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INTRODUCTION |
The posttranslational modification of proteins by ubiquitination
has been shown to play an important role in the regulation of cell
cycle progression, signal transduction, and transcriptional events
within the cell. Covalent attachment of the 76-amino acid polypeptide
ubiquitin to a substrate protein is a catastrophic signal,
targeting the substrate for rapid degradation (1, 2). The specific
enzymes involved in this process,
E1,1 E2, and E3, have
been studied in great detail (3). A human ubiquitin-activating enzyme
(E1) is responsible for the ATP-dependent activation of the
ubiquitin polypeptide. Activated ubiquitin is subsequently transferred
to a downstream ubiquitin carrier protein (E2), and in many cases to a
ubiquitin-protein isopeptide ligase (E3), which mediates the final
transfer of activated ubiquitin to a substrate protein. Evidenced by
the numerous examples of cellular dysregulation resulting from aberrant
ubiquitination (4, 5), this ultimate enzyme-substrate recognition step is crucial for cellular homeostasis. Accordingly, there is of late a
heightened level of interest in defining the mechanisms that govern the
target specificity of the various E3 ligases and how this event is
regulated in target cells.
The experiments that have formed the foundation of our understanding of
the role of E3 ligases were those that describe E6-associated protein and its ability to cooperate with the viral E6 protein in ubiquitinating p53 following human papillomavirus infection (6).
This work led to the discovery of a family of proteins with sequence
homology to E6-associated protein, the homology to
E6-associated protein at the carboxyl
terminus (hect) family of proteins (7), and the observation
that the amino terminus is the primary determinant of target
specificity. Recent work demonstrating RNA polymerase II ubiquitination
by yeast RSP5, Smad ubiquitination by Smurf1, and Notch ubiquitination by Itch confirm the importance of the amino terminus in target selection (8-10).
Nedd4 is a hect E3 ubiquitin ligase enzyme the transcript of which was
originally identified to be developmentally down-regulated in neural
precursor cells (11). The domain structure of hRPF1/Nedd4 places this
E3 ligase in the WWhect subclass of ubiquitin ligases. This subclass of
enzymes is characterized by 2-4 copies of a WW protein-protein
interaction domain (12), followed by a conserved hect domain. The hect
domain is the catalytic domain responsible for ubiquitination, an
activity that is absolutely dependent upon an invariant cysteine
residue located within the active site (7). Similar to many other human
WWhect E3 ligases, hRPF1/Nedd4 contains a C2/CaLB domain at the amino
terminus, which is responsible for mediating membrane localization in
response to calcium (13).
Although hRPF1/Nedd4 contains a hect domain, implying that it is
involved in ubiquitination, many studies performed thus far with
putative targets have not been illuminating with respect to the
function of the enzyme or how the activity is regulated. To date, the
best characterized substrate of Nedd4 is the rat sodium epithelial
channel (14-16). These studies, which demonstrate that Nedd4
can interact with and regulate the turnover of the sodium epithelial
channel, imply a nonnuclear function of this enzyme. This contention is
supported by additional work that indicates that Nedd4 and the
cytoplasmic adapter protein mGrb10 interact (17). However, recent
studies from our laboratories have demonstrated that hRPF1/Nedd4 may
have roles in the nucleus. Specifically, it has been shown that 1) a
Nedd4 homolog, yeast RSP5, is responsible for the ubiquitination of the
large subunit of RNA polymerase II in response to DNA damage (18), and
2) overexpression of hRPF1/Nedd4 alters the transcriptional activity of
the progesterone receptor (19). In addition, an erythroid-specific
transcription factor, NF-E2, has been shown to physically associate
with hRPF1/Nedd4 (20), although the significance of this interaction
remains to be determined. Cumulatively, these data suggest that
hRPF1/Nedd4 can modulate target protein ubiquitination in both the
cytoplasm and the nucleus. However, because hRPF1/Nedd4 is localized
predominantly in the cytoplasm (21), the physiological significance of
the interaction between hRPF1/Nedd4 and nuclear proteins is unclear. In
an effort to evaluate the potential roles of hRPF1/Nedd4 in the nucleus
and how these activities are manifest, we have undertaken a strategy to
identify additional nuclear substrates of hRPF1/Nedd4 with a view to
(a) confirming that this enzyme can interact with and
regulate the stability of nuclear proteins and (b) defining a mechanism by which this cytoplasmic ubiquitin ligase can exert its
activity in the nucleus.
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EXPERIMENTAL PROCEDURES |
Antibodies and Reagents--
Splicing factor SC35
monoclonal antibody was purchased from Sigma. Anti-c-Myc
monoclonal antibody (9E10) was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). All horseradish peroxidase-conjugated secondary
antibodies and ECL reagents were obtained from Amersham Pharmacia
Biotech. Anti-mouse Texas Red-conjugated secondary antibodies, horse serum, and VectaShield with DAPI
(4',6-diamidino-2-phenylindole) were purchased from Vector Laboratories
(Burlingame, CA).
Plasmids--
The complete 5' coding sequence of hRPF1/Nedd4 (aa
1-900) was obtained using 5' rapid amplification of cDNA
ends, polymerase chain reaction-amplified, and subcloned into an
incomplete RPF1/Nedd4 cDNA (derived from pBKC-hRPF1) (19) using
standard subcloning procedures. hRPF1/Nedd4 deletion constructs
encoding C2 (aa 1-192), WW (aa 173-564), hect (aa 507-900), aa
293-900, aa 309-900, and aa 404-900 were subcloned by polymerase
chain reaction from full-length hRPF1/Nedd4 constructs, and all
sequences were verified by sequence analysis. To create the
hRPF1/Nedd4-C867A mutant, we utilized the SacI site just
upstream of amino acid 867 for construct preparation. Briefly, a primer
was designed (5'-gccaagagctcataccgcttttaatcgcc-3') that allowed
polymerase chain reaction amplification of amino acids 862-900,
resulting in the incorporation of a two-base substitution that changed
cysteine 867 to alanine. The SacI-NotI fragment
containing the C867A amino acid change was incorporated into the
context of the full-length hRPF1/Nedd4 using standard subcloning
procedures. Site-directed mutagenesis was used to introduce the PY
mutation in hPRTB (P40A/Y42A), and the mutant nuclear export
sequence (NES) (L307A/I309A) in hRPF1/Nedd4-C867A. Primers used
were as follows: PRTB-PYmut, 5'-ccgatgctccagctgccgcctcagagctc-3'(sense)
and 5'-gagctctgaggcggcagctggagcatcgg-3' (antisense);
hRPF1/Nedd4-C867A-mutNES,
5'-gaattgaatgccagagccaccgcttttggaaattcagccg-3' (sense) and
5'-cggctgaatttccaaaagcggtggctctggcattcaattc-3' (antisense). The
integrity of all constructs was verified by sequencing.
Enhanced green fluorescent protein (EGFP) fusion constructs were the
result of subcloning hPRTB (from the library vector pGADGH) into
the EcoRI-BamHI sites of pEGFP-C1
(CLONTECH). To create Myc-tagged fusions, we
inserted a BamHI-BamHI fragment of hPRTB cDNA
(containing extra 5' BamHI-EcoRI linker sequence:
ggatccccgaattc) into the BamHI site of
pcDNA3-5×Myc vector.
Cell Culture and Transfections--
HeLa cells were cultured in
minimal essential medium (Life Technologies, Inc.) supplemented with
10% fetal bovine serum, 0.1 mM nonessential amino acids,
and 1 mM sodium pyruvate and maintained in a humidified
incubator at 37 °C, 5% CO2. Cells were transiently transfected using Lipofectin (Life Technologies, Inc.) for 4 h and
allowed to recover for 24-48 h prior to harvest and analysis.
Yeast Two Hybrid Screen--
A cDNA encoding the amino
terminus of hRPF1/Nedd4 (NW, aa 26-506) was subcloned into the vector
pGBT9 (CLONTECH) and introduced by standard lithium
acetate protocol into HF7c cells (CLONTECH). A HeLa
Matchmaker library was sequentially transformed, and colonies were
grown on selective plates containing 1 mM 3-aminotriazole. A HeLa cervical carcinoma cDNA library was chosen based upon
previous observations (19) and immunodetection of endogenous
hRPF1/Nedd4 protein product in HeLa cells. His+ clones were isolated
after 10 days of growth at 30 °C, restreaked onto selective plates
(1 mM 3-aminotriazole), and grown for 3 days prior to
-galactosidase filter lift assays. Library and bait plasmids were
subsequently cotransformed into Y190 yeast cells to verify phenotype
and to quantitate interaction using a liquid
-galactosidase assay.
Briefly, a mid-logarithmic phase culture
(A600 = 0.5-0.8) was pelleted, washed,
and subjected to two cycles of freeze-thaw lysis. The
-galactosidase
activity of the lysate was measured at A578 and quantitated using CPRG (chlorophenol
red-
-D-galactopyranoside) as a substrate.
Glutathione S-Transferase (GST)-Pulldown Interaction
Assays--
hPRTB was subcloned into pcDNA3 (Invitrogen), in
vitro transcribed/translated, and radiolabeled in a rabbit
reticulocyte lysate system (TNT, Promega). hRPF1/Nedd4 or deletions
thereof (C2, aa 1-192; WW, aa 173-564; hect, aa 507-900) were fused
to GST, expressed, and purified from bacteria. Purified GST fusion
proteins were bound to glutathione-Sepharose (Amersham Pharmacia
Biotech) and incubated with radiolabeled hPRTB in NETN-A (25 mM Tris 8.0, 75 mM NaCl, 0.1% Nonidet P-40, 2 mM EDTA) overnight at 4 °C. Bound proteins were washed
in NETN-B (150 mM NaCl), eluted, and analyzed by SDS-PAGE
followed by autoradiography.
In Vitro Ubiquitination Assays--
Assays were performed
essentially as described previously (18). Briefly, an in
vitro translated, radiolabeled substrate was incubated with BL21
bacterial extracts overexpressing E1 and an E2 (UbcH5B). Reaction
mixtures contained 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1 mM dithiothreitol, 2 mM ATP, 2 mM MgCl2, 2 µg of
bovine ubiquitin (Sigma), and 500 ng of purified E3 enzyme (either
yRSP5 or hRPF1/Nedd4-whect). In some cases, an ATP regenerating system
was also included, consisting of 0.1 M phosphocreatine, 3.5 units/ml creatine phosphokinase, and 0.6 units/ml inorganic pyrophosphatase. After incubation at 30 °C for 1 h, reactions were terminated with 3× SDS-PAGE sample buffer, resolved, and detected
by SDS-PAGE followed by autoradiography.
Detection of in Vivo Ubiquitin Conjugates--
Following a
previously published procedure (15), HeLa cells were transiently
transfected with mammalian expression plasmids for His-tagged ubiquitin
and a Myc-tagged substrate (hPRTB or PY mutant). Thirty-six hours after
transfection, cells were harvested in lysis buffer (PBS, 1% Triton
X-100, 10% glycerol). The insoluble fraction was removed by a high
speed spin, and the clarified supernatant was denatured by addition of
2% SDS followed by boiling for 5 min. Denatured extract was diluted
with 12 volumes of lysis buffer and incubated with 50 µl of
nickel-nitrilotriacetic acid resin (Qiagen) for 4 h at
4 °C. After thorough washing with lysis buffer containing 300 mM NaCl and 40 mM imidazole, His-tagged
proteins were eluted with 3× SDS sample buffer and analyzed by Western immunoblot analysis using an anti-c-Myc antibody, 9E10.
Pulse-Chase Analysis--
HeLa cells (~70-80% confluency)
that had been plated in 60 mM dishes were transiently
transfected with 2.7 µg of a Myc-hPRTB or Myc-hPRTB-PYmut expression
plasmid and 20 ng of the internal control Myc-EGFP. In experiments
assaying the effect of exogenous hRPF1/Nedd4 on PRTB stability, 1.3 µg of pc:RPF1 or pc:RPF1-C867A (or an equimolar amount of empty
pcDNA3 vector) was cotransfected as well. Twenty-four hours
posttransfection, cells were washed with PBS and incubated in
methionine- and cysteine-free medium for 30 min. A radioactive mixture
of methionine and cysteine (175 µCi of Tran35S-Label,
ICN) was used to metabolically label cells for 2 h, after which
the medium was removed and replaced with cold medium containing an excess of methionine and cysteine (3 and 1 mM,
respectively) to chase for indicated time points. At each time point,
cells were harvested in PBS, 0.5% Triton X-100 plus protease
inhibitors, flash-frozen, and subsequently immunoprecipitated with an
antibody directed against the Myc tag (9E10, Santa Cruz Biotechnology). Immunoprecipitates were analyzed by SDS-PAGE and autoradiography and
quantitated using a phosphorimager.
Indirect Immunolocalization--
HeLa cells were transiently
transfected with an expression plasmid for an EGFP-hPRTB (or PY mutant)
fusion protein and plated onto 25-mm round glass coverslips. Cells were
fixed using 4% paraformaldehyde for 10 min and permeabilized in 0.5%
Triton X-100 for 10 min. Samples were blocked in PBS containing 10%
horse serum and then incubated with anti-SC35 (Sigma) at a dilution of
1:2000 in PBS/2% horse serum, followed by incubation with anti-mouse
Texas Red-conjugated secondary antibody at a 1:75 dilution in PBS/2%
horse serum. Coverslips were mounted in VectaShield plus DAPI
(4',6-diamidino-2-phenylindole). Localization of transfected
Myc-hRPF1/Nedd4 deletion or mutation constructs was performed
essentially as described above, only using the anti-c-Myc antibody
(9E10) at a 1:2000 dilution. Indicated samples were treated with 20 ng/ml of leptomycin B for 4 h prior to fixation and sample
preparation. A Zeiss LSM410 laser scanning confocal microscope with a
krypton/argon laser (Carl Zeiss Inc., Thornwood, NY) was used for
confocal microscopy.
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RESULTS |
Identification of Potential Substrates for hRPF1/Nedd4 Using a
Yeast Two Hybrid Screen--
Several nuclear proteins have been shown
to be ubiquitinated by hRPF1/Nedd4 in vitro; however, a
validated target of this enzyme in the nucleus has not been established
in intact mammalian cells. With the goal of identifying bona
fide cellular substrates of the catalytic activity of hRPF1/Nedd4,
we used the amino terminus of hRPF1/Nedd4 in a yeast two hybrid screen
to identify novel binding partners, a subset of which we would predict
to be ubiquitination substrates.
As a result of our screen, six cDNAs were isolated, all encoding a
17-kDa proline-rich protein, which specifically interact with aa
26-506 of hRPF1/Nedd4 (Fig.
1A). Homology searches using BLAST programs indicated that this 17-kDa protein is identical to
KIAA0058, a cDNA isolated from a myeloid cell line, KG-1. We and
others have termed this human cDNA, hPRTB (Fig. 1B),
based upon its high amino acid identity with mouse proline-rich
transcript, brain-expressed (PRTB) protein, which was isolated
in a gene trap screen as a transcript expressed in the developing mouse
inner ear (22). Although its amino acid sequence does not share
significant sequence homology with that of other proteins, the most
notable feature of hPRTB is its proline-rich composition (18%).

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Fig. 1.
hRPF1/Nedd4 Interacts (via its WW
domains) with the PPAY motif of hPRTB. A, yeast two
hybrid interaction between aa 26-506 of hRPF1/Nedd4 and hPRTB. Yeast
Y190 cells were cotransformed with plasmids encoding Gal4DBD- NW and
hPRTB-AD. Midlogarithmic cultures were analyzed in triplicate for LacZ
activity using chlorophenol red- -D-galactopyranoside
(CPRG) as a substrate. Data are represented as a percentage of the
positive control interaction, pVA3-TD1 (p53 and SV40-T antigen).
B, protein sequence for human PRTB. The PPAY motif, which
was mutated in this study, is indicated by the outlined
rectangle. The numerous proline residues are highlighted by
boldface type. C, hRPF1/Nedd4 deletion constructs
encoding NW (aa 26-506), C2 (aa 1-192), WW (aa 173-564), hect (aa
507-900), and whect (aa 193-900). D, GST-pulldown
interactions between GST-hRPF1/Nedd4 and Myc-hPRTB. GST alone, GST
fusions of hRPF1/Nedd4 (or indicated portions), or GST-yRSP5 was
immobilized on glutathione-Sepharose and incubated with
35S-labeled Myc-PRTB or Myc-PRTB-PYmut. Bead-bound proteins
were analyzed by SDS-PAGE and autoradiography. Fusion proteins used
for GST-pulldown interaction studies are shown in the lower
panel. Approximately equivalent microgram amounts of GST or GST
fusion proteins were resolved by SDS-PAGE and detected by Coomassie
Blue staining. WT, wild type.
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To independently verify that hPRTB does indeed interact with
full-length hRPF1/Nedd4, we assayed the ability of in vitro
translated, [35S]methionine-labeled Myc-hPRTB to interact
with recombinant GST fusions of hRPF1/Nedd4 (see schematic in Fig.
1C). Myc-hPRTB was able to interact with full-length
hRPF1/Nedd4 or yRSP5, a yeast homolog that has a domain structure
similar to that of hRPF1 (containing only three WW domains). The WW
domains of hRPF1/Nedd4 were sufficient for interaction with hPRTB; it
is important to note that the greater hPRTB signal associated with
GST-WW (Fig. 1D) is likely a reflection of the greater molar
amount of GST-WW used. Neither the hect domain nor the C2 domain
resulted in any detectable interaction (Fig. 1D). WW domains
are predicted to interact with proline-containing consensus sequences,
which are either PPXY, PPLP, or PGM (12, 23-25).
Examination of the proline-rich regions within hPRTB revealed a
consensus PPAY motif located in the central portion of the protein (Fig. 1B). It is interesting to note that the WWhect ligase,
Smurf1, similarly binds to a PPAY motif in its substrate, Smad1 (9). With the prediction that this conserved motif may mediate the interaction of hPRTB with hRPF1/Nedd4, we substituted the second proline and subsequent tyrosine with alanine and assayed for the ability of this PY mutant to interact with hRPF1/Nedd4. The two-amino acid substitution within the PPAY motif in hPRTB was able to disrupt the ability of hPRTB to bind to hRPF1/Nedd4 (Fig. 1D),
further demonstrating that the WW domains of hRPF1/Nedd4 directly
interact with the PPAY motif of hPRTB. The direct association of a WW
domain-containing enzyme and a substrate with a PPXY motif is ideal for
an enzyme-substrate interaction in that WW domains typically bind with
high specificity rather than high affinity (26), a property that may
explain our inability to isolate hRPF1/Nedd4-hPRTB complexes from
cellular extracts (data not shown).
hPRTB Colocalizes with Splicing Factors in Nuclear
Speckles--
Given our interest in a possible nuclear function of
hRPF1/Nedd4, we next sought to determine the subcellular localization of the potential ubiquitination substrate hPRTB. To this end, we fused
hPRTB to EGFP and analyzed its subcellular localization using
fluorescence confocal microscopy. EGFP-hPRTB and the corresponding PY
mutant (P40A/Y42A) have identical fluorescence patterns and are
localized to the nucleus in a discrete speckled pattern (Fig. 2). We were intrigued by our observation
of the localization of hPRTB to spots in the nucleus, reminiscent of
"nuclear speckles," which are enriched in splicing factors and
contain a population of hyperphosphorylated RNA polymerase II (27, 28).
Using fluorescent confocal microscopy, we demonstrated that both hPRTB
and the PY mutant indeed colocalize with the splicing factor, SC35, in
nuclear speckles (Fig. 2), suggesting that hPRTB may have a role in the transcription and/or splicing of RNA transcripts. Thus, in addition to
identifying a potential nuclear substrate of hRPF1/Nedd4, we have
localized hPRTB to splicing factor-rich speckles, a subnuclear localization where a population of RNA polymerase II, another WWhect E3
substrate, is known to reside.

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Fig. 2.
hPRTB colocalizes with splicing factor SC35
in nuclear speckles. The EGFP-N1 vector was used to
express hPRTB as a green fluorescent protein fusion protein in HeLa
cells. Twenty-four hours posttransfection, cells were plated onto glass
coverslips, allowed to attach, and subsequently fixed, permeabilized,
and incubated with a monoclonal antibody that recognizes the nuclear
speckle-associated splicing factor SC35 (27). Green fluorescent protein
or mouse-Texas Red fluorescence was detected using fluorescence
confocal microscopy.
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hPRTB Is a Substrate of WW Hect E3 Ubiquitin Ligases in
Vitro--
Given that hPRTB specifically binds to hRPF1/Nedd4, we next
wanted to determine whether it could serve as a substrate for the E3
ubiquitin ligase activity of this enzyme. A recombinant hRPF1/Nedd4
(whect) derivative lacking the amino-terminal C2 domain was used in
enzymatic assays, as full-length protein is not active under the
conditions tested (18). Efficient multi-ubiquitination of hPRTB was
observed when assayed in the presence of purified hRPF1/Nedd4 (whect)
or yRSP5, but not the hect E3 ligase E6-AP, which lacks WW domains in
its amino terminus (Fig. 3A).
Additionally, no ubiquitination was observed when either the E2
(UbcH5B) or E3 (RPF1/Nedd4-whect) enzyme was omitted from the reaction
mixture (data not shown and Fig. 3A). With the prediction
that substrate binding is necessary for E3 ligase activity, we next
tested the hypothesis that the hPRTB-PYmut would not be ubiquitinated
in this assay. Indeed, mutation of two key residues within the PPAY motif of hPRTB was able to completely abrogate ubiquitination of hPRTB
by either hRPF1/Nedd4 (whect) (Fig. 3B) or yeast RSP5 (data
not shown). Thus, a strong correlation between binding of hPRTB to
hRPF1/Nedd4 and its ability to be ubiquitinated in vitro was
established.

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Fig. 3.
hPRTB is an in vitro and
in vivo ubiquitination substrate. A,
hRPF1/Nedd4 ubiquitinates hPRTB in vitro.
35S-Labeled Myc-hPRTB was incubated with purified
hRPF1/Nedd4 (whect), yRSP5, or hE6-AP in the presence of ATP,
ubiquitin, and bacterially expressed E1 and E2 (UbcH5B) enzymes.
B, the hPRTB-PY mutant is unable to be ubiquitinated by
hRPF1/Nedd4 (whect). hPRTB and hPRTB-PYmut, containing a two-amino acid
substitution (P40A/Y42A) of the PPAY motif, were assayed in a standard
ubiquitination assay using E1, E2 (UbcH5B), and hRPF1/Nedd4 (whect) as
the E3 enzyme. C, His-ubiquitin conjugates of hPRTB isolated
from HeLa cells. HeLa cells were transfected with expression plasmids
for His-ubiquitin and Myc-hPRTB or Myc-hPRTB-PYmut. Forty hours
posttransfection, denatured lysates were prepared (lanes
1-3), and His-conjugates were purified on nickel resin
(lanes 4-6). Myc-hPRTB or Myc-hPRTB His-ubiquitin
conjugates were detected by Western immunoblot analysis using an
antibody directed against c-Myc (9E10). WT, wild type.
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hPRTB Requires an Intact PPXY Motif to Support Ubiquitin
Conjugation and Degradation in Cells--
Having confirmed that hPRTB
is an efficient substrate for Nedd4 in vitro, we next turned
to evaluating whether hPRTB is a physiological substrate for
ubiquitination. Specifically, we transfected HeLa cells with plasmids
expressing a His-tagged ubiquitin protein and Myc-tagged hPRTB. In
theory, ubiquitination substrates should be modified by His-ubiquitin,
and the resultant conjugates can be isolated on a
nickel-nitrilotriacetic acid resin. As indicated in Fig.
3C, lanes 4-6, His-ubiquitin conjugates were detected upon
cotransfection of His-ubiquitin and wild type Myc-hPRTB. The migration
of these 40-60-kDa ubiquitin conjugates differs from the slower
migrating multi-ubiquitinated conjugates observed in vitro
(Fig. 3, A and B), perhaps due to the regulated
activity of an endogenous (rather than purified) E3 enzyme, coupled
with the efficient cellular degradation of multi-ubiquitinated species within cells. Transfection of either Myc-hPRTB alone (Fig.
3C, lane 4) or cotransfection of Myc-hPRTB-PYmut and
His-ubiquitin (lane 6) was unable to produce His-ubiquitin
hPRTB conjugates. Western analysis (Fig. 3C, lanes 1-3) was
used to verify that all proteins were expressed in HeLa cellular
lysates. It was observed that Myc-hPRTB-PYmut accumulates to a higher
steady state level than Myc-PRTB (Fig. 3C, compare
lane 3 with lanes 1 and 2), a finding
that led us to consider that the stability of wild type and PY mutant
hPRTB proteins may be different. Consequently, we analyzed the
half-lives of Myc-hPRTB and Myc-hPRTB-PYmut in HeLa cells using
pulse-chase analysis. As shown in Fig.
4A, wild type hPRTB has a
significantly shorter half-life than the hPRTB-PYmut, the mutant that
is unable to support ubiquitination. Averaging the data from several
independent experiments, we conclude that mutation of two key residues
within the PPAY motif of hPRTB results in a 3.5-fold increase in the
half-life of hPRTB (2 h to 7 h) (Fig. 4B).
Cumulatively, these observations provide compelling evidence that hPRTB
is a physiological substrate of an endogenous WW-hect E3 ubiquitin
ligase, such as hRPF1/Nedd4.

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Fig. 4.
Mutation of the PPAY motif in hPRTB prolongs
its half-life. A, pulse-chase analysis of hPRTB and its
PY mutant in HeLa cells. HeLa cells were transfected with Myc-hPRTB or
Myc-hPRTB-PY mut, metabolically labeled, and chased in cold medium.
Lysates from indicated time points were immunoprecipitated using a
c-Myc antibody (9E10) and analyzed by SDS-PAGE followed by
autoradiography. B, half-lives of wild type and PY mutant of
hPRTB. Results from four independent pulse-chase experiments were
quantitated using a phosphorimager, normalized against an internal
Myc-EGFP control, and expressed as percentage of labeled protein at
time 0.
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hRPF1 Regulates the Stability of hPRTB in Cells--
It has
previously been shown that nuclear proteins such as the large subunit
of RNA polymerase II and the transcription factor NF-E2 bind to or are
able to be ubiquitinated by mammalian Nedd4 family members in
vitro (18, 20, 29); however, as of yet, there is no evidence that
such proteins are physiologically regulated by a particular mammalian
E3 ubiquitin ligase within cells. Given our data thus far, we suspected
that the WW-hect E3 ubiquitin ligase responsible for the
ubiquitin-dependent degradation of PRTB was hRPF1/Nedd4.
Thus, we sought to establish that exogenous hRPF1/Nedd4 was able to
alter the ubiquitin-dependent degradation of hPRTB. Using
pulse-chase analysis, we demonstrated that the degradation of PRTB was
accelerated in samples containing transiently transfected hRPF1/Nedd4
but not in samples transfected with the catalytic mutant
hRPF1/Nedd4-C867A or an empty expression plasmid (Fig.
5A). Specifically, the
half-life of PRTB in the presence of overexpressed hRPF1/Nedd4 was 70 min, compared with a t1/2 of 150 min in samples
containing either hRPF1/Nedd4-C867A or no exogenous hRPF1/Nedd4 (Fig.
5B). Western blot analysis confirmed that the total amount
of hRPF1/Nedd4 or hRPF1/Nedd4-C867A protein in transfected cells was at
least 2-3 times the amount normally present within HeLa cells (Fig.
5C). Thus, we have identified a nuclear speckle-associated
protein, hPRTB, as a substrate of the E3 WW-hect ubiquitin ligase
hRPF1/Nedd4 within cells. These data provide direct evidence that
hRPF1/Nedd4 can interact with, ubiquitinate, and regulate the stability
of a confirmed nuclear protein in intact cells.

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Fig. 5.
hRPF1/Nedd4, but not its C867A catalytic
point mutant, accelerates degradation of hPRTB. A, HeLa
cells were cotransfected with Myc-PRTB, the internal control Myc-EGFP,
and pcDNA3, hRPF1/Nedd4, or hRPF1/Nedd4-C867A. Cells were
subsequently radiolabeled and chased in cold medium. Lysates from
indicated time points were immunoprecipitated using a c-Myc antibody
(9E10) and analyzed by SDS-PAGE followed by autoradiography.
B, half-life of hPRTB is decreased in the presence of
exogenous hRPF1/Nedd4. Results from three independent experiments were
quantitated using a phosphorimager, normalized against an internal
Myc-EGFP control, and expressed as percentage of labeled hPRTB protein
at time 0. C, Western analysis of HeLa extracts expressing
hRPF1/Nedd4, hRPF1/Nedd4-C867A, or an empty vector pcDNA3 control.
Affinity-purified rabbit polyclonal antibody used for detection was
raised against the hect domain of hRPF1/Nedd4.
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hRPF1/Nedd4 Contains a Rev-like Nuclear Export Sequence--
To
further substantiate our observations that hRPF1/Nedd4 is able to
target the nuclear protein hPRTB for ubiquitination and degradation, we
finally sought to understand the mechanism by which a primarily
cytoplasmic E3 enzyme, hRPF1/Nedd4, is able to modify the nuclear
protein hPRTB.
hRPF1/Nedd4 has been reported to contain a bipartite nuclear
localization signal between amino acids 534-550; however, it has been
suggested in the past that Nedd4 is primarily a cytoplasmic protein
(21, 30). Indeed, when Myc-tagged RPF1 is expressed in HeLa or
NIH3T3 cells, we have shown that it is primarily cytoplasmic (data not
shown). In an attempt to artificially place hRPF1/Nedd4 into the
nuclear compartment of cells, we fused a strong SV40 nuclear
localization signal to the amino terminus of hRPF1/Nedd4. When cellular
localization of SV40 nuclear localization signal-RPF1/Nedd4 was
assayed, little to no nuclear staining was detected despite significant
cytoplasmic staining (data not shown), raising the possibility that
hRPF1/Nedd4 is a protein that is constitutively exported from the nucleus.
Many nuclear proteins undergo nuclear export via the
CRM1-dependent nuclear export pathway (31-33). To
determine whether the WWhect E3 ligase hRPF1/Nedd4 might be a substrate
of this nuclear export system, we evaluated hRPF1/Nedd4 localization in
the presence of the drug leptomycin B, a specific inhibitor of CRM-1
dependent export (34). Myc-hRPF1/Nedd4-expressing cells or
Myc-hRPF1/Nedd4-C867A-expressing cells were treated with 20 ng/ml
leptomycin B, and the subcellular localization of hRPF1/Nedd4 was
analyzed by immunofluorescence with an antibody directed against the
Myc tag. The results of this analysis (Fig.
6A) indicate that a population
of both wild type and catalytically inactive hRPF1/Nedd4 were localized
within the nucleus after treatment with leptomycin B, indicating that this WWhect E3 ubiquitin ligase, or a complex containing this protein,
is a substrate of CRM-1 dependent nuclear export.

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Fig. 6.
Amino acids 297-307 of hRPF1/Nedd4 contain a
Rev-like nuclear export sequence. A, HeLa cells were
transfected with either Myc-hRPF1/Nedd4 or Myc-hRPF1/Nedd4-C867A,
plated onto glass coverslips, and treated with leptomycin B (20 ng/ml)
for 4 h. Localization of Myc-tagged constructs was detected using
a Myc antibody (9E10), and Texas Red fluorescence was visualized using
confocal microscopy. B, hRPF1/Nedd4 contains a consensus
Rev-like NES. Amino acids 297-307 are aligned with the export
sequences of PKI, HIVrev, human p53, and Rex. Conserved leucines within
the consensus NES are highlighted in blue. C,
mutation of the NES results in a steady state population of
hRPF1/Nedd4-C867A in the nucleus. Two conserved residues within the NES
of hRPF1/Nedd4 were substituted with alanine (L305A/I307A) within the
context of the C867A catalytic mutant of Myc-tagged hRPF1/Nedd4.
cDNAs were transiently transfected into HeLa cells, and cells were
prepared for immunofluorescence as previously described.
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Given the data of others that a hRPF1/Nedd4 derivative encoding aa
404-900 is localized primarily within the nucleus (21), whereas
full-length constructs are cytoplasmic, we next used a series of
amino-terminal deletion constructs to map the region of hRPF1/Nedd4
responsible for its cytoplasmic localization. Full-length hRPF1/Nedd4,
as well as aa 293-900, localized primarily to the cytoplasm, but
proteins encoding aa 309-900 and 404-900 were present in both the
cytoplasm and the nucleus (data not shown), suggesting that a sequence
between amino acids 293 and 309 of hRPF1/Nedd4 is responsible for its
steady state cytoplasmic localization. Therefore, we compared amino
acids 293-309 of hRPF1/Nedd4 with the leucine-rich consensus for
Rev-like nuclear export. Indeed, amino acids 297-307 of hRPF1/Nedd4
contain sequence identity with this NES consensus and share significant
homology with the NESs found in other proteins, such as PKI, HIVrev,
human p53, and Rex (Fig. 6B) (31, 35-37). To prove that
this sequence within hRPF1/Nedd4 is able to act as an NES, we
substituted conserved residues with alanine (L305A and I307A) and
assayed for the cellular localization of this putative NES mutant.
Mutation of these two conserved amino acids within the export sequence
significantly increased the amount of hRPF1/Nedd4 protein that was
detected in the nucleus, as compared with the primarily cytoplasmic
localization of Myc-hRPF1/Nedd4-C867A (Fig. 6C) or Myc-RPF1
(data not shown). Interestingly, when the NES mutant was created in the
context of a wild type hRPF1/Nedd4, extremely low quantities of
exogenous protein were detected by Western blot and immunofluorescence
analysis. Nonetheless, these observations cumulatively demonstrate that
amino acids 297-307 mediate the CRM1-dependent nuclear
export of hRPF1/Nedd4.
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DISCUSSION |
Presented in this study is evidence that hPRTB, a proline-rich
protein that colocalizes with splicing machinery in nuclear speckles,
is a bona fide nuclear substrate of the WW hect E3 ubiquitin ligase, hRPF1/Nedd4. This identification of a nuclear substrate extends
the role of the hRPF1/Nedd4 ubiquitin ligase to nuclear proteins.
Furthermore, deletion and mutational analyses have led to our
subsequent identification of a leucine-rich rev-like nuclear export
sequence within hRPF1/Nedd4. Thus, we propose that nuclear import/export is an important component of the regulation between the
primarily cytoplasmic E3 enzyme, hRPF1/Nedd4, and its nuclear substrate, hPRTB.
An in Vivo Nuclear Substrate of hRPF1/Nedd4--
It has been well
established that Nedd4 can interact with and modulate the level of
several cytoplasmic proteins (14, 15, 17). In this study, we have
explored an additional role for Nedd4 with the identification and
characterization of substrate proteins that are localized within the
nucleus. In every aspect examined, the proline-rich nuclear protein,
hPRTB, is a characteristic substrate for hRPF1/Nedd4 ubiquitination and
degradation within the cell. However, given the lack of known
determinants of in vivo WWhect specificity, we cannot
exclude the possibility that there may be other human WWhect E3
ubiquitin ligases that are able to ubiquitinate this novel nuclear
substrate within cells. Indeed, there are several human WWhect E3
ubiquitin ligases with similar domain structure to hRPF1/Nedd4,
including Smurf1, a human WWhect ligase containing two WW domains,
which targets Smad1 and Smad5 for ubiquitin-dependent
degradation (9). However, our examination of Smurf1 amino acid sequence
failed to identify either a bipartite nuclear localization sequence or
rev-like export sequence similar to those in hRPF1/Nedd4, suggesting
that certain WWhect enzymes, such as Smurf1, may specifically target
substrates in the cytoplasm. Although our work strongly suggests a
nuclear function for hRPF1/Nedd4, additional research into the in
vivo substrate specificity of this and other potentially nuclear
human WWhect proteins is necessary to address precise questions of
overlapping enzyme/substrate choice.
The localization of a ubiquitination substrate such as hPRTB in nuclear
speckles is not surprising, given observations that cellular proteins
modified by the ubiquitin-like protein SUMO-1 are targeted to precise
subnuclear localizations. SUMO-1-modified promyelocytic leukemia gene
product (PML) localizes to nuclear bodies (38); similarly, the
sumoylation of the homeodomain-interacting protein kinase 2 results in
localization to nuclear speckles (dots) that are distinct from either
splicing factor-rich speckles or PML bodies (39). The localization of
hPRTB in nuclear speckles is unaffected by a mutation that blocks
ubiquitination, suggesting that the covalent attachment of a ubiquitin
moiety is not required for nuclear speckle localization. However, in
addition to playing a significant role in subnuclear localization,
SUMO-1 modification also antagonizes the
ubiquitin-dependent degradation of proteins such as I
B,
leading to an increase in protein stability (40). We acknowledge the
possibility that hPRTB may also be targeted (by a distinct enzyme) for
sumoylation, a ubiquitin-like modification, which could either direct
its localization to nuclear speckles or antagonize its
ubiquitin-dependent degradation. Further studies are needed
to address whether hPRTB may be modified by such a ubiquitin-like protein.
Although the precise function of hPRTB remains unknown, its
localization in nuclear speckles may offer clues to a possible function. Nedd4 was first described as a transcript that is
dramatically down-regulated upon the maturation of neural precursor
cells (11). Conversely, PRTB is a transcript that is highly expressed
in the developing mouse inner ear and is present in high amounts in the adult brain (22). These reports of temporal and spatial expression of
the Nedd4 and hPRTB transcripts are provocative given the results of
this study. Given its colocalization with splicing factor-rich nuclear
speckles, could hPRTB function as a developmental specific splicing
factor? Initial attempts to demonstrate colocalization of core Sm
proteins with hPRTB immunoprecipitates or alteration of a splice site
choice in vivo by hPRTB (data not shown) were not
successful. Although its cellular function remains unknown, it
nonetheless remains possible that hPRTB may modulate a splicing or RNA
processing event within cells.
Nuclear Import and Export of hRPF1/Nedd4--
With our
identification of a Rev-like NES within hRPF1/Nedd4, we provide
evidence that Nedd4 can indeed access both cytoplasmic and nuclear
compartments within a cell. However, it is apparent that hRPF1/Nedd4
protein that enters the nucleus has a very strong constitutive export
sequence, resulting in little time spent resident within the nucleus.
For example, exogenous hRPF1/Nedd4 targeted to the nucleus by a strong
SV40 nuclear localization signal or a mutated nuclear export sequence
is not tolerated by the cell, and protein does not accumulate (data not
shown). Thus, although a population of hRPF1/Nedd4 is able to enter the
nucleus, its presence appears to be transient, with the cell having a
strong preference to return it to the cytoplasm. This transient nuclear localization of hRPF1/Nedd4 offers an additional explanation for our
inability to isolate and immunoprecipitate what are presumably nuclear
hRPF1/Nedd4-hPRTB complexes (data not shown).
Although hRPF1/Nedd4 is able to enter the nucleus, it is intriguing
that neither leptomycin B treatment nor mutation of the NES is
sufficient to "trap" all of the hRPF1/Nedd4 protein within the
nucleus. Presumably the remaining cytoplasmic hRPF1/Nedd4 population
has not received a signal for nuclear entry but instead may be poised
to act upon known cytoplasmic Nedd4 targets, such as the sodium
epithelial channel. Accordingly, one logical question raised is what
stimulus or signal targets cytoplasmic hRPF1/Nedd4 to the
nucleus? hRPF1/Nedd4 is cleaved in cells in response to apoptotic
stimuli (41); however, a truncated Nedd4 protein corresponding to the
caspase cleavage product retains a primarily cytoplasmic localization
(data not shown), suggesting that removal of the first 200 amino acids
is not a sufficient signal for nuclear import. Although other factors
such as phosphorylation, acetylation, or a conformational change may
signal hRPF1/Nedd4 nuclear import, regulation of Nedd4 localization may
also occur at the level of nuclear export. For example, the sequences
flanking the NES of hRPF1/Nedd4 could be modified or change
conformation to specifically block accessibility of the NES to the
export receptor, preventing efficient export. Such NES masking has been
proposed to play a role in the blocking of p53 nuclear export upon p53
tetramerization (37). We have not yet observed a set of conditions
under which hRPF1/Nedd4 is exclusively nuclear, but we remain
interested in identifying factors that regulate such nuclear import
and/or export. Experiments aimed at identifying developmental stages,
cell types, or conditions under which hRPF1/Nedd4 may be localized
within the nucleus will likely offer insight into the important role of
nuclear/cytoplasmic localization in substrate recognition.
Given that hRPF1/Nedd4 may spend only a short time in the nucleus, it
is likely that nuclear import of hRPF1/Nedd4 is the limiting step in
enzyme/substrate recognition and catalysis. For example, an activating
signal could effect the transient nuclear localization of hRPF1/Nedd4,
resulting in ubiquitination of a substrate within the nucleus, followed
by rapid export of hRPF1/Nedd4 to the cytoplasm. Alternatively, nuclear
import of hRPF1/Nedd4 could be the limiting step if transient enzyme
entry was needed to allow the enzyme to bind a nuclear substrate and
transport it to the cytoplasm, where ubiquitination and degradation
might occur. Such a piggyback mechanism is one explanation for the
complex regulation of p53 by its RING-domain E3 ubiquitin ligase,
MDM2, a protein demonstrated to shuttle to and from the nucleus
(42, 43). Additional studies aimed at elucidating the location in the
cell at which hRPF1/Nedd4 substrate binding and catalysis occur are
needed to further our understanding of the localization constraints
that affect in vivo enzyme/substrate regulation.
Cumulatively, this work establishes a novel role for Nedd4 within the
nucleus, expanding our understanding of this E3 ubiquitin ligase as a
regulator of both cytoplasmic and nuclear targets. Specifically, we
have identified and characterized a nuclear speckle-associated protein,
hPRTB, as a substrate of hRPF1/Nedd4. Importantly, the identification
of a nuclear export sequence within hRPF1/Nedd4 offers a mechanism by
which a predominantly cytoplasmic enzyme accesses a nuclear substrate.
Despite significant research efforts to date, there remains a paucity
of knowledge of the in vivo determinants of substrate
specificity for the WWhect E3 ubiquitin ligase family. With the
identification of a nuclear import/export mechanism by which Nedd4
accesses a nuclear substrate, we propose that subcellular localization
is an important component of in vivo WWhect enzyme/substrate recognition and regulation.