Role of Exon 2-encoded
-Domain of the von Hippel-Lindau Tumor
Suppressor Protein*
Marie-Eve
Bonicalzi,
Isabelle
Groulx,
Natalie
de Paulsen, and
Stephen
Lee
From the Department of Cellular and Molecular Medicine and Kidney
Research Center, Faculty of Medicine, University of Ottawa,
Ottawa, Ontario K1H 8M5, Canada
Received for publication, September 11, 2000
 |
ABSTRACT |
Sporadic clear cell renal carcinomas
frequently harbor inactivating mutations in exon 2 of the von
Hippel-Lindau (VHL) tumor suppressor gene. Here, we examine the effect
of the loss of exon 2-encoded
-domain function on VHL biochemical
properties. Exon 2-encoded residues are required for VHL-mediated NEDD8
conjugation on cullin-2 and assembly with hypoxia-inducible
factor
(HIF
) and fibronectin. These residues are not essential
for VHL ability to assemble with elongin BC/cullin-2, to display E3
ubiquitin ligase activity in vitro and to confer
energy-dependent nuclear import properties to a reporter
protein. Localization studies in HIF-1
-null embryonic cells suggest
that exon 2-encoded
-domain mediates
transcription-dependent nuclear/cytoplasmic shuttling of
VHL independently of assembly with HIF-1
and oxygen concentration. Exon 3-encoded 
helical domain is required for VHL complex
formation with BC/cullin-2 and E3 ubiquitin ligase activity, for
binding to HIF
/fibronectin, but this domain is not essential for
transcription-dependent nuclear/cytoplasmic trafficking.
VHL
/
renal carcinoma cells expressing 
domain
mutants failed to produce an extracellular fibronectin matrix and to
degrade HIF
, which accumulated exclusively in the nucleus of
normoxic cells. These results demonstrate that exon 2-encoded residues
are involved in two independent functions: substrate protein
recognition and transcription-dependent nuclear/cytoplasmic
trafficking. They also suggest that
-domain mutations inactivate VHL
function differently than
-domain mutations, potentially providing
an explanation for the relationship between different mutations of the
VHL gene and clinical outcome.
 |
INTRODUCTION |
Inactivating mutations of the von Hippel-Lindau
(VHL)1 tumor suppressor gene
are associated with inherited VHL cancer syndrome, of which afflicted
individuals are at risk to develop a wide variety of tumors including
clear cell renal cell carcinoma (RCC) (1-3). Biallelic inactivating
mutations of the VHL gene are also associated with sporadic RCC, the
most common form of kidney cancer in humans (4, 5). Reintroduction of
wild-type VHL in VHL
/
RCC cells is sufficient to
suppress their ability to form tumors in nude mice (6). The VHL gene
contains three exons that code for a 213-residue protein. VHL protein
assembles with at least four other associated proteins: elongin B,
elongin C, cullin-2, and Rbx (the complex will be hereafter referred to
as VBC/Cul-2) (7-11). VBC/Cul-2 is an E3 ubiquitin ligase that targets
the
-subunits of hypoxia-inducible factor (HIF
) for
oxygen-dependent degradation (12-18). HIF
are
stabilized by hypoxia and play an important role in the activation of
hypoxia-inducible genes such as the vascular endothelial growth factor
and glucose transport-1 (Glut-1) (8, 13, 19-22). HIF
are stable in
VHL
/
RCC cells bringing about a constitutive
"hypoxia-like" response, regardless of oxygen concentration. The
exact mechanism by which VHL can mediate the degradation of HIF
is
still unknown but might be related to its ability to shuttle between
the nucleus and the cytoplasm (6, 23-27). Another key functional
characteristic of VHL is that it binds to fibronectin and is involved
in the assembly of an extracellular fibronectin matrix (28).
The crystal structure of VHL has been reported. VHL mainly consists of
two independent domains: a large
-domain that spans residues 64-154
and an
-helical domain (
-domain) that encompasses most of the
C-terminal part of the protein (residues 157-189) (29). Tumor-derived
inactivating mutations (279 entries; Ref. 30) are found across the VHL
protein, indicating that both domains play a critical role in VHL tumor
suppressor function (29). There is, however, an interesting correlation
between the nature and localization of inactivating mutations and the
clinical consequences in patients afflicted with inherited VHL
syndrome. Individuals with type II VHL syndrome develop
pheochromocytoma and have generally inherited a mutation in the exon
3-encoded
-domain. Type I VHL syndrome differs from type II in that
patients do not develop pheochromocytoma and are likely to have
inherited a mutation in the hydrophobic core of the
-domain (31).
There is also an intriguing disparity in the distribution of
tumor-derived missense mutations between the inherited and sporadic
form of RCC. Mutations in the
-domain of VHL are much more frequent
in the inherited form of RCC (5). The role of a few of these residues
is well understood, since they correspond to the ones required for VHL binding to elongin C and formation of the VBC/Cul-2 complex (7, 8, 10,
32). In contrast to inherited RCC, sporadic RCC frequently harbor
inactivating point mutations in exon 2. This includes point mutations
at the exon 2 boundary that cause a splice defect producing a mRNA
that lacks exon 2 sequences altogether (5). Exon 2 mutations are rare
in VHL patients, and it has been suggested that such mutations might
not be easily tolerated and thus not transmitted in the germ line (1).
The discrepancy in the distribution of mutations between sporadic and
inherited RCC suggests that exon 2-associated mutations might
inactivate VHL function in a different way than exon 3-associated
mutations. Exon 2-encoded residues 114-154 are mostly hydrophobic and
form three of the seven
-strands of the
-domain (29). These
residues are hypothesized to play a role in substrate protein
recognition, although recent in vitro experiments have
revealed that they might not be required for VHL binding to HIF
(33). Therefore, the role that exon 2-encoded sequences play in
VHL-mediated tumor suppression is still poorly understood. Here, we
further examine the role of these sequences in cells by comparing a
tumor-derived VHL mutant that lacks residues 114-154 with the known
biochemical properties of wild-type VHL and mutant VHL lacking the exon
3-encoded
-domain. We show that the exon 2-encoded
-domain plays
two independent roles in binding to HIF
and fibronectin and
mediating transcription-dependent nuclear/cytoplasmic
trafficking of the VBC/Cul-2 complex. The use of a novel method to
examine the energy requirement for nuclear import of proteins will also
be discussed in this report. The results presented here support the
model that the
-domain of VHL is involved in substrate recognition
and nuclear/cytoplasmic trafficking.
 |
MATERIALS AND METHODS |
Cell Culture, Transfections, and Adenoviral Infections
The VHL
/
786-0 RCC cells and HeLa cells were
obtained from the American Type Culture Collection (Manassas, VA). The
VHL-GFP cell line corresponds to 786-0 cells stably transfected with
the VHL-GFP fusion protein as described elsewhere (25). The 117-4 (VHL
/
; referred to as 117) cells were a kind gift from
Dr. James R. Gnarra (LSU Health Sciences Center, New Orleans, LA). The
mouse embryonic fibroblasts (MEFs) nullizygous for HIF-1
were a kind gift from Dr. Randy Johnson (Department of Biology, University of
California, San Diego) (34). Cell lines were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% (v/v) fetal calf serum
in a 37 °C, humidified, 5% CO2-containing atmosphere incubator. Transient transfections were performed overnight using a
standard calcium phosphate method. Viruses were used as freeze/thaw lysates, and all infections were also performed overnight.
Expression Vectors and Constructs
The human VHL cDNA, which codes for a 213-amino acid VHL
protein, was subcloned into pcDNA3.1(
) (Invitrogen) vector. A
FLAG epitope tag (DYKDDDDK) was added to the N terminus of the VHL cDNA open reading frame. A cDNA coding for an enhanced
fluorescence version of GFP (Fred 25; Ref. 35) was subcloned at the C
terminus VHL to produce the VHL-GFP fusion protein. A deletion mutant
of the last 56 amino acids was fused to GFP to produce the
E3-GFP fusion protein. Another deletion mutant of VHL lacking amino acids 114-154 was fused to GFP to produce the
E2-GFP fusion protein. Two
GFPs in tandem were cloned into pcDNA 3.1(
) to produce the GFP-GFP fusion protein. VHL-GFP-NES,
E2-GFP-NES, and GFP-GFP-NES were produced by fusion of VHL-GFP,
E2-GFP, and GFP-GFP at their C
terminus to the strong nuclear export signal (NES) of human immunodeficiency virus REV, LPPLERLTL (NES) (36). All constructs were
verified by standard DNA sequencing.
Construction of Adenovirus Vectors through Cre-lox
Recombination
CRE8 and 293 cells were obtained from Dr. David Park (University
of Ottawa, Ottawa, Ontario, Canada) and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS). The
construction and properties of CRE8 cells are described elsewhere (37).
The pAdlox vector and the
5 viral DNA were also obtained from Dr.
Park. The three different VHL constructions (VHL-GFP,
E2-GFP, and
E3-GFP) were previously subcloned in the pAdlox vector.
Transfections were done according to Graham and van der Eb (38).
Typically, a confluent 10-cm diameter dish of CRE8 cells (1.6 × 107) was split into 5-6-cm diameter dishes for
transfection 2-4 h later. Each dish received 3 µg of pAdlox vector
(containing the foreign VHL construction) and 3 µg of
5 viral DNA
in a final volume of 0.5 ml of CaPO4, which was applied to
the cells for 16 h. The 10% fetal calf serum DMEM was changed
for 2% fetal calf serum DMEM 16 h following the transfection.
Cells were fed with fresh 2% DMEM after 64 h. Between day 8 and
10, we had a sizable infection in each dish; almost all of the cells
were rounded up or floating. Cells were harvested and subjected to
freeze/thaw three times with an alternating liquid
N2/37 °C water bath. The virus was then passed
sequentially through CRE8 cells twice. Finally, a plaque purification
assay was performed in order to isolate the recombinant virus
expressing adVHL-GFP, ad
E2-GFP, or ad
E3-GFP. The recombinant
viruses were then amplified in CRE8 cells to high titer.
Nuclear Import Assay in Living Cells
HeLa cells were plated on a 35-mm dish with a hole at the bottom
replaced by a glass coverslip and transfected overnight with VHL-GFP-NES,
E2-GFP-NES, and GFP-GFP-NES. Cells were washed with PBS
and incubated for 2 h in DMEM at 37 °C with or without
metabolic poisons (6 mM 6-deoxyglucose and 0.02% sodium
azide), at 4 °C, or at 37 °C with 10 µM leptomycin
B (39-40).
In Vitro Ubiquitination Assay
VHL
/
786-0 RCC cells infected with the three
different adenoviruses and 786-0 cells stably expressing VHL-GFP were
lysed in the presence of 1% Triton X-100, 20 mM Tris-HCl,
pH 8.0, 137 mM NaCl with protease mixture for 30 min. at
4 °C. Whole cell lysates were first immunoprecipitated with
anti-FLAG M2 monoclonal antibody. Precipitates were washed five times
with a buffer containing 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM dithiothreitol. The
total volume of the reaction mixture was adjusted to 20 µl. E1
ubiquitin-activating enzyme (100 ng), E2 ubiquitin-conjugating enzyme
(200 ng), 0.5 µg of ubiquitin aldehyde, 0.5 µg of ubiquitin, and an
ATP-regenerating system (0.5 mM ATP, 10 mM
creatine phosphate, and 10 µg of creatine phosphokinase) were added
to the reaction mixture (complete mixture). The reaction was stopped
after 2 h of incubation at 37 °C by adding 4× SDS loading
buffer. Samples were boiled 10 min and separated on an 8% SDS-PAGE and
blotted onto a PVDF membrane. Blots were blocked and incubated in the
presence of a mouse anti-ubiquitin antibody (Berkeley Antibody
Company). The E1 ubiquitin-activating enzyme and the E2
ubiquitin-conjugating enzyme were a kind gift from Dr. Kazuhiro Iwai
(Kyoto University, Kyoto, Japan).
Immunoprecipitations and Immunoblotting
Immunoprecipitation of HIF-1
and
HIF-2
--
VHL
/
786-0 cells expressing endogenous
HIF-2
or 117-4 cells expressing endogenous HIF-1
were exposed for
4 h to hypoxia (0.1% O2) 16 h after infection.
Proteasomal inhibition was performed with 100 µM calpain
inhibitor I (CI) for 2 h. When still in the hypoxic chamber, cells
were washed several times with PBS and scraped from the Petri dishes in
lysis buffer containing 100 mM NaCl, 0.5% Igepal CA630, 20 mM Tris-HCl (pH 7.6), 5 mM MgCl2, and 1 mM sodium orthovanadate with 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin A, and 1 mM
4-(2-aminoethyl) benzene sulfonyl fluoride. Tubes were put back in
normoxia and kept at 4 °C for 30 min. with rocking. After clearance
by centrifugation, 1-mg aliquots of lysate were incubated for 2 h
at 4 °C with anti-FLAG M2 beads (Scientific Imaging Systems, Eastman
Kodak Co.). Beads were washed, boiled, and loaded on an 8% SDS-PAGE
and blotted onto PVDF membranes using standard methods. Blots were
blocked with 3% milk powder in PBS containing 0.2% Tween 20 and were
then incubated in the presence of anti-HIF-1
(Transduction
Laboratories), anti-HIF-2
antibody (Novus Biologicals), or an
anti-FLAG M2 monoclonal antibody (Sigma).
Immunoprecipitation of Cullin-2, NEDD8, and
Fibronectin--
VHL-GFP cells and infected 786-0 cells were lysed in
100 mM NaCl, 0.5% Igepal CA630, 20 mM Tris-HCl
(pH 7.6), 5 mM MgCl2, and 1 mM
sodium orthovanadate with 2 µg/ml leupeptin, 2 µg/ml aprotinin, and
1 µg/ml pepstatin A. After clearance by centrifugation, 1-mg aliquots
of lysate were incubated for 2 h at 4 °C with anti-FLAG M2
beads. Beads were washed, boiled, and loaded on an 8% SDS-PAGE and
blotted onto PVDF membranes using standard methods. Blots were blocked
with 3% milk powder in PBS containing 0.2% Tween 20 and were then
incubated in the presence of anti-cullin-2 (Ref. 41; provided by Dr.
Arnim Pause, Max-Plank Institute, Germany), anti-NEDD8 (Alexis),
anti-fibronectin (Dako Diagnostic), or anti-FLAG M2 monoclonal antibody
(Sigma). For total cell lysates, cells were washed several times in
PBS, scraped from the Petri dishes, centrifuged, and resuspended in 4%
SDS in PBS (24). The samples were boiled for 5 min, and the DNA was
sheared by passage of lysates through 19-gauge needles. Protein
concentration was determined by bicinchoninic acid method (Pierce) and
was used to normalize protein loading in a whole-cell immunoblot assay.
Immunofluorescence Staining
For Fibronectin--
VHL
/
RCC 786-0 cells or
VHL-GFP cells were infected and were grown on coverslips for 6 days,
washed three times with PBS, and fixed/permeabilized in prechilled 95%
ethanol at
20 °C for 30 min. Ethanol was then aspirated, and the
residual ethanol was allowed to air dry at 4 °C. Cells were stained
with polyclonal anti-fibronectin antibody (5 µg/ml) (Dako Diagnostic)
for 1 h at room temperature. The coverslips were then washed with
PBS three times and incubated with CyTM3-conjugated
anti-rabbit antibody (Jackson ImmunoResearch, PA) diluted 1:1000 for
1 h at room temperature. Coverslips were washed three times with
PBS, incubated for 2 min with Hoechst 33342, and mounted with
fluoromount-G (Southern Biotechnology Associates) on slides.
For HIF-1
--
117 cells or transiently transfected 786-0 with HIF-1
were grown on coverslip and infected with the three
different adenoviruses overnight. Cells were washed three times with
PBS, fixed/permeabilized in PBS containing 4% formaldehyde for 30 min
at room temperature, washed again three times with PBS, and incubated
for 1 h at room temperature with anti-HIF-1
antibody
(Transduction Laboratories, Lexington, KY) diluted 1:1000 in PBS, 1%
Triton X-100, 10% FCS. The cells were washed in PBS and incubated for
60 min in the presence of a CyTM3-conjugated anti-mouse
antibody (Jackson ImmunoResearch) diluted 1:1000. The cells were washed
in PBS, incubated for 2 min in Hoechst 33342, and mounted with
fluoromount-G on slides.
Fluorescence Analysis and Image Processing
GFP fluorescence images were captured using a Zeiss Axiovert
S100TV microscope with a C-Apochromat 40 × water immersion
objective, equipped with an Empix digital charge-coupled device camera
using Northern Eclipse software. Images were manipulated with Northern Eclipse and Adobe Photoshop software as described elsewhere (25). GFP
images were always taken before Hoechst images to minimize any possible
bleaching effect.
 |
RESULTS |
Biochemical Characterization of the Exon 2-Encoded
-Domain of
VHL--
The VHL protein, encoded by the VHL gene that contains three
exons, can be divided into three independent domains: an acidic domain,
a
-domain, and an
-domain (Fig.
1A). Sporadic RCC frequently harbor inactivating mutations in the exon 2-encoded part of the
-domain, whereas these mutations are relatively rare in individuals afflicted with inherited VHL syndrome (5). To study the role of exon
2-encoded
-domain in VHL tumor suppressor function, a cDNA
encoding a tumor-derived truncation of residues 114-154 was fused to
GFP to produce the
E2-GFP fusion protein (Fig. 1B). This
truncation mutant is the consequence of point mutations that cause a
splice defect producing a mRNA that lacks exon 2 sequences altogether (5).
E2-GFP is predicted to have a partial, if not total,
loss of
-domain function while retaining an intact, exon 3-encoded
-helical domain. A tumor-derived truncation of the exon 3-encoded
-helical domain (last 56 C-terminal residues), which retained intact
the sequences of the
-domain, was also fused to GFP (
E3-GFP)
(Fig. 1B).
E2-GFP,
E3-GFP, and wild-type VHL-GFP were
cloned in pAdlox vector, and adenoviruses (ad
E2-GFP, ad
E3-GFP,
and adVHL-GFP) were produced to high titers (Fig. 1B) (42).
Adenovirus was chosen as a method to reintroduce VHL, since it
eliminates the necessity to produce stable clones of different
VHL
/
RCC cell lines. VHL
/
RCC cells
were infected with very high efficiency, with essentially 100% of
cells displaying GFP fluorescence (Fig. 1C). In
adenovirus-infected cells, adVHL-GFP was mostly localized to the
cytoplasm with some nuclear signal, consistent with data obtained with
stable transfectants. In contrast to VHL alone (without GFP; Ref. 43),
adVHL-GFP did not restrain proliferation of VHL
/
RCC
cells or other cell lines such as 293 cells, even when expressed to
very high levels (data not shown). Glut-1 protein levels were significantly decreased in VHL
/
786-0 RCC infected with
adVHL-GFP in normoxia compared with uninfected cells or cells infected
with an adenovirus that expressed GFP alone (data not shown). Western
blot analysis indicated that ad
E2-GFP accumulated to levels similar
to those of adVHL-GFP and ad
E3-GFP, suggesting that ad
E2-GFP is a
stable protein (Fig. 1D). We conclude that the adVHL-GFP
protein produced from an adenovirus is a functional molecule and shares
similar characteristics with VHL.

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic diagram of VHL protein and
characterization of adenovirus-mediated expression of VHL-GFP.
A, schematic diagram of VHL protein. VHL has three exons
that code for a 213-amino acid protein containing an acidic domain, a
-domain, and an -domain. Exon 2 encodes for the latter part of
the -domain and is the site of frequent mutations in sporadic RCC
but not in the inherited RCC of VHL syndrome. B, schematic
diagram of VHL fusions to GFP. The GFP was fused at the C terminus
(black box (not to scale)), resulting in VHL-GFP
fusion protein. A cDNA encoding a tumor-derived truncation of
residues 114-154 (strike box) was fused
to GFP to produce E2-GFP. A tumor-derived truncation of the exon
3-encoded -domain (strike box) was also fused to GFP to produce
E3-GFP. A FLAG tag (shadow box) was fused to the N-termini of
all three constructs. E2-GFP, E3-GFP, and VHL-GFP were cloned in
pAdlox vector to prepare adenoviruses. ad E2-GFP, ad E3-GFP, and
adVHL-GFP refer to proteins obtained following the infection with the
corresponding adenovirus. C, 100% of VHL /
RCC cells (786-0) displayed GFP fluorescence following infection with
the adenovirus adVHL-GFP. 786-0 cells were infected overnight (16 h),
and adVHL-GFP pictures were obtained by a charge-coupled device camera
(left panel). Counterstaining with Hoechst 33342 dye (2 µg/ml for 2 min) provided staining of all nuclei
(right panel). D, ad E2-GFP,
ad E3-GFP, and adVHL-GFP accumulated to similar levels following
adenoviral infection of 786-0. Total cell lysates were run on an 8%
SDS-PAGE and transferred to a PVDF membrane. The membrane was then
blocked and incubated in the presence of a mouse anti-FLAG M2
antibody.
|
|
We next examined the biochemical properties of ad
E2-GFP in
comparison with adVHL-GFP and ad
E3-GFP. The
-domain mutant
ad
E2-GFP still retained the ability to assemble with cullin-2 (Fig.
2A) and to exhibit E3
ubiquitin ligase activity in vitro (Fig. 2B) to
levels similar to those observed for adVHL-GFP. The
-helical domain
deletion mutant (ad
E3-GFP) failed to assemble with cullin-2 and to
display E3 ubiquitin ligase activity in vitro, as expected. While the experiments described above were being performed, it was
noticed that a second band, which migrated slower than cullin-2, was
found in the adVHL-GFP lane but was lacking from the ad
E2-GFP lane
(Fig. 2A). NEDD8 is a ubiquitin-like molecule, which is
conjugated to cullin-2 in a VHL-dependent manner (41, 44).
Western blotting with an anti-NEDD8 antibody revealed that the slower
migrating form of cullin-2 is conjugated to NEDD8 (Fig. 2C).
Therefore, an intact exon 2-encoded
-domain is not required for VHL
ability to assemble with cullin-2 and to function as an E3 ubiquitin
ligase in vitro but is necessary for VHL-mediated NEDD8
conjugation on cullin-2.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 2.
Biochemical characterization of an exon
2-encoded -domain mutant of VHL.
A, an intact -domain is not required for VHL ability to
assemble with cullin-2. Stable VHL / RCC 786-0 cells
stably expressing FLAG-tagged VHL-GFP or 786-0 cells infected or not
infected with the adenoviruses ad E2-GFP, ad E3-GFP, and adVHL-GFP
were lysed and immunoprecipitated with anti-FLAG M2 beads. Precipitated
proteins were run on SDS-PAGE (8% acrylamide) and transferred on PVDF
membranes. The membranes were then blocked and incubated in the
presence of a rabbit anti-cullin-2 (top panel) or
a mouse anti-FLAG M2 (bottom panel) antibody.
Notice that a second band migrates slower than cullin-2 in the VHL-GFP
and adVHL-GFP lanes only. This represents NEDD8 conjugation
to cullin-2 (further confirmed in C). B, an
intact -domain is not required for VHL ability to function as an E3
ubiquitin ligase in vitro. In vitro ubiquitination reactions
were performed as described under "Materials and Methods" (complete
mixture) except for two negative controls; adVHL-GFP was
immunoprecipitated and incubated with the complete mixture except the
E1 enzyme (first lane from the left)
or the E2 enzyme (third lane from the
left). Reactions were stopped by adding 4× sample buffer.
Samples were electrophoresed in 8% SDS-PAGE and transferred to a PVDF
membrane. The membrane was then blocked and incubated in the presence
of a mouse anti-ubiquitin antibody. C) Exon 2-encoded -domain is
required for VHL-mediated NEDD8 conjugation to cullin-2.
Immunoprecipitations were performed exactly like for cullin-2.
Immunoprecipitated proteins were run on an 8% SDS-PAGE and transferred
to a PVDF membrane. The membrane was then blocked and incubated in the
presence of a rabbit anti-NEDD8 antibody.
|
|
Exon 2-encoded
-Domain Is Required for VHL Binding to
Fibronectin and Proper Assembly of a Fibronectin Extracellular
Matrix--
VHL
/
RCC cells are unable to promote
assembly of an extracellular fibronectin matrix, and the reintroduction
of VHL was shown to be sufficient to correct this defect (28).
Adenovirus-mediated reintroduced adVHL-GFP displayed similar activity
as VHL and restored the ability of VHL
/
RCC cells to
properly produce a fibronectin extracellular matrix (Fig.
3A; VHL-GFP). In contrast,
ad
E2-GFP was unable to rescue this defect (Fig. 3A).
Fibronectin was observed in an endoplasmic reticulum-like intracellular
distribution in uninfected cells as well as in cells expressing
ad
E2-GFP. Immunoprecipitation analysis revealed that adVHL-GFP was
able to assemble with fibronectin, whereas ad
E2-GFP failed to do so
(Fig. 3B). The ad
E3-GFP was also unable to bind to
fibronectin and correct the fibronectin deposition defect of
VHL
/
RCC. Therefore, VHL requires an exon 2-encoded
-domain to bind to fibronectin and mediate proper extracellular
matrix formation.

View larger version (63K):
[in this window]
[in a new window]
|
Fig. 3.
VHL requires an intact
-domain to bind fibronectin and mediate proper
extracellular matrix formation. A, ad E2-GFP is
unable to produce a fibronectin extracellular matrix. Uninfected
VHL / 786-0 cells, VHL / RCC 786-0 cells
stably expressing FLAG-tagged VHL-GFP, or cells infected with
adVHL-GFP, ad E2-GFP, or ad E3-GFP were grown on coverslips for 6 days. Cells were washed, fixed, incubated with Hoechst for 2 min
(blue), and stained with a rabbit anti-fibronectin
antibody (red). Pictures were obtained by charge-coupled
device camera, and superposition of fibronectin and Hoechst-stained
nuclei frames was done with Adobe Photoshop. The arrows are
pointing at fibronectin deposition. B, ad E2-GFP is unable
to bind fibronectin. Stable VHL / RCC 786-0 cells stably
expressing FLAG-tagged VHL-GFP or 786-0 cells uninfected or infected
with the adenoviruses ad E2-GFP, ad E3-GFP, and adVHL-GFP were
lysed and immunoprecipitated with anti-FLAG M2 beads. Precipitated
proteins were run on an 8% SDS-PAGE and transferred on PVDF membranes.
The membranes were then blocked and incubated in the presence of a
rabbit anti-fibronectin (top panel) or a mouse
anti-FLAG M2 (bottom panel) antibody.
|
|
Role of Exon 2-Encoded
-Domain of VHL in
Oxygen-dependent Degradation of HIF
--
It was
recently shown that one of the major defects of VHL
/
RCC cells is their inability to mediate oxygen-dependent
degradation of HIF
, and reintroduction of wild-type VHL was
sufficient to correct this defect (13). In vitro studies
have also revealed that truncation mutants of exon 2 and exon 3 of VHL
are still able to bind to HIF
(33), which probably assemble with
sequences encoded by exon 1 (residues 64-113) (12).
Adenovirus-mediated reintroduction of adVHL-GFP was sufficient to
restore VHL
/
RCC cell line 117 (HIF-1
) and 786-0 (HIF-2
) ability to mediate degradation of HIF
in normoxia (Fig.
4A). HIF
levels were not affected by expression of ad
E2-GFP or ad
E3-GFP (Fig.
4A). We notice that adVHL-GFP assembled with a significant
amount of HIF
(1
and 2
) in hypoxia and in the presence of the
proteasome inhibitor CI. In contrast to data obtained in
vitro, immunoprecipitation analysis revealed that ad
E2-GFP and
ad
E3-GFP failed to bind to HIF
in adenovirus-infected cells (Fig.
4B, top panels). We did not detect
binding of HIF
to ad
E2-GFP and ad
E3-GFP in cells expressing
low to very high levels of the fusion proteins (data not shown). These
results indicate that an intact exon 2-encoded
-domain, as well as
the
-domain, is required for VHL assembly with HIF
in cells.

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 4.
HIF fails to bind to
an exon 2-encoded -domain mutant of VHL and
accumulates in the nucleus of normoxic cells. A,
ad E2-GFP failed to mediate oxygen-dependent degradation
of HIF . VHL / 786-0 RCC cells (HIF-2 ) and 117-4 cells (HIF-1 ) were uninfected or infected with adVHL-GFP,
ad E2-GFP, and ad E3-GFP. Total cell lysates were run on an 8%
SDS-PAGE, transferred to PVDF membranes. The membranes were blocked and
incubated in presence of a mouse anti-HIF-1 (top
panel) or a rabbit anti-HIF-2 (bottom
panel) antibody. B, adVHL-GFP assembled with
endogenous HIF-1 (117 cells) or with endogenous HIF-2 (786-0 cells), but ad E2-GFP and ad E3-GFP failed to do so. Cells were put
under hypoxic conditions (0.1% O2) for 4 h in the
presence of the calpain inhibitor I. Cells were lysed, and
immunoprecipitation was performed with anti-FLAG M2 beads for 2 h.
Immunoprecipitated proteins were run on an 8% SDS-PAGE and blotted
onto PVDF membranes. Membranes were blocked and incubated in the
presence of a mouse anti-HIF-1 (top left
panel), a rabbit anti-HIF-2 (top
right panel), or a mouse anti-FLAG M2 antibody
(bottom panels). C, nuclear import of
HIF occurs regardless of oxygen tension. VHL / RCC
117 cells (endogenous HIF-1 ) (a-l) or 786-0 cells
transiently transfected with HIF-1 (m-p) were uninfected
or infected with the adenoviruses adVHL-GFP, ad E2-GFP, and
ad E3-GFP. Cells were washed, fixed, and stained with a mouse
anti-HIF-1 antibody. Counterstaining of cells with Hoechst 33342 dye
provided staining of all 117 cells' nuclei (i-l).
D, line drawing of mutant VHL-GFP, which were co-transfected
with HIF-1 cDNA in VHL / RCC 786-0 cells. The
arrows indicate single amino acid substitutions at residues
98 and 117, whereas black bars indicate small
deletion mutants within exon 2. HIF-1 accumulated in the nucleus in
normoxia when co-transfected with these VHL mutants.
|
|
It has been hypothesized that HIF-1
requires a hypoxic environment
to import in the nucleus most likely assembled into complexes that
contain VBC/Cul-2 (33, 45). To further examine the role of hypoxia and
VHL in nuclear import of HIF
, the subcellular localization of
endogenous HIF-1
was examined by immunofluorescence in
VHL
/
117 RCC cells uninfected or infected with
different VHL constructs. Data shown in Fig. 4C revealed
that endogenous HIF-1
accumulated exclusively in the nucleus of
uninfected VHL
/
RCC 117 cell line although these cells
were incubated in normoxia (Fig. 4C, a,
e, and i). This demonstrates that HIF-1
is
able to import in the nucleus even in the presence of oxygen and in the
absence of VHL. A strong HIF-1
nuclear signal was also observed in
cells expressing ad
E2-GFP (Fig. 4C, c,
g, and k) as well as ad
E3-GFP (Fig.
4C, d, h, and l), whereas
it was essentially undetectable in cells expressing reintroduced
adVHL-GFP (Fig. 4C, b, f, and j). We then examined the subcellular localization of
overexpressed HIF-1
in RCC VHL
/
786-0 cells (which
do not express endogenous HIF-1
). A strong HIF-1
signal was
detected exclusively in the nucleus of normoxic RCC
VHL
/
786-0 cells transiently transfected with HIF-1
cDNA that were either uninfected (Fig. 4C,
m), infected with GFP alone (data not shown), or infected
with ad
E2-GFP (Fig. 4C, o) and ad
E3-GFP (Fig. 4C, p). The addition of proteasome
inhibitors or incubation in hypoxia led to nuclear accumulation of
endogenous or overexpressed HIF
regardless of the presence of
adVHL-GFP or mutants, as expected (data not shown). HIF-1
was also
detected in the nucleus of normoxic RCC VHL
/
786-0 cells when co-transfected with different smaller deletion mutants of
exon 2, with a substitution at residue 117 in exon 2 or at residue 98 in exon 1, fused to GFP (Fig. 4D). These results demonstrate
that HIF-1
is able to import in the nucleus regardless of oxygen
concentration or assembly with VHL.
Exon 2-encoded Residues Mediate Transcription-dependent
Nuclear/Cytoplasmic Trafficking of VHL Independently of Assembly with
HIF
and Oxygen Concentration--
We recently demonstrated that VHL
mediates transcription-dependent nuclear/cytoplasmic
trafficking of the VBC/Cul-2 complex (25, 46). The addition of
5,6-dichlorobenzimidazole riboside (DRB), an inhibitor of RNA
polymerase II activity, causes an important increase of nuclear
VBC/Cul-2 by blocking VHL-mediated nuclear export of the complex. The
dependence on transcription for trafficking is abolished by a
deletion on exon 2-encoded sequences (25). We next wanted to determine
if exon 2-encoded residues also regulate subcellular trafficking of VHL
in conditions known to affect HIF
stability, such as oxygen
concentration, and if it is able to do so independently of assembly
with HIF
. Since ad
E2-GFP is a small molecule (40 kDa), its
presence in the nucleus (Fig.
5c) might be simply the
outcome of unregulated passive diffusion through the nuclear pore
complex rather than by the utilization of signal-mediated and
-regulated energy-dependent processes. Therefore, the first step consisted of determining if the
-domain mutant required energy
expenditure for nuclear import before further investigating its role in
VHL-mediated shuttling of BC/Cul-2. To do so, we developed a new assay
to test for energy requirement for nuclear import in living cells based
on fusing proteins to the energy-dependent human
immunodeficiency virus REV NES. NES confers strong nuclear export
properties to fusion proteins, leading to their cytoplasmic accumulation at steady state (Fig. 5; compare a with
d, b with e, and c with
f; see Ref. 36). GFP-GFP-NES rapidly accumulated in the
nucleus upon inhibition of NES function at 4 °C or with metabolic
poisons, as expected, since this fusion protein is able to passively
diffuse in and out of the nucleus (Fig. 5, g and j, and Ref. 46). In contrast, VHL-GFP-NES and
E2-GFP-NES
strictly remained in the cytoplasm at 4 °C or in the presence of
metabolic poisons (Fig. 5, h, i, k,
and l), indicating that both fusion proteins are unable to
passively diffuse in the nucleus.
E2-GFP-NES and VHL-GFP-NES (Fig.
5, n and o) accumulated in the nucleus upon incubation with leptomycin B, a drug that specifically inhibits NES
function (39, 40) at 37 °C, but not at 4 °C, indicating that both
fusion proteins contain energy-dependent nuclear import signals. These observations demonstrate that VHL ability to confer energy-dependent nuclear import properties to a reporter
GFP is independent of assembly with HIF
and exon 2-encoded
-domain residues.

View larger version (80K):
[in this window]
[in a new window]
|
Fig. 5.
Nuclear import of
ad E2-GFP is energy-dependent.
HeLa cells were transiently transfected for 24 h with GFP-GFP,
VHL-GFP, and E2-GFP (a-c) or with fusion to NES:
GFP-GFP-NES, VHL-GFP-NES, and E2-GFP-NES (d-o), as
described under "Materials and Methods." Cells were either
incubated at 37 °C (d-f), at 4 °C for 2 h
(g-i), at 37 °C in the presence of 6 deoxyglucose
(6-DOG) and sodium azide (SA) for 2 h
(j-l), or at 37 °C with leptomycin B (LMB; 10 µM; m-o).
|
|
Exon 2-encoded
-domain mediates transcription-dependent
trafficking of VHL and VBC/Cul-2, and the next step was to test if this
domain was sensitive to conditions known to affect HIF
stabilization. GFP fluorescence analysis of living cells indicated that
the steady state distribution of adVHL-GFP was unaffected by oxygen
tension (Fig. 6, a and
j). The addition of the RNA polymerase II inhibitor DRB
caused nuclear accumulation of adVHL-GFP, regardless of oxygen concentration (Fig. 6, b and k). It has been
recently suggested that proteasome inhibitors, which prevent
proteasome-mediated degradation of ubiquitinated proteins, might also
act as general inhibitors of nuclear export (47, 48). Interestingly, a
strong shift in the steady state distribution toward the nucleus of
adVHL-GFP was observed upon incubation with the proteasome inhibitor
CI, or lactacystin (data not shown) in normoxia and hypoxia (Fig. 6,
c and l). ad
E3-GFP steady state distribution
is more nuclear than adVHL-GFP and is unaffected by oxygen
concentration (Fig. 6, g and p). The addition of
DRB or CI also caused an important nuclear accumulation of ad
E3-GFP
with few cells displaying exclusive nuclear signal (Fig. 6,
h, i, q, and r). In
contrast, the localization of the
-domain mutant ad
E2-GFP
remained unchanged regardless of oxygen tension, proteasome inhibitors,
or RNA polymerase II inhibitors (Fig. 6, d-f and
m-o). One possible explanation for ad
E2-GFP
insensitivity to DRB and CI is that this mutant is unable to bind to
HIF
. These observations led us to test if the effect of DRB and CI
on shuttling of VHL are intrinsic to exon 2-encoded residues or if this
activity is mediated by HIF
. To test this, VHL shuttling was
analyzed in mouse embryonic fibroblasts that do not express endogenous
HIF
(Fig. 7). We noticed that
adVHL-GFP steady state subcellular localization was unaffected by the
absence of HIF-1
(Fig. 7, a and c). Likewise,
the addition of DRB caused nuclear accumulation of adVHL-GFP in
HIF-1
/
as well as in HIF-1
+/+ cells
(Fig. 7, b and d). The localization of both
mutants was unaffected by the absence or presence of HIF-1
(Fig. 7,
e, g, i, and k). The
-domain mutant ad
E3-GFP accumulated in the nucleus upon
incubation with DRB, whereas ad
E2-GFP was unaffected by this
treatment in HIF-1
/
and HIF-1
+/+
cells. The effect of CI was essentially the same as DRB (data not
shown) on the three fusion proteins (data not shown). The same data
were obtained in hypoxia (data not shown). Put together, these results
demonstrate that oxygen tension and HIF
have no affect on VHL
nuclear/cytoplasmic shuttling properties. They also indicate that exon
2-encoded
-domain plays a role in nuclear/cytoplasmic trafficking of
VHL, which is independent of its role in binding to HIF
.

View larger version (100K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of oxygen tension and proteasome
inhibitors on the subcellular localization and nuclear/cytoplasmic
trafficking properties of adVHL-GFP. Subcellular localization of
adVHL-GFP and mutants in cells grown in normoxia and hypoxia in the
presence or absence of DRB or CI. VHL / RCC 786-0 cells
were infected with adVHL-GFP, ad E2-GFP and ad E3-GFP and incubated
in normoxia (a-i) or for 4 h in hypoxia
(j-r). Cells were grown without further treatments
(a, d, g, j, m,
and p) or were treated with DRB (25 µM) for
2 h (b, e, h, k,
n, and q) or with CI (100 µM) for
2 h (c, f, i, l,
o, and r).
|
|

View larger version (105K):
[in this window]
[in a new window]
|
Fig. 7.
Exon 2-encoded residues mediate
transcription-dependent trafficking of VHL independently of
assembly with HIF .
HIF-1 / or HIF-1 +/+ MEF cells were
infected with adVHL-GFP, ad E2-GFP, and ad E3-GFP and incubated in
normoxia in the presence or absence of DRB (25 µM) for
2 h. The addition of CI (100 µM) for 2 h
essentially gave the same results as DRB (data not shown). The exact
same data were also obtained for cells incubated in hypoxia (data not
shown).
|
|
 |
DISCUSSION |
Inactivating mutations of the VHL tumor suppressor gene are
distributed equally between the
- and
-domains, suggesting that both domains play a key role in tumor suppression (29). Yet, the nature
and localization of the mutations has a profound effect on the clinical
manifestations in inherited VHL syndrome (31). Likewise, sporadic RCC
tumors are much more likely to harbor mutations in exon 2, mutations
that are rarely found in individuals afflicted with inherited VHL
syndrome (5). The discrepancy in the distribution of inactivating
mutations between sporadic and inherited RCC implies that exon
2-associated mutations might inactivate VHL function in different ways
than exon 3-associated mutations. We show here that loss of exon 2 or
exon 3 function essentially gives rise to the same cellular defects in
RCC, which includes aberrant nuclear accumulation of HIF
in normoxia
and inability to produce an extracellular fibronectin matrix. However,
loss of exon 2 function appears to have a lesser effect on the overall
activity of the VHL protein compared with loss of
-domain activity.
The major defects of the
-domain mutant that we were able to
identify were its inability to bind to HIF
and fibronectin and to
mediate transcription-dependent shuttling of VHL. The
binding results are similar to those recently reported by two other
groups, which demonstrated that missense mutations in exon 1-encoded
portion of the
-domain also abrogated VHL assembly to HIF
but not
to BC/Cul-2 (12, 18). A deletion of the
-domain caused a more
complete loss of function, since this mutant failed to assemble with
BC/Cul-2 as well as with substrate proteins and act as an E3 ubiquitin
ligase. This is not the consequence of a truncation of the
-domain,
since a missense mutation at residue 162 in the elongin C-binding box
has recently been reported to cause similar defects (8, 29). There is a
discrepancy between data obtained in vitro and in culture
inasmuch as truncations of exon 2- and exon 3-encoded sequences of VHL
are still able to assemble with HIF
in vitro (12, 18,
33). Either
E2-GFP and
E3-GFP fold in a different way in
vivo compared with in vitro, or these mutants have a
yet uncharacterized defect that prevents their assembly with HIF
in
cells. Interestingly, an alternative spliced mRNA of the VHL gene
that lacks exon 2 sequences has been reported to be produced in several
independent tissues and cell lines (1). A VHL protein without exon 2 sequences might change substrate specificity from HIF
to another
unidentified protein while still acting as an E3 ubiquitin ligase. An
endogenous protein product originating from a mRNA lacking exon 2 sequences still remains to be identified. Nevertheless, the data
presented in this report are in good agreement with the proposed model
predicted by the crystal structure of VHL that the
-domain of VHL is
involved in substrate protein, as well as fibronectin, recognition
(29). They also demonstrate that tumor-derived mutations inactivate VHL
functions in different ways, which may lead to distinct cellular phenotypes.
The study of ad
E2-GFP has also revealed other interesting
biochemical aspects of the function of exon 2-encoded sequences, one of
which is that it is required for VHL-mediated NEDD8 conjugation on
cullin-2. The functional relevancy of this post-translational modification is still unknown, but it has been suggested that it might
play a role in protecting cullin-2 from self-ubiquitination (49). Data
shown here are somewhat in disagreement with this model, since equal
amounts of cullin-2 can be found bound to VHL and ad
E2-GFP,
regardless of conjugation to NEDD8. NEDD8 conjugation is reported to be
a nuclear event (44). ad
E2-GFP can be detected in the nuclear
compartment at steady state, and the lack of NEDD8 conjugation activity
cannot be simply explained by a defect in nuclear import of the
VBC/Cul-2 complex. This argument is supported by a novel assay
presented here, which enables the analysis of energy requirement for
nuclear import of proteins in living cells. Energy expenditure for
nuclear import is a hallmark of signal-mediated and -regulated
nuclear/cytoplasmic trafficking processes (50-52). The observation
that ad
E2-GFP retains the ability to import in the nucleus in an
energy-dependent manner suggest that other protein/protein interactions involved in nuclear import of the VBC/Cul-2 complex are
not affected by loss of function of exon 2-encoded sequences. Likewise,
we noticed HIF
signal exclusively in the nucleus of normoxic
VHL
/
cells, indicating that HIF
is able to import
even in the absence of hypoxic conditions and assembly with VHL. These
data are somewhat surprising, since it is generally believed that
HIF
contains a nuclear import signal that is activated only in
hypoxia (45). One possible interpretation of these data is that the
hypoxia-inducible nuclear import of HIF
is regulated by VHL, which
might play a role in retaining HIF
in the cytoplasm in normoxia.
Results shown here suggest that transcription-dependent
nuclear/cytoplasmic shuttling and steady state distribution of VHL are
not affected by oxygen tension and do not require assembly with HIF
.
However, we did find that adVHL-GFP accumulated in the nucleus upon
incubation with proteasome inhibitors, similar to the effect obtained
with DRB treatment. Drugs that inhibit proteasome-mediated degradation
of proteins have been hypothesized to also interfere with general
nuclear export processes (47, 48). Sensitivity to proteasome inhibitors
is mediated by exon 2-encoded
-domain in a manner reminiscent of
DRB. We have previously shown that VHL
transcription-dependent shuttling domain acts dominantly on
the VBC/Cul-2 complex and that DRB is a good inhibitor of VHL-mediated VBC-Cul-2 nuclear export in living cells and in vitro (46). It is conceivable that CI also blocks exon 2-mediated nuclear export of
VHL, leading to nuclear accumulation of VBC/Cul-2. It is unlikely that
the observed nuclear accumulation of adVHL-GFP is the consequence of
HIF
-mediated nuclear retention, since proteasome inhibitors and DRB
have similar effects on VHL in HIF-null MEFs. The presence of
ad
E2-GFP in the cytoplasm at steady state might be explained by a
fraction of VHL that is not importable at a given time. Alternatively,
the existence of other nuclear export signals within the complex
might gain dominance upon loss of function of exon 2-encoded residues.
Taken together, these results support the model that exon 2-encoded
residues are involved in two independent functions: mediating nuclear
export of the VBC/Cul-2 complex and binding to substrate proteins. We
are still in the process of identifying relevant sequences involved in
signal-mediated and Ran-dependent nuclear/cytoplasmic
trafficking of the VBC/Cul-2 complex. Identification of these sequences
will surely provide important clues in the elucidation of VHL-mediated
tumor suppressor function.
 |
ACKNOWLEDGEMENTS |
We sincerely thank Dr. David Park, Ruth
Slack, and Steve Callaghan for their help with the adenovirus system
and Dr. Randy Johnson for the HIF-1
/
MEF cell line.
 |
FOOTNOTES |
*
This work was supported by an Operating Grant from the
Medical Research Council of Canada (MRC) (to S. L.).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.
Scholar of the MRC. To whom correspondence should be addressed:
Dept. of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario K1H 8M5, Canada.
Tel.: 613-562-5800 (ext. 8385); Fax: 613-562-5636; E-mail: slee@uottawa.ca.
Published, JBC Papers in Press, October 9, 2000, DOI 10.1074/jbc.M008295200
 |
ABBREVIATIONS |
The abbreviations used are:
VHL, von
Hippel-Lindau;
RCC, renal cell carcinoma(s);
E1, ubiquitin-activating
enzyme;
E2, ubiquitin carrier protein;
E3, ubiquitin-protein isopeptide
ligase;
HIF, hypoxia-inducible factor;
HIF
,
-subunit(s) of
hypoxia-inducible factor;
MEF, mouse embryonic fibroblast;
GFP, green
fluorescent protein;
NES, nuclear export signal;
DMEM, Dulbecco's
modified Eagle's medium;
FCS, fetal calf serum;
PVDF, polyvinylidene
difluoride;
CI, calpain inhibitor I;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel electrophoresis;
DRB, 5,6-dichlorobenzimidazole riboside.
 |
REFERENCES |
1.
|
Gnarra, J. R.,
Duan, D. R.,
Weng, Y.,
Humphrey, J. S.,
Chen, D. Y.,
Lee, S.,
Pause, A.,
Dudley, C. F.,
Latif, F.,
Kuzmin, I.,
Schmidt, L.,
Duh, F. M.,
Stackhouse, T.,
Chen, F.,
Kishida, T.,
Wei, M. H.,
Lerman, M. I.,
Zbar, B.,
Klausner, R. D.,
and Linehan, W. M.
(1996)
Biochim. Biophys. Acta
1242,
201-210[Medline]
[Order article via Infotrieve]
|
2.
|
Latif, F.,
Tory, K.,
Gnarra, J.,
Yao, M.,
Duh, F. M.,
Orcutt, M. L.,
Stackhouse, T.,
Kuzmin, I.,
Modi, W.,
Geil, L.,
Schmidt, L.,
Zhou, F.,
Li, H.,
Wei, M. H.,
Chen, F.,
Glenn, G.,
Choyke, P.,
Walther, M. M.,
Weng, Y.,
Duan, D. S. R.,
Dean, M.,
Glavac, D.,
Richards, F. M.,
Crossey, P. A.,
Ferguson-Smith, M. A.,
Le Paslier, D.,
Chumakov, I.,
Cohen, D.,
Chinault, A. C. R.,
Maher, E. R.,
Linehan, W. M.,
Zbar, B.,
and Lerman, M. I.
(1993)
Science
260,
1317-1320[Medline]
[Order article via Infotrieve]
|
3.
|
Linehan, W. M.,
Lerman, M. I.,
and Zbar, B.
(1995)
J. Am. Med. Assoc.
273,
564-570[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Foster, K.,
Prowse, A.,
van den Berg, A.,
Fleming, S.,
Hulsbeek, M. M. F.,
Crossey, P. A.,
Richards, F. M.,
Cairns, P.,
Affara, N. A.,
Ferguson-Smith, M. A.,
Buys, C. H.,
and Maher, E. R.
(1994)
Hum. Mol. Genet.
3,
2169-2173[Abstract]
|
5.
|
Gnarra, J. R.,
Tory, K.,
Weng, Y.,
Schmidt, L.,
Wei, M. H.,
Li, H.,
Latif, F.,
Liu, F. S.,
Chen, F.,
Duh, F. M.,
Lubensky, I.,
Duan, D. R.,
Florence, C.,
Pozzatti, R.,
Walther, M. M.,
Bander, N. H.,
Grossman, H. B.,
Brauch, H.,
Pomer, S.,
Brooks, J. D.,
Isaacs, W. B.,
Lerman, M. I.,
Zbar, B.,
and Linehan, W. M.
(1994)
Nat. Genet.
7,
85-90[Medline]
[Order article via Infotrieve]
|
6.
|
Iliopoulos, O.,
Kibel, A.,
Gray, S.,
and Kaelin, W. G.
(1995)
Nat. Med.
1,
822-826[Medline]
[Order article via Infotrieve]
|
7.
|
Duan, D. R.,
Pause, A.,
Burgess, W. H.,
Aso, T.,
Chen, D. Y. T.,
Garrett, K. P.,
Conaway, R. C.,
Conaway, J. W.,
Linehan, W. M.,
and Klausner, R. D.
(1995)
Science
269,
1402-1406[Medline]
[Order article via Infotrieve]
|
8.
|
Lonergan, K. M.,
Iliopoulos, O.,
Ohh, M.,
Kamura, T.,
Conaway, R. C.,
Conaway, J. W.,
and Kaelin, W. G.
(1998)
Mol. Cell. Biol.
18,
732-741[Abstract/Free Full Text]
|
9.
|
Kamura, T.,
Koepp, D. M.,
Conrad, M. N.,
Skowyra, D.,
Moreland, R. J.,
Iliopoulos, O.,
Lane, W. S.,
Kaelin, W. G.,
Elledge, S. J.,
Conaway, R. C.,
Harper, J. W.,
and Conaway, J. W.
(1999)
Science
284,
657-661[Abstract/Free Full Text]
|
10.
|
Kibel, A.,
Iliopoulos, O.,
DeCaprio, J. A.,
and Kaelin, W. G.
(1995)
Science
269,
1444-1446[Medline]
[Order article via Infotrieve]
|
11.
|
Pause, A.,
Lee, S.,
Worrell, R. A.,
Chen, D. Y. T.,
Burgess, W. H.,
Linehan, W. M.,
and Klausner, R. D.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2156-2161[Abstract/Free Full Text]
|
12.
|
Cockman, M. E.,
Masson, N.,
Mole, D. R.,
Jaakkola, P.,
Chang, G. W.,
Clifford, S. C.,
Maher, E. R.,
Pugh, C. W.,
Ratcliffe, P. J.,
and Maxwell, P. H.
(2000)
J. Biol. Chem.
275,
25733-25741[Abstract/Free Full Text]
|
13.
|
Maxwell, P. H.,
Wiesener, M. S.,
Chang, G. W.,
Clifford, S. C.,
Vaux, E. C.,
Cockman, M. E.,
Wykoff, C. C.,
Pugh, C. W.,
Maher, E. R.,
and Racliff, P. J.
(1999)
Nature
399,
271-275[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Huang, L. E.,
Gu, J.,
Schau, M.,
and Bunn, H. F.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7987-7992[Abstract/Free Full Text]
|
15.
|
Salceda, S.,
and Caro, J.
(1997)
J. Biol. Chem.
272,
22642-22647[Abstract/Free Full Text]
|
16.
|
Iwai, K.,
Yamanaka, K.,
Kamura, T.,
Minato, N.,
Conaway, R. C.,
Conaway, J. W.,
Klausner, R. D.,
and Pause, A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12436-12441[Abstract/Free Full Text]
|
17.
|
Lisztwan, J.,
Imbert, G.,
Wirbelauer, C.,
Gstaiger, M.,
and Krek, W.
(1999)
Genes Dev.
13,
1822-1833[Abstract/Free Full Text]
|
18.
|
Ohh, M.,
Park, C. W.,
Ivan, M.,
Hoffman, M. A.,
Kim, T. Y.,
Huang, L. E.,
Pavletich, N.,
Chau, V.,
and Kaelin, W. G.
(2000)
Nat. Cell Biol.
2,
423-427[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Gnarra, J. R.,
Zhou, S.,
Merrill, M. J.,
Wagner, J. R.,
Krumm, A.,
Papavassiliou, E. O.,
Idfield, E. H.,
Klausner, R. D.,
and Linehan, W. M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10589-10594[Abstract/Free Full Text]
|
20.
|
Iliopoulos, O.,
Levy, A. P.,
Jiang, C.,
Kaelin, W. G.,
and Goldberg, M. A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10595-10599[Abstract/Free Full Text]
|
21.
|
Knebelmann, B.,
Ananth, S.,
Cohen, H. T.,
and Sukhatme, V. P.
(1998)
Cancer Res.
58,
226-231[Abstract]
|
22.
|
Seimeister, G.,
Weidel, K.,
Mohrs, K.,
Barleon, B.,
Martiny-Baron, G.,
and Marne, D.
(1996)
Cancer Res.
56,
2299-2301[Abstract]
|
23.
|
Corless, C. L.,
Kibel, A.,
Iliopoulos, O.,
and Kaelin, W. G.
(1997)
Hum. Pathol.
28,
459-464[Medline]
[Order article via Infotrieve]
|
24.
|
Lee, S.,
Chen, D. Y. T.,
Humphrey, J. S.,
Gnarra, J. R.,
Linehan, W. M.,
and Klausner, R. D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
1770-1775[Abstract/Free Full Text]
|
25.
|
Lee, S.,
Neumann, M.,
Stearman, R.,
Stauber, R.,
Pause, A.,
Pavlakis, G. N.,
and Klausner, R. D.
(1999)
Mol. Cell. Biol.
19,
1486-1497[Abstract/Free Full Text]
|
26.
|
Los, M.,
Jansen, G. H.,
Kaelin, W. G.,
Lips, C. J.,
Blijham, G. H.,
and Voest, E. E.
(1996)
Lab. Invest.
75,
231-238[Medline]
[Order article via Infotrieve]
|
27.
|
Tsuchiya, H.,
Iseda, T.,
and Hino, O.
(1996)
Cancer Res.
56,
2881-2885[Abstract]
|
28.
|
Ohh, M.,
Yauch, R. L.,
Lonergan, K. M.,
Whaley, J. M.,
Stemmer-Rachamimov, O. D.,
Louis, A. N.,
Gavin, B. J.,
Kley, N.,
Kaelin, W. G.,
and Iliopoulos, O.
(1998)
Mol. Cell
1,
959-968[Medline]
[Order article via Infotrieve]
|
29.
|
Stebbins, C. E.,
Kaelin, W. G.,
and Pavletich, N. P.
(1999)
Science
284,
455-461[Abstract/Free Full Text]
|
30.
|
Beroud, C.,
Joly, D.,
Gallou, C.,
Staroz, F.,
Orfanelli, M. T.,
and Junien, C.
(1998)
Nucleic Acids Res.
26,
256-258[Abstract/Free Full Text]
|
31.
|
Chen, F.,
Kishida, T.,
Yao, M.,
Hustad, T.,
Glavac, D.,
Dean, M.,
Gnarra, J. R.,
Orcutt, M. L.,
Duh, F. M.,
Glenn, G.,
Green, J.,
Hsia, Y. E.,
Lamiell, J.,
Li, H.,
Wei, M. H.,
Schmidt, L.,
Tory, K.,
Kuzmin, I.,
Stackhouse, T.,
Latif, F.,
Linehan, W. M.,
Lerman, M. I.,
and Zbar, B.
(1995)
Hum. Mutat.
5,
66-75[Medline]
[Order article via Infotrieve]
|
32.
|
Kishida, T.,
Stackhouse, T. M.,
Chen, F.,
Lerman, M. I.,
and Zbar, B.
(1995)
Cancer Res.
55,
4544-4548[Abstract]
|
33.
|
Tanimoto, K.,
Makino, Y.,
Pereira, T.,
and Poellinger, L.
(2000)
EMBO J.
19,
4298-4309[Abstract/Free Full Text]
|
34.
|
Ryan, H. E.,
Poloni, M.,
McNulty, W.,
Elson, D.,
Gassmann, M.,
Arbeit, J. M.,
and Johnson, R. S.
(2000)
Cancer Res.
60,
4010-4015[Abstract/Free Full Text]
|
35.
|
Stauber, R.,
Gaitanaris, G. A.,
and Pavlakis, G. N.
(1995)
Virology
213,
439-449[CrossRef][Medline]
[Order article via Infotrieve]
|
36.
|
Fischer, U.,
Huber, J.,
Boelens, W. C.,
Mattaj, I. W.,
and Luhrmann, R.
(1995)
Cell
82,
475-483[Medline]
[Order article via Infotrieve]
|
37.
|
Chen, L.,
Anton, M.,
and Graham, F. L.
(1996)
Somat. Cell Mol. Genet.
6,
477-488
|
38.
|
Graham, F. L.,
and van der Eb, A. J.
(1973)
Virology
52,
456-467[Medline]
[Order article via Infotrieve]
|
39.
|
Nishi, K.,
Yoshida, M.,
Fujiwara, D.,
Nishikawa, M.,
Horinouchi, S.,
and Beppu, T.
(1994)
J. Biol. Chem.
269,
6320-6324[Abstract/Free Full Text]
|
40.
|
Wolff, B.,
Sanglier, J. J.,
and Wang, Y.
(1997)
Chem. Biol.
4,
139-147[Medline]
[Order article via Infotrieve]
|
41.
|
Liakopoulos, D.,
Büsgen, T.,
Brychzy, A.,
Jentsch, S.,
and Pause, A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
5510-5515[Abstract/Free Full Text]
|
42.
|
Hardy, S.,
Kitamura, M.,
Harris-Stansil, T.,
Dai, Y.,
and Phipps, M. L.
(1996)
J. Virol.
71,
1842-1849[Abstract]
|
43.
|
Kim, M.,
Katayose, Y.,
Li, Q.,
Rakkar, A. N.,
Li, Z.,
Hwang, S. G.,
Katayose, D.,
Trepel, J.,
Cowan, K. H.,
and Seth, P.
(1998)
Biochem. Cell Biol. Commun.
253,
672-677
|
44.
|
Kamitani, T.,
Kito, K.,
Nguyen, H. P.,
and Yeh, E. T. H.
(1997)
J. Biol. Chem.
272,
28557-28562[Abstract/Free Full Text]
|
45.
|
Kallio, P.,
Okamoto, K.,
O'Brien, S.,
Carrero, P.,
Makino, Y.,
Tanaka, H.,
and Poellinger, L.
(1998)
EMBO J.
17,
6573-6586[Abstract/Free Full Text]
|
46.
|
Groulx, I.,
Bonicalzi, M. E.,
and Lee, S.
(2000)
J. Biol. Chem.
275,
8991-9000[Abstract/Free Full Text]
|
47.
|
Scheffner, M.
(1999)
Nature
398,
103-104[CrossRef][Medline]
[Order article via Infotrieve]
|
48.
|
Tomoda, K.,
Kubota, Y.,
and Kato, J.
(1999)
Nature
398,
160-165[CrossRef][Medline]
[Order article via Infotrieve]
|
49.
|
Schoenfeld, A. R.,
Davidowitz, E. J.,
and Burk, R. D.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
8507-8512[Abstract/Free Full Text]
|
50.
|
Moore, M. S.,
and Blobel, G.
(1992)
Cell
69,
939-950[Medline]
[Order article via Infotrieve]
|
51.
|
Görlich, D.,
and Mattaj, W.
(1996)
Science
271,
1513-1518[Abstract]
|
52.
|
Nigg, E. A.
(1997)
Nature
386,
779-787[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.