Role of Exon 2-encoded beta -Domain of the von Hippel-Lindau Tumor Suppressor Protein*

Marie-Eve Bonicalzi, Isabelle Groulx, Natalie de Paulsen, and Stephen LeeDagger

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 alpha  (HIFalpha ) 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-1alpha -null embryonic cells suggest that exon 2-encoded beta -domain mediates transcription-dependent nuclear/cytoplasmic shuttling of VHL independently of assembly with HIF-1alpha and oxygen concentration. Exon 3-encoded alpha -helical domain is required for VHL complex formation with BC/cullin-2 and E3 ubiquitin ligase activity, for binding to HIFalpha /fibronectin, but this domain is not essential for transcription-dependent nuclear/cytoplasmic trafficking. VHL-/- renal carcinoma cells expressing beta -domain mutants failed to produce an extracellular fibronectin matrix and to degrade HIFalpha , 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 beta -domain mutations inactivate VHL function differently than alpha -domain mutations, potentially providing an explanation for the relationship between different mutations of the VHL gene and clinical outcome.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -subunits of hypoxia-inducible factor (HIFalpha ) for oxygen-dependent degradation (12-18). HIFalpha 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). HIFalpha 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 HIFalpha 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 beta -domain that spans residues 64-154 and an alpha -helical domain (alpha -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 alpha -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 beta -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 alpha -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 beta -strands of the beta -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 HIFalpha (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 alpha -domain. We show that the exon 2-encoded beta -domain plays two independent roles in binding to HIFalpha 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 beta -domain of VHL is involved in substrate recognition and nuclear/cytoplasmic trafficking.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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-1alpha 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 Delta E3-GFP fusion protein. Another deletion mutant of VHL lacking amino acids 114-154 was fused to GFP to produce the Delta E2-GFP fusion protein. Two GFPs in tandem were cloned into pcDNA 3.1(-) to produce the GFP-GFP fusion protein. VHL-GFP-NES, Delta E2-GFP-NES, and GFP-GFP-NES were produced by fusion of VHL-GFP, Delta 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 psi 5 viral DNA were also obtained from Dr. Park. The three different VHL constructions (VHL-GFP, Delta E2-GFP, and Delta 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 psi 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, adDelta E2-GFP, or adDelta 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, Delta 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-1alpha and HIF-2alpha -- VHL-/- 786-0 cells expressing endogenous HIF-2alpha or 117-4 cells expressing endogenous HIF-1alpha 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-1alpha (Transduction Laboratories), anti-HIF-2alpha 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-1alpha -- 117 cells or transiently transfected 786-0 with HIF-1alpha 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-1alpha 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Biochemical Characterization of the Exon 2-Encoded beta -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 beta -domain, and an alpha -domain (Fig. 1A). Sporadic RCC frequently harbor inactivating mutations in the exon 2-encoded part of the beta -domain, whereas these mutations are relatively rare in individuals afflicted with inherited VHL syndrome (5). To study the role of exon 2-encoded beta -domain in VHL tumor suppressor function, a cDNA encoding a tumor-derived truncation of residues 114-154 was fused to GFP to produce the Delta 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). Delta E2-GFP is predicted to have a partial, if not total, loss of beta -domain function while retaining an intact, exon 3-encoded alpha -helical domain. A tumor-derived truncation of the exon 3-encoded alpha -helical domain (last 56 C-terminal residues), which retained intact the sequences of the beta -domain, was also fused to GFP (Delta E3-GFP) (Fig. 1B). Delta E2-GFP, Delta E3-GFP, and wild-type VHL-GFP were cloned in pAdlox vector, and adenoviruses (adDelta E2-GFP, adDelta 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 adDelta E2-GFP accumulated to levels similar to those of adVHL-GFP and adDelta E3-GFP, suggesting that adDelta 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.



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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 beta -domain, and an alpha -domain. Exon 2 encodes for the latter part of the beta -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 Delta E2-GFP. A tumor-derived truncation of the exon 3-encoded alpha -domain (strike box) was also fused to GFP to produce Delta E3-GFP. A FLAG tag (shadow box) was fused to the N-termini of all three constructs. Delta E2-GFP, Delta E3-GFP, and VHL-GFP were cloned in pAdlox vector to prepare adenoviruses. adDelta E2-GFP, adDelta 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, adDelta E2-GFP, adDelta 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 adDelta E2-GFP in comparison with adVHL-GFP and adDelta E3-GFP. The beta -domain mutant adDelta 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 alpha -helical domain deletion mutant (adDelta 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 adDelta 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 beta -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.



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Fig. 2.   Biochemical characterization of an exon 2-encoded beta -domain mutant of VHL. A, an intact beta -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 adDelta E2-GFP, adDelta 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 beta -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 beta -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 beta -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, adDelta 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 adDelta E2-GFP. Immunoprecipitation analysis revealed that adVHL-GFP was able to assemble with fibronectin, whereas adDelta E2-GFP failed to do so (Fig. 3B). The adDelta 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 beta -domain to bind to fibronectin and mediate proper extracellular matrix formation.



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Fig. 3.   VHL requires an intact beta -domain to bind fibronectin and mediate proper extracellular matrix formation. A, adDelta 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, adDelta E2-GFP, or adDelta 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, adDelta 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 adDelta E2-GFP, adDelta 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 beta -Domain of VHL in Oxygen-dependent Degradation of HIFalpha -- It was recently shown that one of the major defects of VHL-/- RCC cells is their inability to mediate oxygen-dependent degradation of HIFalpha , 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 HIFalpha (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-1alpha ) and 786-0 (HIF-2alpha ) ability to mediate degradation of HIFalpha in normoxia (Fig. 4A). HIFalpha levels were not affected by expression of adDelta E2-GFP or adDelta E3-GFP (Fig. 4A). We notice that adVHL-GFP assembled with a significant amount of HIFalpha (1alpha and 2alpha ) in hypoxia and in the presence of the proteasome inhibitor CI. In contrast to data obtained in vitro, immunoprecipitation analysis revealed that adDelta E2-GFP and adDelta E3-GFP failed to bind to HIFalpha in adenovirus-infected cells (Fig. 4B, top panels). We did not detect binding of HIFalpha to adDelta E2-GFP and adDelta 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 beta -domain, as well as the alpha -domain, is required for VHL assembly with HIFalpha in cells.



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Fig. 4.   HIFalpha fails to bind to an exon 2-encoded beta -domain mutant of VHL and accumulates in the nucleus of normoxic cells. A, adDelta E2-GFP failed to mediate oxygen-dependent degradation of HIFalpha . VHL-/- 786-0 RCC cells (HIF-2alpha ) and 117-4 cells (HIF-1alpha ) were uninfected or infected with adVHL-GFP, adDelta E2-GFP, and adDelta 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-1alpha (top panel) or a rabbit anti-HIF-2alpha (bottom panel) antibody. B, adVHL-GFP assembled with endogenous HIF-1alpha (117 cells) or with endogenous HIF-2alpha (786-0 cells), but adDelta E2-GFP and adDelta 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-1alpha (top left panel), a rabbit anti-HIF-2alpha (top right panel), or a mouse anti-FLAG M2 antibody (bottom panels). C, nuclear import of HIFalpha occurs regardless of oxygen tension. VHL-/- RCC 117 cells (endogenous HIF-1alpha ) (a-l) or 786-0 cells transiently transfected with HIF-1alpha (m-p) were uninfected or infected with the adenoviruses adVHL-GFP, adDelta E2-GFP, and adDelta E3-GFP. Cells were washed, fixed, and stained with a mouse anti-HIF-1alpha 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-1alpha 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-1alpha accumulated in the nucleus in normoxia when co-transfected with these VHL mutants.

It has been hypothesized that HIF-1alpha 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 HIFalpha , the subcellular localization of endogenous HIF-1alpha 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-1alpha 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-1alpha is able to import in the nucleus even in the presence of oxygen and in the absence of VHL. A strong HIF-1alpha nuclear signal was also observed in cells expressing adDelta E2-GFP (Fig. 4C, c, g, and k) as well as adDelta 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-1alpha in RCC VHL-/- 786-0 cells (which do not express endogenous HIF-1alpha ). A strong HIF-1alpha signal was detected exclusively in the nucleus of normoxic RCC VHL-/- 786-0 cells transiently transfected with HIF-1alpha cDNA that were either uninfected (Fig. 4C, m), infected with GFP alone (data not shown), or infected with adDelta E2-GFP (Fig. 4C, o) and adDelta E3-GFP (Fig. 4C, p). The addition of proteasome inhibitors or incubation in hypoxia led to nuclear accumulation of endogenous or overexpressed HIFalpha regardless of the presence of adVHL-GFP or mutants, as expected (data not shown). HIF-1alpha 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-1alpha 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 HIFalpha 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 HIFalpha stability, such as oxygen concentration, and if it is able to do so independently of assembly with HIFalpha . Since adDelta 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 beta -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 Delta 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. Delta 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 HIFalpha and exon 2-encoded beta -domain residues.



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Fig. 5.   Nuclear import of adDelta E2-GFP is energy-dependent. HeLa cells were transiently transfected for 24 h with GFP-GFP, VHL-GFP, and Delta E2-GFP (a-c) or with fusion to NES: GFP-GFP-NES, VHL-GFP-NES, and Delta 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 beta -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 HIFalpha 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). adDelta 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 adDelta E3-GFP with few cells displaying exclusive nuclear signal (Fig. 6, h, i, q, and r). In contrast, the localization of the beta -domain mutant adDelta 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 adDelta E2-GFP insensitivity to DRB and CI is that this mutant is unable to bind to HIFalpha . 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 HIFalpha . To test this, VHL shuttling was analyzed in mouse embryonic fibroblasts that do not express endogenous HIFalpha (Fig. 7). We noticed that adVHL-GFP steady state subcellular localization was unaffected by the absence of HIF-1alpha (Fig. 7, a and c). Likewise, the addition of DRB caused nuclear accumulation of adVHL-GFP in HIF-1alpha -/- as well as in HIF-1alpha +/+ cells (Fig. 7, b and d). The localization of both mutants was unaffected by the absence or presence of HIF-1alpha (Fig. 7, e, g, i, and k). The alpha -domain mutant adDelta E3-GFP accumulated in the nucleus upon incubation with DRB, whereas adDelta E2-GFP was unaffected by this treatment in HIF-1alpha -/- and HIF-1alpha +/+ 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 HIFalpha have no affect on VHL nuclear/cytoplasmic shuttling properties. They also indicate that exon 2-encoded beta -domain plays a role in nuclear/cytoplasmic trafficking of VHL, which is independent of its role in binding to HIFalpha .



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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, adDelta E2-GFP and adDelta 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).



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Fig. 7.   Exon 2-encoded residues mediate transcription-dependent trafficking of VHL independently of assembly with HIFalpha . HIF-1alpha -/- or HIF-1alpha +/+ MEF cells were infected with adVHL-GFP, adDelta E2-GFP, and adDelta 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inactivating mutations of the VHL tumor suppressor gene are distributed equally between the beta - and alpha -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 HIFalpha 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 alpha -domain activity. The major defects of the beta -domain mutant that we were able to identify were its inability to bind to HIFalpha 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 beta -domain also abrogated VHL assembly to HIFalpha but not to BC/Cul-2 (12, 18). A deletion of the alpha -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 alpha -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 HIFalpha in vitro (12, 18, 33). Either Delta E2-GFP and Delta 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 HIFalpha 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 HIFalpha 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 beta -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 adDelta 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 adDelta E2-GFP, regardless of conjugation to NEDD8. NEDD8 conjugation is reported to be a nuclear event (44). adDelta 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 adDelta 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 HIFalpha signal exclusively in the nucleus of normoxic VHL-/- cells, indicating that HIFalpha 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 HIFalpha 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 HIFalpha is regulated by VHL, which might play a role in retaining HIFalpha 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 HIFalpha . 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 beta -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 HIFalpha -mediated nuclear retention, since proteasome inhibitors and DRB have similar effects on VHL in HIF-null MEFs. The presence of adDelta 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-1alpha -/- 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.

Dagger 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; HIFalpha , alpha -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
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ABSTRACT
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DISCUSSION
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