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Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8,1 Department of Medical Biophysics, University of Toronto and Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Ontario M5G 2M9, Canada,3 Washington University School of Medicine, St. Louis, Missouri 63110,2 Department of Molecular Oncology, Dana-Farber Cancer Institute, Harvard Medical School, and Howard Hughes Medical Institute, Boston, Massachusetts 021154
Received 25 June 2003/ Returned for modification 13 August 2003/ Accepted 8 January 2004
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ABSTRACT |
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(HIF-) subunits via ubiquitin-mediated proteolysis and in the regulation of fibronectin matrix assembly. Although most disease-causing pVHL mutations hinder the regulation of the HIF pathway, every disease-causing pVHL mutant tested to date has failed to promote the assembly of the fibronectin matrix, underscoring its potential importance in VHL disease. Here, we report that a ubiquitin-like molecule called NEDD8 covalently modifies pVHL. A nonneddylateable pVHL mutant, while retaining its ability to ubiquitylate HIF, failed to bind to and promote the assembly of the fibronectin matrix. Expression of the neddylation-defective pVHL in RCC cells, while restoring the regulation of HIF, failed to promote the differentiated morphology in a three-dimensional growth assay and was insufficient to suppress the formation of tumors in SCID mice. These results suggest that NEDD8 modification of pVHL plays an important role in fibronectin matrix assembly and that in the absence of such regulation, an intact HIF pathway is insufficient to prevent VHL-associated tumorigenesis. |
INTRODUCTION |
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The VHL protein (pVHL) is a component of an E3 ubiquitin ligase complex, VEC, comprising elongins B and C, Rbx1, and Cul2 (14). VEC binds and targets specifically prolyl-hydroxylated subunits of hypoxia-inducible factor 1 (HIF-1), HIF-2, and HIF-3 for polyubiquitylation (2, 10, 12, 19, 20, 22). The hydroxylation of HIF- by the newly identified class of prolyl-hydroxylases occurs on conserved proline residues within the oxygen-dependent degradation domain (ODD) in the presence of oxygen and iron (5, 10-12). The polyubiquitin-tagged HIF- subunits are subsequently captured by the 26S proteasome for degradation. Thus, under hypoxia, HIF- subunits remain unhydroxylated and consequently escape ubiquitin-mediated proteolysis. The HIF- subunits bind to constitutively stable HIF-ß (also known as ARNT [aryl-hydrocarbon receptor nuclear translocator]) subunits, forming an active heterodimeric HIF transcription factor that binds to the hypoxia-responsive elements in the promoters of numerous hypoxia-inducible genes, triggering the physiologic responses to hypoxia. Tumor cells devoid of functional pVHL produce inordinate amounts of HIF-regulated hypoxia-inducible genes, such as vascular endothelial growth factor, irrespective of the ambient oxygen tension (14). The expression of this and other hypoxia-inducible angiogenic factors likely accounts for the vascular phenotype of VHL-associated tumors.
pVHL also binds directly to fibronectin (1, 9, 23). More importantly, all disease-associated pVHL mutants examined to date exhibit reduced or undetectable binding to fibronectin (21). The physical interaction of fibronectin with the wild-type pVHL has been linked to the proper assembly of an extracellular fibronectin matrix (23). Lieubeau-Teillet and coworkers demonstrated that RCC cells lacking pVHL grow as tightly packed spheroids in a three-dimensional growth assay, indicative of an undifferentiated phenotype (17). In contrast, RCC cells reconstituted with pVHL form loose aggregates which, upon microscopic and ultrastructural examination, exhibit evidence of epithelial differentiation, such as trabecular and tubular structures (17). These observations correlate with differences in fibronectin matrix deposition, with pVHL-expressing cells producing appreciable fibronectin arrays in the extracellular space, suggesting that fibronectin is actively involved in the growth and differentiation of cells through the interaction with pVHL (17). Furthermore, Davidowitz and colleagues demonstrated that pVHL-expressing RCC cells grown on extracellular matrix (ECM) differentiate into organized epithelial monolayers, whereas pVHL-deficient cells are branched and disorganized and fail to arrest under high cell density (4). The reintroduction of pVHL in RCC cells markedly increases the expression of hepatocyte nuclear factor 1, a transcription factor and global activator of proximal tubule-specific genes in the nephron (4). These observations strengthen the notion that pVHL plays a causal role in renal cell differentiation and mediates growth arrest through the integration of cell-ECM signals.
Here, we show that pVHL becomes covalently conjugated by NEDD8. This modification is not required for the E3 activity of VEC to target HIF- for ubiquitylation. However, a nonneddylateable pVHL mutant failed to bind to and promote the assembly of fibronectin. The expression of the neddylation-defective pVHL in RCC cells, while restoring the regulation of HIF, failed to promote the differentiated morphology in a three-dimensional growth assay and was insufficient to suppress the formation of tumors in SCID mice. Thus, the neddylation-defective pVHL mutant clearly separates the functions of E3 ligase activity and fibronectin matrix assembly. The study below demonstrates that NEDD8 modification of pVHL plays an important role in the proper assembly of fibronectin and that in the absence of such regulation, an intact HIF pathway is insufficient to prevent tumorigenesis.
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MATERIALS AND METHODS |
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Antibodies. Monoclonal anti-Gal4 (DBD; RK5C1) and anti-hemagglutinin (HA) (12CA5) antibodies were from Santa Cruz and Boehringer, respectively. Monoclonal anti-T7 antibody was from Novagen (Madison, Wis.). Polyclonal anti-GLUT1 and monoclonal anti-Cul2 antibodies were from Alpha Diagnostics and Zymed, respectively. Polyclonal anti-fibronectin antibody was from Calbiochem. Monoclonal anti-pVHL antibody (IG32) was previously described (13).
Plasmids.
Mammalian expression plasmids pRc-CMV-HA-VHL(WT) and pRc-CMV-HA-VHL(C162F) were described previously (18). pRc-CMV-HA-VHL(RRR), -(KRR), -(RKR), -(RRK), and -(K159E) and pRc-CMV-HA-VHL(R200K) and -(R210K), which are in the background of pVHL(RRR), were generated the same way, by using a Stratagene site-directed mutagenesis kit according to the manufacturer's instructions. Expression plasmids pcDNA3-T7-NEDD8, pcDNA3-T7-NEDD8
GG, and pcDNA3-T7-SUMO were described previously (24). All plasmids were confirmed by direct DNA sequencing.
Immunoprecipitation and immunoblotting. Immunoprecipitation and Western blotting were performed as described previously (23). In brief, cells were lysed in EBC buffer (50 mM Tris [pH 8.0], 120 mM NaCl, 0.5% NP-40) supplemented with protease and phosphatase inhibitors (Roche). Immunoprecipitates, immobilized on protein A-Sepharose (Amersham Biosciences, Uppsala, Sweden), were washed five times with NETN buffer (20 mM Tris [pH 8.0], 120 mM NaCl, 1 mM ETDA, 0.5% NP-40), eluted by boiling in sodium dodecyl sulfate (SDS)-containing sample buffer, and separated by SDS-polyacrylamide gel electrophoresis (PAGE).
In vitro ubiquitylation assay.
An in vitro ubiquitylation assay was performed as previously described (22). [35S]methionine-labeled reticulocyte lysate HA-HIF1-[ODD] translation products (4 µl) were incubated in RCC 786-O S100 extracts (100 to 150 µg), prepared as previously described (22), supplemented with 8 µg of ubiquitin/µl (Sigma), 100 ng of ubiquitin-aldehyde/µl (BostonBiochem, Cambridge, Mass.), and an ATP-regenerating system (20 mM Tris [pH 7.4], 2 mM ATP, 5 mM MgCl2, 40 mM creatine phosphate, 0.5 µg of creatine kinase/µl) in a reaction volume of 20 to 30 µl for 1.5 h at 30°C.
Purification of HIF prolyl-hydroxylase. Extracts containing enriched prolyl-hydroxylases were purified from rabbit reticulocyte lysate as previously described (11).
Fibronectin ELISA. The enzyme-linked immunosorbent assay (ELISA) for measuring the deposition of fibronectin was performed as described previously (23).
Immunofluorescence microscopy. Fibronectin immunofluorescence microscopy was performed as described previously (23). Briefly, 1 x 105 RCC cells were seeded on sterilized coverslips in six-well plates and grown for 5 days. Cells on coverslips were washed three times in 1x phosphate-buffered saline (PBS) prior to ethanol fixation at -20°C for 30 min. Fixed cells were hybridized with polyclonal anti-fibronectin antibody at 4°C for 16 h. Cells were washed twice in 1x PBS and then hybridized with rhodamine-conjugated goat anti-rabbit antibody for 1 h at room temperature. Cells were washed twice in 1x PBS, rinsed briefly with DAPI (4',6'-diamidino-2-phenylindole) stain at 1:1,000 in 1x PBS, and mounted on glass slides with glycerol-PBS (1:1). Fluorescence images were captured on a Carl Zeiss Axiovert 200 microscope imaging system.
Three-dimensional spheroid assay. Multicellular spheroids were prepared as described previously (17). Briefly, 24-well tissue culture plates (Nunc) were coated with 250 µl of prewarmed 1% SeaPlaque agarose (Cambrex BioScience Rockland, Inc.) in serum-free medium. A total of 105 cells in 1 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum was added to the agarose-coated wells. The cells were grown in a humidified 5% CO2 atmosphere at 37°C for 72 h. Digital images were taken at x50 magnification.
SCID mouse xenograft assay. Multiple 786-O RCC subclones expressing pVHL(WT), pVHL(RRR), or plasmid alone were grown to approximately 90% confluence in a humidified 5% CO2 atmosphere at 37°C. The cells were harvested with 0.25% trypsin-1 mM EDTA solution. Doses of 5 x 106 cells in 50 µl of 1x PBS were injected intramuscularly into the left hind leg of SCID male mice (Charles River Lab). Tumor growth was assessed and measured weekly by carefully passing the tumor-bearing leg through a series of holes of decreasing diameter (0.5-mm decrements) in a plastic rod.
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RESULTS |
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GG) (Fig. 1C). The cells were lysed and immunoprecipitated and immunoblotted with an anti-HA antibody. A slower-migrating form of pVHL of approximately 40 kDa was observed only in cells transfected with both HA-pVHL and full-length T7-NEDD8 (Fig. 1C, lane 4). Elongin B, like elongin C, binds pVHL noncovalently. Thus, under the denaturing condition of SDS-PAGE, a slower-migrating form of pVHL was not observed. Furthermore, as an internal negative control, T7-NEDD8GG failed to associate with pVHL (Fig. 1C, lane 5). The expression of T7-tagged ubiquitin-like proteins in cells was confirmed by Western blotting (Fig. 1C, lanes 6 to 10). These results suggest that pVHL is covalently modified by NEDD8. To address whether the neddylation of pVHL occurs under physiologic conditions, the PC-3 prostatic cell line was lysed and immunoprecipitated with a monoclonal anti-pVHL antibody. The upper pVHL of around 40 kDa reacted with both anti-pVHL antibody and anti-NEDD8 antibody (Fig. 1D). A similar result was also observed in the 293 embryonic kidney cell line (data not shown). In control experiments, we confirmed that the anti-NEDD8 antibody recognized NEDD8 but not other ubiquitin-like proteins such as SUMO, elongin B, and ubiquitin (24; data not shown).
NEDD8 conjugation of pVHL is dependent on lysine 159. Analogous to the ubiquitin pathway, the neddylation of proteins involves the concerted actions of the NEDD8-activating enzyme (E1 [APP-BP1/Uba3]) and a NEDD8-conjugating enzyme (E2). Neddylation occurs invariably on lysine residues. pVHL contains three potential NEDD8 acceptor sites at lysines 159, 171, and 196. We tested whether neddylation indeed occurs on these sites by generating a triple K (lysine) to R (arginine) substitution mutant, pVHL(RRR). As a specificity control, we generated pVHL(RRR) mutants with R200K and R210K substitutions. Plasmids encoding these mutants were transfected into U2OS cells in combination with T7-NEDD8 (Fig. 2A). pVHL(RRR), as well as pVHL(R200K) and pVHL(R210K), failed to become neddylated (Fig. 2A), suggesting that the neddylation of pVHL occurs specifically on lysines 159, 171, and/or 196.
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subunit. S100 extracts made from 786-O RCC cells devoid of pVHL supplemented with in vitro-translated empty plasmid alone (MOCK) or tumor-derived pVHL(C162F) failed to support the ubiquitylation of HIF-1(ODD) (Fig. 3C). However, S100 extracts supplemented with either in vitro-translated pVHL(WT) or pVHL(RRR) ubiquitylated HIF-1(ODD) (Fig. 3C). Furthermore, multiple 786-O subclones stably transfected with pVHL(RRR), like stable subclones expressing pVHL(WT), down-regulated the expression of the HIF-targeted gene GLUT1 under normal oxygen tension (Fig. 3D). As expected, 786-O subclones expressing empty plasmid alone (MOCK) or pVHL(C162F) showed overexpression of GLUT1 even under normoxia (Fig. 3D). These results demonstrate that neddylation of pVHL is not required for the proper E3 ligase activity of VEC.
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domain pVHL(C162F) mutant failed to bind fibronectin. pVHL(RRR) showed negligible binding to fibronectin (Fig. 4A). Furthermore, 786-O cells expressing pVHL(RRR) deposited a small quantity of fibronectin, which was comparable to that of 786-O expressing plasmid alone (MOCK) or pVHL(C162F) (Fig. 4B). 786-O cells expressing pVHL(WT) deposited high concentrations of fibronectin (Fig. 4B) and were capable of forming strands of extracellular fibronectin (Fig. 4C). However, pVHL(RRR)-expressing cells failed to form fibrillar fibronectin arrays in the extracellular compartment (Fig. 4C).
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pVHL(K159E), which contains a mutation in the domain, still retained the ability to bind Cul2 (Fig. 7A, upper panel), as well as elongins B and C, albeit with weaker affinity (data not shown). Thus, we asked whether pVHL(K159E) can support the ubiquitylation of HIF-1(ODD). S100 extracts made from 786-O RCC cells devoid of pVHL supplemented with in vitro-translated empty plasmid alone (MOCK) or disease-causing pVHL(C162F), which does not form an E3 ligase complex, failed to support the ubiquitylation of HIF-1(ODD) (Fig. 7B). However, S100 extracts supplemented with either in vitro-translated pVHL(WT), pVHL(RRR), pVHL(K159E), or pVHL(KRR) ubiquitylated HIF-1(ODD) (Fig. 7B), and concordantly, 786-O stable subclones expressing pVHL(WT), pVHL(K159E), and pVHL(KRR) down-regulated the expression of the HIF-targeted gene product GLUT1 (Fig. 7C).
To ascertain whether pVHL(K159E) is able to support binding to fibronectin, 786-O cells stably expressing empty plasmid alone (MOCK), HA-pVHL(WT), HA-pVHL(K159E), and HA-pVHL(KRR) were metabolically labeled with [35S]methionine. The cells were lysed, immunoprecipitated with an anti-HA antibody, and resolved with SDS-PAGE (Fig. 7D). pVHL(WT), as expected, and pVHL(KRR) bound fibronectin. However, pVHL(K159E) failed to bind fibronectin (Fig. 7D). These results, taken together, suggest that although NEDD8-modification of pVHL is not required for forming an active E3 ligase complex, it is required for the interaction with fibronectin.
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DISCUSSION |
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and ß (22, 31). The domain is required for binding elongin C, which allows the nucleation of the E3 complex. The ß domain acts as a substrate recognition and binding region. Most disease-causing mutations on pVHL affect the ability of pVHL either to form an active E3 ligase complex or to bind its substrates (14). Thus, the development of VHL disease has been strongly suggested to be due to the failure of the pVHL's E3 ligase activity, implying that normal HIF regulation is critical for the tumor suppressor function of pVHL.
However, the notion of HIF regulation being the sole essential function of pVHL that is critical for tumor suppression came into question when type 2C disease-causing pVHL mutants were shown to properly regulate HIF but failed to promote the assembly of fibronectin (1, 9). Here, we show that another type 2C-causing pVHL(K159E) is not modified by NEDD8 but is capable of ubiquitylating the ODD of HIF-1. Moreover, this neddylation-defective pVHL(K159E) fails to bind fibronectin. The potential significance of fibronectin assembly for the ability of pVHL to suppress tumor development is further inferred by the finding that every disease-causing pVHL mutant, covering the entire spectrum of VHL disease (i.e., types 1, 2A, 2B, and 2C), tested to date has failed to bind fibronectin (1, 9, 23). Furthermore, Clifford and colleagues demonstrated that the impairment of HIF regulation was potentially required for hemangioblastoma and RCC development, while the loss of fibronectin binding was observed for all disease-causing pVHL mutants (1). However, it should be noted that the functional significance of the majority of pVHL mutations has not been formally defined.
Dysregulated ECM, as well as abnormal cell-matrix interactions, are hallmarks of solid tumors (28). Alterations in the fibronectin component of the ECM have been correlated with cellular transformation (6, 7, 28). Conversely, multimeric forms of fibronectin have been shown to promote differentiation and suppress the proliferative and metastatic potentials of transformed cells in various model systems (27). Here, we have shown that a small fraction of pVHL is covalently conjugated by NEDD8. In the absence of such modifications, pVHL failed to bind fibronectin, and the assembly of extracellular fibronectin was severely compromised. It should be noted that fibronectin matrix assembly is also dependent on other cellular events such as the interplay between fibronectin receptors and cytoskeletal organization (29, 30). Furthermore, since disease-causing pVHL mutants with intact triple lysine residues fail to bind fibronectin, there are likely other mechanisms that influence the assembly of fibronectin. Moreover, the potential requirement of the E3 ligase activity of VEC for proper fibronectin assembly has not been determined. Understanding the precise underlying mechanisms, including the NEDD8 modification of pVHL, that influence the proper extracellular fibronectin matrix assembly will be important in understanding the initial steps of the VHL disease progression.
The E3 activity of VEC was demonstrated to be independent of pVHL neddylation status. Thus, the E3 ligase function was separable from the fibronectin assembly function by blocking the NEDD8 modification of pVHL. In other words, RCC cells expressing nonneddylateable pVHL, while retaining the ability to regulate HIF, grew as compact, undifferentiated spheroids and formed tumors in SCID mice. Thus, an intact HIF pathway in the absence of proper pVHL-dependent fibronectin regulation was insufficient to prevent tumorigenesis.
The specific subtype of VHL disease may depend on the failure of pVHL to perform various functions ascribed to pVHL, including ubiquitylation of HIF-1, -2, or -3, as well as atypical protein kinase C, the deubiquitylating enzyme VDU-1, and the homeodomain-containing protein Jade-1 (2, 15, 19, 20, 22, 26, 33). pVHL has also been shown to affect transcription by binding Sp1 and the KRAB-A domain protein (3, 16). Recently, pVHL was found to associate with microtubules to prevent depolymerization and, in effect, regulated the stability of microtubules (8). Our results, in light of the fact that every disease-causing pVHL mutant fails to bind fibronectin, strongly suggest that a defect in fibronectin assembly is a common event in VHL-associated tumorigenesis. Further mechanistic insights into the various ascribed functions of pVHL will undoubtedly aid in unraveling the mystery of the genotype-phenotype correlation that exists in VHL disease.
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ACKNOWLEDGMENTS |
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This work has been supported by the National Cancer Institute of Canada with funds from the Terry Fox Foundation (grant no. 13030). N.H.S. is a recipient of the Natural Science and Engineering Research Council of Canada scholarship. W.G.K. is an investigator of the Howard Hughes Medical Institute. M.O. is a recipient of the Canada Research Chair.
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FOOTNOTES |
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| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
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| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
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