. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Leon, Z. Erpapazoglou, and R. Haguenauer-Tsapis Ear1p and Ssh4p Are New Adaptors of the Ubiquitin Ligase Rsp5p for Cargo Ubiquitylation and Sorting at Multivesicular Bodies Mol. Biol. Cell, June 1, 2008; 19(6): 2379 - 2388. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Usami, S. Popov, E. Popova, and H. G. Gottlinger Efficient and Specific Rescue of Human Immunodeficiency Virus Type 1 Budding Defects by a Nedd4-Like Ubiquitin Ligase J. Virol., May 15, 2008; 82(10): 4898 - 4907. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Oberst, M. Malatesta, R. I. Aqeilan, M. Rossi, P. Salomoni, R. Murillas, P. Sharma, M. R. Kuehn, M. Oren, C. M. Croce, et al. The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch PNAS, July 3, 2007; 104(27): 11280 - 11285. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Ingham, K. Colwill, C. Howard, S. Dettwiler, C. S. H. Lim, J. Yu, K. Hersi, J. Raaijmakers, G. Gish, G. Mbamalu, et al. WW Domains Provide a Platform for the Assembly of Multiprotein Networks Mol. Cell. Biol., August 15, 2005; 25(16): 7092 - 7106. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Plafker, K. S. Plafker, A. M. Weissman, and I. G. Macara Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import J. Cell Biol., November 22, 2004; 167(4): 649 - 659. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. Xu, B. Liao, Q. J. Zhang, B. B. Wang, H. Li, X. M. Zhong, H. Z. Sheng, Y. X. Zhao, Y. M. Zhao, and Y. Jin Wwp2, an E3 Ubiquitin Ligase That Targets Transcription Factor Oct-4 for Ubiquitination J. Biol. Chem., May 28, 2004; 279(22): 23495 - 23503. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Feng, C. W. Scott, N.-H. Cho, H. Nakamura, Y.-H. Chung, M. J. Monteiro, and J. U. Jung Kaposi's Sarcoma-Associated Herpesvirus K7 Protein Targets a Ubiquitin-Like/Ubiquitin-Associated Domain-Containing Protein To Promote Protein Degradation Mol. Cell. Biol., May 1, 2004; 24(9): 3938 - 3948. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Magnifico, S. Ettenberg, C. Yang, J. Mariano, S. Tiwari, S. Fang, S. Lipkowitz, and A. M. Weissman WW Domain HECT E3s Target Cbl RING Finger E3s for Proteasomal Degradation J. Biol. Chem., October 31, 2003; 278(44): 43169 - 43177. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. L. Xu, Y. Shi, G. Petrovics, C. Sun, M. Makarem, W. Zhang, I. A. Sesterhenn, D. G. McLeod, L. Sun, J. W. Moul, et al. PMEPA1, an Androgen-regulated NEDD4-binding Protein, Exhibits Cell Growth Inhibitory Function and Decreased Expression during Prostate Cancer Progression Cancer Res., August 1, 2003; 63(15): 4299 - 4304. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Watanabe, S. Wachi, and T. Fujita Identification and Characterization of BCL-3-binding Protein: IMPLICATIONS FOR TRANSCRIPTION AND DNA REPAIR OR RECOMBINATION J. Biol. Chem., July 3, 2003; 278(28): 26102 - 26110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Suzuki, G. Murakami, M. Fukuchi, T. Shimanuki, Y. Shikauchi, T. Imamura, and K. Miyazono Smurf1 Regulates the Inhibitory Activity of Smad7 by Targeting Smad7 to the Plasma Membrane J. Biol. Chem., October 11, 2002; 277(42): 39919 - 39925. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |