Mechanism of conditional regulation of the hypoxia-inducible factor-1 by the von Hippel-Lindau tumor suppressor protein

Alterations to the transactivation (N-TAD) domain of HIF-1 alpha demonstrate this domain contains structures necessary for hypoxia-inducible transactivation, oxygen-dependent degradation, and VHL-HIF-1 alpha interaction. HIF-1 alpha sequences with alterations of the N-TAD domain, fragments and analogs thereof are useful in modifying or regulating the activity of bioentities. Agonists and antagonists of the N-TAD domain of HIF-1 alpha are also useful in modifying or regulating the activity of bioentities.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
 EXAMPLE 1 Regulation of HIF-1 Alpha Protein Stability by the VHL Tumor Suppressor Protein HIF-1 alpha is normally not detectable by immunoblot analysis of cellular extracts under normoxic conditions due to the pronounced lability of the HIF-1 alpha protein under normoxic conditions. Thus, it is not currently possible to investigate the effect of expression of VHL on endogenous HIF-1 alpha protein levels. A. To establish experimental conditions to examine the effect of VHL on HIF-1 alpha protein stability under normoxic conditions, COS7 cells were transiently transfected with 0.2, 0.5 or 1.0&mgr; grams of FLAG epitope-tagged HIF-1 alpha expression plasmids in 6 cm diameter plastic dishes. A pFLAG-CMV2/HIF-1 alpha (1-826) expression plasmid was constructed as described in Kallio, P. J. et al., Proc. Natl. Acad. Sci., 94: 5667-5672, 1997. COS7 cells (obtained from ATCC) were routinely maintained in Dulbecco's minimal essential medium supplemented with 10% fetal calf serum plus penicillin (50 IU/ml) and Streptomycin (50 &mgr;g/ml). The plasmids were mixed with two volumes of FuGene6 (Boehringer Mannhein) and added to the culture medium. After 12 hours of incubation, cells were treated for 12 hours under normoxic (21% O 2 ) or hypoxic (1.0% O 2 ) conditions. For the detection of HIF-1 alpha fusion protein expression, whole cell extracts were prepared essentially as described in Kallio, P. J. et al., Proc. Natl. Acad. Sci., 94: 5667-5672, 1997. Briefly, cells were harvested in TEN buffer (40 mM Tris-HCl pH 7.9, 10 mM EDTA, 150 mM NaCl), and the cell pellet was resuspended in 20 microliters of lysis buffer (150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 0.05 M Tris-HCl pH 8.0), followed by centrifugation for 30 minutes at 14,000 rpm. Protein concentrations of the extracts were measured using the Bio-Rad protein assay reagent. Fifty micrograms of total cell proteins were blotted onto nitrocellulose filters following SDS-polyacrylamide gel electrophoresis and blocked overnight with 5% non-fat milk in Tris-buffered saline (TBS). Anti-FLAG M2 (Kodak) antibodies, used as a primary antibody, were diluted 1:500 in TBS containing 0.1% Tween-20 (TBS-T) and 1% non-fat milk and incubated with the sample for 1 hour. After several washes, anti-mouse Ig-horseradish peroxidase conjugate (Amersham Life Science), used as a secondary antibody, was diluted 1:1000 in TBS-T buffer containing 1% non-fat milk and incubated with the sample for 1 hour at room temperature. After extensive washing with TBS-T buffer, immunocomplexes were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech) according to the manufacturer's instructions. FIG. 1 shows the results using increasing amounts of HIF-1 alpha protein (0.2 &mgr;g (lanes 1 and 2), 0.5 &mgr;g (lanes 3 and 4), 1.0 &mgr;g (lanes 5 and 6)) at normoxia (21% O 2 , lanes 1, 3 and 5) or hypoxia (1.0% O 2 , lanes 2, 4 and 6). The mobility of molecular weight markers are shown on the right hand side of the blot. At the lowest concentration of expression vector, HIF-1 alpha was not detected at normoxic conditions but was detected under hypoxic conditions ( FIG. 1 , lanes 1 and 2). However, at higher concentrations of expression vector, HIF-1 alpha protein expression could be detected under normoxic conditions ( FIG. 1 , lane 3). At the highest concentrations of expression vector tested, significant HIF-1 alpha expression levels were observed both under normoxic and hypoxic conditions ( FIG. 1 , lanes 5 and 6). Thus, these experiments indicate that the mechanism of degradation of HIF-1 alpha at normoxia had become saturated under these conditions and that one or several components of the degradation machinery were limiting. B. To determine the effect of degradation of the HIF-1 alpha protein in the presence of the VHL tumor suppressor protein, 1.0 &mgr;g of pFLAG CMV2/HIF-1 alpha was co-transfected with either 2.0 &mgr;g of empty vector or wild-type VHL expression vector (pCMX/VHL)into COS7 cells. The pCMX/VHL plasmid was constructed by inserting an NheI-EcoRI (following blunting of both sites with Klenow polymerase) fragment of pCI/VHL wild-type (generously provided by Dr. Joan W. Conaway, Howard Hughes Medical Institute, USA) into an EcoRV-digested pCMX vector. The plasmid mixtures were mixed with two volumes of FuGene6 (Boehringer Mannhein) and added to the culture medium in a 6-cm dish. After 12 hours of incubation, cells were treated for 12 hours under hypoxic (1% O 2 ) or normoxic (21% O 2 ) conditions. Whole cell extracts were prepared as above and analyzed by immunoblotting as carried out above using anti-FLAG M2 (Kodak) or anti-VHL (Pharmingen) antibodies as the primary antibody. As seen in FIG. 2 , co-transfection of the cells with FLAG/HIF-1 alpha and VHL expression plasmids resulted in significant reduction of the HIF-1 alpha protein signal under normoxic conditions (upper panel, compare lanes 1 and 3), indicating that VHL may have been limiting under the conditions of overexpression of HIF-1 alpha alone. Interestingly, transiently expressed VHL failed to induce reduction of the HIF-1 alpha protein levels under hypoxic conditions (compare lanes 2 and 4). In control experiments similar levels of VHL expression were detected in extracts from either normoxic or hypoxic cells (lower panel, compare lanes 3 and 4). The mobility of molecular weight markers are shown on the right hand side of the blot. C. To determine if VHL mediates proteasomal degradation of HIF-1, the above experiment was repeated except the co-transfected cells were incubated in the absence or presence of 5 &mgr;M of the proteasome inhibitor MG-132 for 6 hours before harvesting. Cellular extracts were prepared and analyzed as in Example 1B. As shown in FIG. 3 , VHL-induced reduction of HIF-1 alpha protein levels under normoxic conditions was inhibited by treatment of the cells with the proteasome inhibitor MG-132 (compare lanes 1-3). Taken together with the above experiments, these results indicate that VHL mediates proteasomal degradation of HIF-1 alpha. D. To determine if VHL is specific for HIF-1 alpha, an experiment was conducted to see if overexpression of VHL affects dioxin receptor (DR) protein levels. The dioxin receptor is a basic helix-loop-helix/PAS (Per/Arnt/Sim) protein belonging to the same class of transcriptional regulators as HIF-1. One (1) &mgr;g of a FLAG-tagged dioxin receptor expression vector (pCMV/DR/FLAG) was co-transfected with 2.0 &mgr;g of either empty vector or wild-type VHL expression vector (pCMX/VHL) into COS7 cells. The FLAG-tagged dioxin receptor expression plasmid, pCMV/DR/FLAG, was provided by Dr. J. McGuire (Karolinska Institutet, Sweden). Cotransection, incubation and analysis was carried out as in Example 1B. As seen in FIG. 4 , transiently expressed VHL did not affect the protein levels of the FLAG-tagged dioxin receptor. This indicates the effect of VHL was specific for HIF-1 alpha. 
 EXAMPLE 2 Two Domains of VHL are Required for Inducing Protein Degradation of HIF-1 To determine if VHL physically interacts with HIF-1 alpha, 35 S-labeled, in vitro translated HIF-1 alpha was incubated with wild-type or mutant GAL4-VHL fusion proteins or the minimal GAL4 DNA binding domain (DBD) alone prior to immunoprecipitation assays. FIG. 5 provides a schematic representation of the GAL4 fused wild-type (1-213) and deletion or single amino acid point mutant (pmt) forms of VHL. The wild-type or mutant forms of VHL and GAL4 DBD fusion VHLs were assembled by inserting an NheI-EcoRI (following blunting of both sites with Klenow polymerase) fragment of pCI/VHL wild-type or mutant/FLAG (generously provided by Dr. Joan W. Conaway, Howard Hughes Medical Institute, USA) into an EcoRV-digested pCMX-GAL4. In in vitro immunoprecipitation assays, GAL4 fusion proteins containing full length HIF-1 were translated in the presence of &lsqb; 35 S&rsqb;methionine in rabbit reticulocyte lysate (Promega). &lsqb; 35 S&rsqb;methionine-labeled translation products were separated by SDS-PAGE and analyzed by phosphorimager (Fuji) for calculation of the protein concentration on the basis of incorporated &lsqb; 35 S&rsqb;methionine. Immunoprecipitation experiments were performed as described in Gradin, K. et al., Mol. Cell. Biol., 16:5221-5231, 1996). Briefly, radio-labeled HIF-1 alpha was incubated with equal concentrations of unlabeled GAL4-VHL or VHL mutants at room temperature for 1 hour. Cells were treated with 5 &mgr;M MG-132 for 6 h and then harvested in TEN buffer. Protein concentrations of the extracts were measured using the Bio-Rad protein assay reagent. Five (5) microliters of anti-GAL4 (Upstate Biotech) or pre-immune rabbit serum was added to the protein mixtures and incubated for another 1 hour at room temperature. Thirty (30) microliters of a 50% slurry of protein G-Sepharose in TEG buffer (20 mM Tris-HCl (pH7.4), 1 mM EDTA, 10% glycerol, 1 mM DTT) containing 150 mM NaCl and 0.1 % Triton X-100, was then added to the reaction mixtures and incubated for 12 hours at 4° C. under rotation. After rapid centrifugation, the resulting Sepharose pellets were washed three times with supplemented TEG buffer, and coimmunoprecipitated proteins were analyzed by SDS-PAGE and subsequent autoradiography. Equal concentrations of in vitro translated 35 S-labeled full length HIF-1 alpha was incubated with in vitro translated wild-type GAL4/VHL ( FIG. 6 , lane 2) or VHL deletion mutants ( FIG. 6 , lane 3-5) or GAL4-DBD spanning the GAL4 DNA binding domain alone ( FIG. 6 , lane 6). For loading controls, 10% of input 35 S-labeled labeled HIF-1 alpha was added (FIG. 6 , lane 1). Coimmunoprecipitation with anti-GAL4 antibody is shown in the upper panel of FIG. 6 while the control preimmune serum is found in the lower panel. As seen in FIG. 6, 35 S-labeled HIF-1 alpha was coimmunoprecipitated in the presence of GAL4/VHL by GAL4-specific antibodies, whereas no interaction was observed between HIF-1 alpha and the minimal GAL4 DNA binding domain (upper panel, compare lanes 2 and 6). Non-specific pre-immune rabbit antiserum did not precipitate HIF-1 alpha protein in the presence of either VHL or GAL4 alone ( FIG. 6 , lower panel), indicating that wild-type VHL specifically interacted with HIF-1 alpha in vitro. With respect to the deletion mutants, the VHL &Dgr;114-154 deletion mutant showed interaction with HIF-1 alpha, whereas the VHL 114-154 fragment failed to do so ( FIG. 6 , compare lanes 3 and 4). However the VHL 91-154 deletion mutant was able to interact with HIF-1 alpha ( FIG. 6 , compare lanes 4 and 5). Thus, a structure located between residues 91 and 113 of VHL appeared to be critical for interaction with HIF-1 alpha. Interestingly, this region of VHL is contained not only within the putative macromolecular binding observed in the crystal structure of the VHL-BC complex (Stebbins, C. E. et al., Science, 284:455-461, 1999), but also represents one of the mutational hot spots in tumors (Gnarra, J. R. et al., Biochim. Biophys. Acta., 1242: 201-210, 1996). To determine whether tumor derived mutations of VHL would affect its ability to interact with HIF-1 alpha and/or to induce HIF-1 alpha degradation, experiments were performed using GAL4-VHL fusion proteins containing either a Y98N (the most frequent tumor mutation in this region) or a C162F single amino acid mutation (see FIG. 5 for schematic representation of the single amino acid point mutant (pmt) forms of VHL). The C162F mutation has been demonstrated to render VHL unable to bind the elongin B-C complex (Lonergan, K. M. et al., Mol. Cell. Biol., 18:732-741, 1998; Lisztwan, J. et al., Genes & Dev., 13:1822-1833, 1999), and inhibit ubiquitin ligase activity in vitro (Lisztwan, J. et al., Genes & Dev., 13:1822-1833, 1999). In these coimmunoprecipitation experiments, equal concentrations of in vitro-translated 35 S-labeled full-length HIF-1 alpha were incubated with in vitro-translated GAL4 fused wild-type VHL ( FIG. 7 , lane 3) or mutant VHL Y98N ( FIG. 7 , lane 4), C162F ( FIG. 7 , lane 5) or GAL4-DBD alone ( FIG. 7 , lane 2). The results of the coimmunoprecipitation are shown in the upper panel of FIG. 7 while the control preimmune serum is found in the lower panel. These coimmunoprecipitation experiments were carried out and analyzed as in FIG. 6 . As seen in FIG. 7 , upper panel, VHL Y98N was unable to interact with HIF-1, whereas VHL C162F showed wild-type levels of interaction with HIF-1 alpha (compare lanes 3-5). These results indicate that a tumor-derived point mutation in the beta domain impairs the interaction between HIF-1 alpha and VHL. Next a cellular degradation assay was carried out. pFLAG CMV2/HIF-1 alpha was transiently coexpressed in COS7 cells in the absence or presence of GAL4 fused wild-type or mutant forms of VHL as indicated in FIG. 8 . The cells were incubated at normoxia for 24 h. Whole cell extracts were prepared and analyzed by immunoblotting. The blots were developed with anti-FLAG (upper panel) or anti-VHL (lower panel) antibodies. As seen in FIG. 8 , immunoblot analysis demonstrated that, in contrast to wild-type VHL, both the VHL Y98N and VHL C162F mutants failed to induce degradation of HIF-1 alpha at normoxia ( FIG. 8 , compare lanes 2-4). Accordingly, these results demonstrate that both the HIF-1 alpha interaction domain and the elongin C binding domain of VHL are necessary to mediate degradation of HIF-1, and that regulation of HIF-1 alpha may be involved in the tumor suppressor function of VHL. 
 EXAMPLE 3 The Oxygen-dependent Degradation Domain of HIF-1 Alpha is Targeted for Regulation by VHL To identify the domain of HIF-1 alpha which is targeted by VHL to mediate proteasomal degradation at normoxia, either wild-type FLAG/HIF-1 alpha or a series of FLAG-tagged HIF-1 alpha deletion mutants were transiently expressed in COS7 cells under normoxic conditions in the presence or absence of VHL. FIG. 9A provides a schematic representation of the series of FLAG-tagged HIF-1 alpha deletion mutants. In FIG. 9 A, bHLH is an acronym for basic helix-loop-helix domain; PAS is an acronym for Per/Arnt/Sim domain; ODD is an acronym for oxygen-dependent degradation domain; N-TAD is an acronym for N-terminal transactivation domain; and C-TAD is an acronym for C-terminal transactivation domain. The N-terminal transactivation domain has been mapped to reside between amino acids 531-575 (Jiang, B. H. et al., J. Biol. Chem., 272:19253-19260, 1997), or 549-582 (Pugh, C. W. et al., J. Biol. Chem., 272:11205-11214, 1997), respectively. The FLAG epitope-tagged deletion mutant HIF-1 alpha (1-652) was made by inserting a BamHI-SpeI fragment (the SpeI site was filled-in with Klenow polymerase) of pGFP/HIF-1(1-652) into a BamHI-SmaI-digested pFLAG-CMV2 (Kodak). pFLAG-CMV2/HIF-1(1-330) was constructed by inserting an EcoRI fragment of pFLAG-CMV2/HIF-1(1-826) into a EcoRI-digested pFLAG-CMV2. pFLAG-CMV2/HIF-1(526-826) was assembled by inserting a SalI-Hind III (the HindIII site was filled-in with Klenow polymerase) fragment of pCMX-GAL4/HIF-1(526-826) into a SalI-BamH I (BamH I was site was filled-in with Klenow polymerase) digested pFLAG-CMV2. Whole cell extracts were prepared and used for immunoblot analysis as described in Example 1B. As seen in FIG. 9 B, the GAL4/HIF-1 alpha 1-652 and HIF-1 alpha 526-826 fragments show VHL-mediated degradation in normoxic cells. However, deletion mutant HIF-1 alpha 1-330 was not degraded upon overexpression of VHL in COS7 cells under normoxic conditions. To map the domain of HIF-1 alpha required to interact with VHL, full length HIF-1 alpha or a set of HIF-1 alpha deletion mutants fused to the GAL4 DNA binding domain were expressed and coimmunoprecipitation assays were performed following incubation with 35 S-labeled VHL. 3 5S-labeled VHL was translated in the presence of 35 S-methionine in rabbit reticulocyte lysate (Promega). The 35 S-methionine-labeled translation products were separated by SDS-PAGE and analyzed by phosphorimager (Fuji) for calculation of the protein concentration on the basis of incorporated 35 S-methionine. Equal concentrations of in vitro translated 35 S-labeled full-length VHL was incubated with in vitro-translated GAL4-fusion proteins spanning the indicated HIF-1 alpha deletion mutants ( FIG. 10 , lanes 2-7) or GAL4-DBD alone ( FIG. 10 , lane 8). For loading controls, 10% of input 35 S-labeled VHL is shown ( FIG. 10 , lane 1). The precipitated material was analyzed by SDS-PAGE and autoradiography. The results of the coimmunoprecipitation assays performed with anti-GAL4 antibodies are shown in the upper panel of FIG. 10 . The assays performed with the control non-specific rabbit antiserum are shown in the lower panel of FIG. 10 . As seen in FIG. 10 , GAL4/HIF-1 alpha 1-330 failed to physically interact in vitro with 35 S-labeled VHL (upper panel, lane 2). Moreover, GAL4/HIF-1 alpha 778-826 spanning the C-terminal transactivation domain of HIF-1 alpha did not show any interaction with VHL (upper panel, lane 7). In contrast, the GAL4/HIF-1 alpha 1-652, GAL4/HIF-1 alpha 331-641, GAL4/HIF-1 alpha 526-641 and HIF-1 alpha 526-826 fragments clearly interacted with VHL (upper panel, lanes 3-6). In fact, when compared to full length HIF-1, all these latter fragments of HIF-1 alpha interacted with VHL with very similar efficacies. In control precipitation reactions, non-specific pre-immune rabbit antiserum did not precipitate VHL protein in the presence of any of the used GAL4/HIF-1 alpha fragments or GAL4 alone (lower panel). Taken together, these results indicate that a region of HIF-1 alpha spanning residues 526-641 was essential for physical interaction with VHL. Interestingly, this region contains the oxygen/redox-dependent degradation domain of HIF-1 alpha which has previously been demonstrated to mediate proteasomal degradation of HIF-1 alpha in normoxic cells and which has broadly been defined to be located between amino acid residues 401 and 603 of hHIF-1 alpha (Huang, L. E. et al., Proc. Natl. Acad. Sci., 95:7987-7992, 1998; Kallio, P. J. et al., J. Biol. Chem., 274:6519-6525, 1999). 
 EXAMPLE 4 The Minimal N-terminal Transactivation Domain of HIF-1 Alpha is a Target for Ubiquitination and Proteasomal Degradation by VHL The VHL-interacting fragment, GAL4/HIF-1 alpha 526-641, contains not only the core of the oxygen-dependent degradation domain of HIF-1 alpha but also the N-terminal transactivation domain, N-TAD (see FIG. 9A ). Within the N-TAD of HIF-1 alpha, a sequence motif of about 19 amino acid residues (located between amino acids 556-574 of human HIF-1 alpha) shows the strongest conservation between species and is also conserved in the related hypoxia-inducible factor EPAS-1/HLF. The related hypoxia-inducible factor EPAS-1/HLF is expressed in a more tissue-restricted fashion (Ema, M. et al., Proc. Natl. Acad. Sci., 94:4273-4278, 1997; Tian, H. et al., Genes & Dev., 11:72-82, 1997). This motif is also highly conserved in the hypoxia-responsive Drosophila Similar protein (Taylor, B. L. et al., Microbiol. Mol. Biol. Rev., 63:479-506, 1999). This sequence motif has recently been reported to be important for the function of the oxygen-dependent degradation domain of HIF-1 alpha since alanine substitutions within this domain impair hypoxia-dependent protein stabilization and stabilize the protein in normoxic conditions (Srinivas, V. et al., Biochem. Biophys. Res. Commun., 260:557-561, 1999). FIG. 11 shows an alignment of the conserved core motif of N-TAD sequences of human (h) and mouse (m) HIF-1 alpha and EPAS-1. To determine if VHL could directly interact with the conserved core motif of N-TAD of HIF-1 alpha, coimmunoprecipitation assays were conducted using in vitro translated 35 S-labeled VHL proteins and FLAG-tagged wild-type or mutant N-TAD fragments. pFLAG/HIF-1/NTAD (amino acids 532-585 of HIF-1 alpha) was constructed by inserting an EcoRI-BamHI fragment made by PCR into EcoRI-BamHI digested pFLAG-CMV2. Next a mutant N-TAD was made where the central PYI triplet with the N-TAD domain was substituted with three aspartic acid residues. Amino acid substitutions within FLAG/HIF-1/NTAD were generated using a QuikChange site-directed mutagenesis kit (Stratagene). The positions of the amino acid substitutions are shown in FIG. 11 . The in vitro translated 35 S-labeled VHL protein was incubated with equal concentrations of in vitro translated FLAG-tagged wild-type ( FIG. 12 , lane 3) or mutant ( FIG. 12 , lanes 4) N-TAD or FLAG epitope alone ( FIG. 12 , lane 2). Coimmunoprecipitation assays were carried out using anti-FLAG antibodies, and the resulting precipitates were analyzed by SDS-PAGE and autoradiography. For loading controls, 10% of input 35 S-labeled VHL was used ( FIG. 12 , lane 1). As shown in FIG. 12 , FLAG antibodies could precipitate 35 S-labeled VHL in the presence of the FLAG epitope-tagged minimal wild-type N-TAD of HIF-1 alpha (lane 3). This demonstrates that this region of HIF-1 alpha is sufficient to mediate interaction with VHL. Substitution of the central PYI triplet with aspartic acid totally abolished interaction between VHL and the N-TAD (compare lanes 3 and 4). These experiments indicate that the highly conserved core motif of the N-TAD is critical for interaction with VHL. Because of the central role of this portion of the sequence, any amino acid substitution that changes positions 564-566 will effectively abrogate interaction with the VHL protein. It will create stabilization against VHL mediated degradation and renders transactivation function of the protein hypoxia-independent. A more detailed mutation analysis of residues within the PYI triplet or in its vicinity demonstrated that mutation of residues YI564-565 to GG, and DDD569-571 to AAA also totally abolished interaction between VHL and the N TAD, whereas mutation of residues PM567-568 resulted in partial inhibition of interaction between VHL and the N-TAD ( FIG. 27 ). Amino acid substitutions at Y565 can abrogate interaction with VHL. Y565 was replaced with G resulting in stabilization against VHL-mediated degradation. Y565 replaced with F, however, does not give the same effect. F565 does not alter the properties of the degradation box, instead, it maintains VHL interaction, mediates degradation at normoxia, and allows for hypoxia-dependent transactivation. Therefore, substitutions at Y565 should alter the hydrophobic mature of the residue, so substitutions with hydrophilic or neutral amino acids will result in a disruption of VHL binding, but a substitution at Y565 with a hydrophobic amino acid may not. Amino acid substitutions at I566 can also abrogate interaction with VHL. For example, replacing I566 with G stabilizes the protein against VHL-mediated degradation. YI565-566 can be replaced with substitute amino acids. For example, YI565-566 can be replaced with GG to abrogate interaction with VHL, to stabilize the protein against VHL-mediated degradation, and to remove the hypoxic dependency of transactivation. Because neutral or hydrophilic amino acid substitutions at Y565 appear to produce this effect, similarly a neutral or hydrophilic substitution at YI565-566 should also produce this effect. The replacement of amino acids DDD569-57l to AAA abrogated interaction with VHL. It is expected that all amino acid substitutions at DDD569-571 should have this effect. A replacement at FQL572-574 to AQA also abrogated the interaction with VHL protein. This substitution stabilized the protein against VHL-mediated degradation and rendered the transactivation function of the protein constitutive, or no longer hypoxia-dependent. Other substitutions to FQL572-574 should have this effect. Next immunoblot assays were conducted to determine the affect on wild-type or mutant N-TAD in the absence or presence of increasing concentrations of VHL. 1 &mgr;g per 6-cm dish of FLAG ( FIG. 13 , lane 1) or FLAG-tagged wild-type ( FIG. 13 , lanes 2-4) or mutant ( FIG. 13 , lanes 5-7) N-TAD was transiently coexpressed in COS7 cells in the absence (−) or presence (&plus;, 1.0 &mgr;g; &plus;&plus;, 2.0 &mgr;g/6-cm dish) of VHL as indicated. The cells were incubated at normoxia for 24 hours. Whole cell extracts were prepared as in Example 1A and analyzed by immunoblotting. The blots were developed with anti-FLAG ( FIG. 13 , upper panel) or anti-VHL ( FIG. 13 , lower panel) antibodies. As seen in FIG. 13 , wild-type N-TAD protein was degraded in a dose-dependent manner by VHL (compare lanes 2-4). In contrast, the stability of the mutant N-TAD was not affected by identical concentrations of VHL (compare lanes 5-7). To investigate the mechanism of VHL-mediated degradation of HIF-1 alpha, in vivo ubiquitination experiments were conducted using FLAG-tagged wild-type or mutant N-TAD. The FLAG epitope alone ( FIG. 14 , lane 1), FLAG-tagged wild-type N-TAD ( FIG. 14 , lanes 2 and 3) or mutant N-TAD ( FIG. 14 , lanes 4 and 5) and HA-tagged ubiquitin were transiently coexpressed in COS7 cells in the presence ( FIG. 14 , lanes 1, 3, 5) or absence ( FIG. 14 , lanes 2, 4, 6) of VHL under normoxic conditions in combination with incubation with the proteasome inhibitor MG132 for 6 hours. After anti-FLAG immunoprecipitation of whole cell extracts, ubiquitinated -TAD forms were detected by anti-HA immunoblotting analysis. As FIG. 14 shows, ubiquitination of wild-type N-TAD was strongly enhanced by coexpression of VHL (upper panel, compare lanes 2 and 3). On the other hand, the mutant N-TAD showed very low levels of ubiquitination even in the presence of VHL (upper panel, lanes 5-7). 10% of input whole cell extracts are shown in the lower panels. Importantly, these results clearly demonstrate that VHL mediates ubiquitination of HIF-1 alpha via physical interaction with the minimal N-TAD motif. Given the ubiquitination of the minimal N-TAD motif, it was determined which of the three lysines of N-TAD were targeted for regulation by VHL. FLAG-tagged wild-type or single lysine mutants (K532R, K538R, and K547R, respectively) of N-TAD were transiently expressed in 293 cells. The cells were incubated at normoxia or hypoxia for 12 hours, and whole cell extracts were prepared as in Example 1A and analyzed by immunoblotting. In analogy to full length HIF-1 alpha, the minimal wild-type GAL4/N-TAD fusion protein showed significant degradation under normoxic conditions, and was stabilized by hypoxia. Interestingly, mutation of K547 stabilized the protein at normoxia, whereas expression of the two other lysine mutants was hardly detectable at normoxia ( FIG. 15 ). These results indicate that lysine 547 is significant for degradation of HIF-1 alpha. 
 EXAMPLE 5 Subcellular Localization of VHL at Normoxia and Hypoxia Previously it was demonstrated that hypoxia induces nuclear translocation of HIF-1 alpha (Kallio, P. J. et al., EMBO J., 17:6573-6586, 1998). In the case of VHL, nuclear-cytoplasmic trafficking has been suggested to be required for VHL function (Lee, et al., 1999). To study the intracellular localization of VHL in relation to its function in normoxic versus hypoxic cells, VHL was transiently expressed in COS7 cells. COS7 cells grown on cover slips were transiently transfected with FLAG-VHL expression plasmid (pCMX/VHL) and incubated for 24 hours. After incubation for 6 hours under hypoxic (1% O 2 ) or normoxic (21% O 2 ) conditions, the cells were fixed with 4% (w/v) paraformaldehyde in PBS at room temperature for 30 minutes, followed by incubation with anti-VHL monoclonal antibody in PBS containing 0.1% Triton X-100 at 4° C. for 9 hours. Indirect immunofluorescence was obtained by using biotinylated anti-mouse IgG antibodies and FITC-conjugated streptavidin (Amersham) in PBS and 0.1% Triton X-100. For quantitative purposes, 150-200 fluorescent cells were analyzed for compartmentalization and subdivided into four categories as described in Kallio, P. J. et al., EMBO J., 17:6573-6586, 1998. As seen in FIG. 16 , at normoxia, immunofluorescence by an anti-VHL antibody was detected throughout the cells with some preference toward localization in the cytoplasmic compartment of the cells. A very similar distribution of VHL immunoreactivity was observed under hypoxic conditions. Semiquantitive analysis of the subcellular localization of VHL-immunoreactivity following the method disclosed in Kallio, P. J. et al., EMBO J., 17:6573-6586, 1998, under normoxic conditions, reveals that 47% of the transfected cells had equal distribution of fluorescence in the cytoplasm and the nucleus (category N−C), whereas in 45% of the transfected cells cytoplasmic fluorescence predominated over that detected in the nucleus (category N<C). Only 3% of the transfected cells showed nuclear fluorescence dominating over the cytoplasmic signal (category N>C), and no transfected cell showed exclusive nuclear staining (category N). Hypoxic treatment of the cells had no effect on the intracellular distribution of VHL since under these conditions 47%, 51% and 6% of the transfected cells fell into categories N&equals;C, N<C, and N>C, respectively. Consistent with the results obtained at normoxia exclusive nuclear fluorescence was also not observed under hypoxic conditions. Thus, in contrast to HIF-1 alpha which shows hypoxia-inducible nuclear import (Kallio, P. J. et al., EMBO J., 17:6573-6586, 1998), the subcellular localization of VHL was not altered upon exposure to hypoxia. 
 EXAMPLE 6 Protection of HIF-1 Alpha from VHL-dependent Proteasomal Degradation is a Multi-step Pathway Requiring Hypoxia-induced Nuclear Translocation of HIF-1 Alpha and a Hypoxia-induced Activation Signal Next the subcellular localization of wild-type HIF-1 alpha was compared to that of a HIF-1 alpha mutants, HIF-1 alpha K719T and HIF-1 alpha &Dgr;178-390 mutant. The HIF-1 alpha K719T mutant fails to enter the nucleus at hypoxia and is thus is constitutively localized in the cytoplasm (Kallio, P. J. et al., EMBO J., 17:6573-6586, 1998). The HIF-1 alpha &Dgr;178-390 mutant lacks a portion of the PAS domain and shows constitutive nuclear localization (Kallio, P. J. et al., EMBO J., 17:6573-6586, 1998). Expression plasmid for the green fluorescent protein (GFP) fused to the wild-type full-length HIF-1 alpha was generated by cutting the HIF-1 alpha coding region from pGEX-4T3-HIF-1 alpha as a BamHI-NotI fragment (the NotI site filled-in with Klenow polymerase) and ligating this in-frame into a BamHI-NheI digested pCMX-SAH-Y145F (Kallio, P. J. et al., EMBO J., 17:6573-6586, 1998). The pCMX-SAH-Y145F expression vector encodes a modified and highly chromophoric form of GFP under the control of CMV immediate early promoter which contains an S65A mutation that confers a wavelength shift and temperature resistance to the protein as well as a Y145F substitution increasing the intracellular stability of GFP. The GFP-HIF-1 alpha (K719T) was constructed by site-directed mutagenesis of the C-terminal nuclear localization signal by overlap PCR as described in Ausubel, F. M. et al., Current Protocols in Molecular Biology, J. Wiley and Sons, NY, 1994. The desired mutation (codon 719 AAG to ACA) was introduced into a PCR product and then inserted as an EcoRI-PstI subfragment into pGFP-HIF-1 alpha (526-826). A GFP fusion of full-length HIF-1 alpha carrying the K719T mutation was thereafter assembled by inserting the N-terminal BamHI-SpeI fragment of HIF-1 alpha into pGFP-HIF-1 alpha (526-826 K719T) (Kallio, P. J. et al., EMBO J., 17:6573-6586, 1998). The plasmids were transiently expressed in COS7 cells. COS7 cells grown on cover slips were transiently transfected with the respective GFP-fusion expression plasmids and incubated for 24 hours. Following 24 hours of expression, the transfected cells were further incubated under either normoxic (21% O 2 ) or hypoxia (1% O 2 ) conditions for 6 hours before observation. Subcellular distribution of fluorescence activity was examined and photographs taken using a Zeiss Axiovert 135 microscope with an FITC-filter set, and epifluorescence with illumination from a Gixenon burner (Carl Zeiss Jena GmbH, Jena, Germany). As expected, HIF-1 alpha &Dgr;178-390 shows constitutive nuclear localization under both normoxic and hypoxic conditions; whereas, HIF-1 alpha K719T, which fails to enter the nucleus at hypoxia and is thus constitutively localized in the cytoplasm ( FIG. 17 ). In FIG. 18 , the effect of VHL on the stability of the wild-type HIF-1 alpha full-length protein was compared to the stability of the HIF-1 alpha K719T mutant. pFLAG-CMV2/HIF-1 alphaK719T was made by inserting a BamHI-NheI (the NheI site was filled-in with Klenow polymerase) fragment of pGFP/HIF-1 alphaK719T into a BamHI-SmaI digested pFLAG-CMV2. Following 24 hours of expression, the transfected cells were further incubated under either normoxic (21% O 2 , FIG. 18 , lanes 1, 2) or hypoxia (1% O 2 , FIG. 18 , lanes 3, 4) conditions for 12 hours before observation. Whole cell extracts were prepared as in Example 1A and assayed by immunoblot analysis as in Example 1B. Under normoxic conditions, both wild-type HIF-1 alpha and HIF-1 alpha K719T were degraded in the presence of overexpressed VHL ( FIG. 18 , lane 2). Interestingly, the wild-type HIF-1 alpha showed significant (albeit not total) resistance to VHL under hypoxic conditions ( FIG. 2 ; FIG. 18 , lane 4), whereas the HIF-1 alpha K719T mutant was potently degraded upon exposure to VHL in hypoxic cells ( FIG. 18 , lane 4). These results indicate that hypoxia-induced nuclear translocation of HIF-1 alpha protects HIF-1 alpha from VHL-mediated proteasomal degradation. To test if nuclear localization is sufficient to protect HIF-1 alpha from VHL-mediated proteasomal degradation, the effect of VHL on the stability of the HIF-1 alpha &Dgr;178-390 mutant was examined. pFLAG-CMV2/HIF-1 alpha (&Dgr;178-390) was constructed by inserting a SalI fragment of pCMX-GAL4/HIF-1 alpha(&Dgr;178-390) into a SalI-digested pFLAG-CMV2. Following 24 hours of expression, the transfected cells were further incubated under either normoxic (1% O 2 , FIG. 18 , lanes 1, 2) or hypoxic (1% O 2 , FIG. 18 , lanes 3, 4) conditions for 12 hours before observation. Whole cell extracts were prepared as in Example 1A and assayed by immunoblot analysis as in Example 1B. As seen in FIG. 18 , the HIF-1 alpha &Dgr;178-390 mutant was degraded at normoxia upon exposure to VHL (compare lanes 1 and 2). Thus, nuclear localization per se cannot explain the protection of HIF-1 alpha against degradation by VHL. However, this mutant showed significant resistance against VHL-mediated degradation in hypoxic cells (compare lanes 2 and 4). These results indicate that, in addition to nuclear translocation, a distinct intranuclear event or signal is required for protection of HIF-1 alpha against VHL-induced proteolysis. Given the striking overlap within the N-TAD of HIF-1 alpha between the structures that mediate oxygen-dependent degradation, physical interaction with VHL, and hypoxia-inducible transactivation, it is possible that this putative intranuclear stabilizing signal may be linked to the transactivation function of the protein. 
 EXAMPLE 7 VHL is not Released from HIF-1 Alpha in Hypoxic Cells In view that both nuclear translocation of HIF-1 alpha and an hypoxia-induced activation signal were necessary to protect HIF-1 alpha from VHL-mediated degradation, the mechanistically important question of whether VHL was released from HIF-1 alpha under hypoxic conditions was next examined. To test whether VHL was released from HIF-1 alpha under hypoxic conditions, GAL4 DBD (4 &mgr;g) ( FIG. 19 , lane 1) or GAL4/HIF-1 alpha (4 &mgr;g) ( FIG. 19 , lanes 2-6) were transiently expressed in COS7 cells in the absence ( FIG. 19 , lane 2) or presence ( FIG. 19 , lanes 1, 3-6) of VHL in 10 cm diameter plastic dishes. After 12 hours of incubation, cells were treated for 1, 3 or 6 hours under normoxic ( FIG. 19 , lanes 1-3, respectively) or hypoxic conditions ( FIG. 19 , lanes 4-6, respectively). Cells were incubated with 5 &mgr;M MG-132 proteasome inhibitor (Calbiochem) for 6 hours before harvesting the cells in TEN buffer. The cell pellet was resuspended in 200 liters of whole cell extract buffer (25 mM Hepes, 100 mM NaCl, 5 mM EDTA, 20 mM beta-glycerophosphate, 20 mM para-nitrophenyl-phosphate, 0.5% Triton X-100, 100 M sodium orthovanadate) supplemented with 20 &mgr;M N-ethylmaleimide, followed by centrifugation for 30 minutes at 14,000 rpm. Protein concentrations of the extracts were measured using the Bio-Rad protein assay reagent. Coimmunoprecipitated proteins were analyzed by SDS-PAGE followed by immunoblotting. Following anti-GAL4 antibody—( FIG. 19 , upper panel) or control anti serum—( FIG. 19 , middle panel) mediated immunoprecipitation of whole cell extracts, VHL was detected by immunoblotting using VHL antibodies. As a control, 10% of input whole cell extracts were used ( FIG. 19 , lower panel). As expected, VHL was specifically coimmunoprecipitated together with GAL4/HIF-1 alpha under normoxic conditions ( FIG. 19 , lanes 1-3). However, VHL was also coimmunoprecipitated under hypoxic conditions at levels similar to those observed at normoxia ( FIG. 19 , lanes 4-6). These results indicate that dissociation of the HIF-1 alpha-VHL complex is not necessary for protection of HIF-1 alpha from VHL-mediated degradation and that there exists a mechanism for hypoxia-dependent inactivation of VHL function when remaining associated with HIF-1. Next it was determined whether Arnt was associated with the HIF-1 alpha-VHL complex. GAL4/HIF-1 alpha or the minimal GAL4 DNA binding domain transiently expressed in COS7 cells in the absence or presence of VHL and/or Arnt at normoxia or hypoxia. As expected, Arnt was specifically coimmunoprecipitated together with GAL4/HIF-1 alpha under hypoxic conditions in the absence of VHL ( FIG. 20 ). Moreover, in the presence of VHL, Arnt and GAL4/HIF-1 alpha were also coimmunoprecipitated in a hypoxia-dependent fashion ( FIG. 20 ), demonstrating that these proteins formed a ternary complex in hypoxic cells. 
 EXAMPLE 8 Role of Protein Stabilization in Regulation of the Hypoxia-dependent Transactivation Function of the HIF-1 Alpha N-TAD Domain Transcriptional activation by HIF-1 alpha in hypoxic cells is mediated by two distinct transactivation domains, N-TAD and C-TAD (Arany, Z. et al., Proc. Natl. Acad. Sci., 93:12969-12973, 1996; Kallio, P. J. et al., EMBO J., 17:6573-6586, 1998; Ema, M. et al., EMBO J., 18:1905-1914, 1999; Carrero, P. et al., Mol. Cell. Biol., 20:402-415, 2000). Given the fact that VHL interacted with the minimal N-TAD structure, a comparison of both the stability and transactivation functions of GAL4 fusion proteins harboring either the wild-type or the PYI mutant N-TAD motifs ( FIG. 11 ) was made under normoxic and hypoxic conditions. Four (4) micrograms of FLAG-tagged wild-type or PYI mutant N-TAD were transiently expressed in COS7 cells in 10 cm diameter plastic dishes under normoxic ( FIG. 21 , lanes 1, 3) or hypoxic ( FIG. 21 , lanes 2, 4) conditions for 12 hours. Whole cell extracts were prepared as in Example 1A and assayed by immunoblot analysis as in Example 1B. In analogy to full length HIF-1, the minimal wild-type GAL4/N-TAD fusion protein showed significant degradation under normoxic conditions, and was stabilized by hypoxia ( FIG. 21 , lanes 1-2). PYI mutant GAL4/N-TAD protein levels were readily detectable by immunoblot analysis of extracts from normoxic cells, and were not significantly increased following exposure of the cells to hypoxia ( FIG. 21 , lanes 3-4). A minimal N-TAD fragment comprising residues 547-575 behaved similar to the N-TAD domain: it showed significant degradation under normoxic conditions, and was stabilized by hypoxia ( FIG. 30 ). In contrast, mutation of the single P564 resulted in striking protection against normoxia-dependent degradation of the protein fragment, and was not further stabilized by hypoxia treatment of the cells ( FIG. 31 ). Thus, the failure of these mutants to interact with VHL, as assessed by coimmunoprecipitation experiments ( FIG. 12 ; FIG. 27 ), correlated with constitutively stable protein expression levels which were similar to those generated by wild-type GAL4/N-TAD under hypoxic conditions. The transcriptional activity of wild-type or PYI mutant GAL4/N-TAD (0.2 &mgr;g/30-mm dish) was analyzed in COS7 cells in a cotransfection assay with a luciferase reporter gene under the control of thymidine kinase minimal promoter and five tandem copies of GAL4-responsive elements (GAL4-luc) (0.5 &mgr;g/30-mm dish) and a beta-galactosidase expression plasmid (0.05 &mgr;g/30-mm dish) as an internal control. After 6 hours of transfection, cells were incubated for 36 hours under hypoxic (1% O 2 ) or normoxic (21% O 2 ) conditions prior to analysis of reporter gene activity. Reporter gene activities are expressed relative to the activity in the presence of the GAL4-DNA binding domain alone at normoxia. Values represent the mean±SD of three independent experiments. As observed in FIG. 22 and FIG. 30 , in reporter gene transactivation assays using a GAL4-driven luciferase reporter gene, a rather modest (about 3-fold) activation of the function of the minimal wild-type GAL4/N-TAD by hypoxia. This result was expected in view of results disclosed in Carrero, P. et al., Mol. Cell. Biol., 20:402-415, 2000. In the context of full length HIF-1 alpha, mutation of the PYI motif rendered the protein stable under normoxic conditions and resistant to VHL-mediated degradation ( FIG. 23 ), demonstrating that this motif is the critical determinant for VHL-dependent degradation of HIF-1 alpha. Although the mutant protein showed an increased constitutive transcriptional activity in comparison to wild-type HIF-1 alpha, it was still hypoxia-inducible ( FIG. 24 ). Within the minimal N-TAD domain, mutation of the PYI motif reduced the transactivation function with regard to both the activity observed at normoxia and hypoxia ( FIG. 22 ). These results indicate that the mutation may generally have altered the structure important for transactivation, possibly impairing some of the protein contacts that may be required for the full activation response. Nevertheless, this mutated construct was inducible by hypoxia, yielding an about two-fold increase in transcriptional activity ( FIG. 22 ). These data indicate that the stabilized protein resistant to degradation by VHL is still capable of mediating a hypoxia-dependent activation response, and that protein stabilization per se does not bypass the need of the hypoxic signal for transactivation. In contrast to the broad PYI-AAA mutation described above the transactivation function of a GAL4-N-TAD fusion protein containing a single P564A mutation resulted in a dramatic increase in transactivation function by the protein. In fact, it was comparable to the activity of the maximally hypoxia-induced function of the wild-type construct ( FIG. 30 ). Importantly, the activity of the P564A mutant construct was not further enhanced by hypoxia treatment, demonstrating that it was rendered constitutively active at high levels by this single point mutation ( FIG. 30 ). In a similar fashion the mutations YI565-566, and DDD-AAA not only abolished VHL-N-TAD interaction ( FIG. 27 ) but also rendered constitutively active mutants of GAL4-N-TAD fusion protein constructs, as assessed in reporter gene assays performed as described above in 293 cells ( FIG. 28 ). In addition to the activity of the P564A mutant construct, additional substitutions of any amino acid at P564 will yield similarly active mutant constructs. For example, P564H also abrogates interaction with VHL, stabilizes the protein against VHL-mediated degradation, and renders the transactivation function of the protein constitutive. That is, the protein is no longer hypoxia dependent. Specific mutations destroying the function of the degradation box are useful applications of the present invention. Further, directing protein degradation through fusion of a normal degradation box to proteins other than HIF-1 alpha has utility with regard to all cellular proteins. 
 EXAMPLE 9 Model of Conditional Regulation of HIF-1 Alpha Function Under Normoxia and Hypoxia The data presented here indicate that under normoxic conditions VHL functions by targeting HIF-1 alpha for ubiquitin-proteasomal degradation by recruiting HIF-1 alpha to the VHL-BC-Cul-2 complex. The interaction between the two proteins occurs via the beta-domain of VHL and the minimal N-TAD of HIF-1. Hypoxic conditions lead to inhibition of degradation of HIF-1 alpha by induction of nuclear translocation and a regulatory signal that may be linked to recruitment of a partner DNA binding factor, Arnt, transcriptional coactivators and/or the redox regulator Ref-1. The shaded areas in VHL in FIG. 25 indicate mutational hot spots in tumors that coincide with the beta-domain or the elongin C binding domain (CBD) of VHL, respectively. Mutations in either of these two domains stabilize the HIF-1 alpha protein. The point mutagenesis experiments indicated that the highly conserved central PYI motif of the N-TAD of HIF-1 alpha, and, in particular, the P564 and Y565 residues were critical for interaction with VHL and for VHL-induced proteasomal degradation. In fact, it was demonstrated that the minimal HIF-1 alpha N-TAD structure was specifically ubiquitinated by VHL, and that this process required the conserved central PYI motif. Given the conservation of this core motif between HIF-1 alpha and the related, tissue-restricted hypoxia-inducible factor EPAS-1/HLF (Ema, M. et al., Proc. Natl. Acad. Sci., 94:4273-4278, 1997; Tian, H. et al., Genes & Dev., 11:72-82, 1997), it is likely that both factors are regulated by VHL-mediated ubiquitination in a similar fashion. In support of this EPAS-1/HLF has recently been reported to also contain an oxygen-dependent degradation domain overlapping with the N-TAD (Ema, M. et al., EMBO J., 18:1905-1914, 1999), and to interact with VHL in vitro (Maxwell, P. H. et al., Nature, 399:271-275, 1999). A second point mutation has also been demonstrated to play a critical role, that being lysine residue K547. This lysine residue was shown to be critical for HIF-1 alpha protein stabilization. As schematically outlined in FIG. 25 , hypoxia-induced protection of HIF-1 alpha against regulation by VHL involves two distinct and successive steps: nuclear translocation of HIF-1 alpha and an intranuclear event or signal required for protecting HIF-1 alpha against VHL-induced proteolysis. This model is based on the observation that a mutant of HIF-1 alpha which fails to enter the nucleus showed VHL-induced degradation even in hypoxic cells. On the other hand, a mutant form of HIF-1 alpha which shows constitutive nuclear localization was degraded in normoxic cells but showed hypoxia-inducible stabilization in the presence of VHL. These data indicate that nuclear compartmentalization does not suffice to protect HIF-1 alpha against VHL function but that HIF-1 alpha requires the hypoxic signal for resistance against degradation. Given the striking overlap within the N-TAD of HIF-1 alpha between the structures that mediate oxygen-dependent degradation, physical interaction with VHL, and hypoxia-inducible transactivation, it is possible that the putative intranuclear stabilizing signal may be linked to the transactivation function of the protein. It was previously observed that hypoxia-inducible function of both the N-TAD and C-TAD is critically dependent on the recruitment of the transcriptional coactivator CBP/p300 and SRC-1/pl60 (Arany, Z. et al., Proc. Natl. Acad. Sci., 93:12969-12973, 1996; Kallio, P. J. et al., EMBO J., 17:6573-6586, 1998; Ema, M. et al., EMBO J., 18:1905-1914, 1999; Carrero, P. et al., Mol. Cell. Biol., 20:402-415, 2000). This recruitment appears to be facilitated by the redox regulator Ref-1 (Ema, M. et al., EMBO J., 18:1905-1914, 1999; Carrero, P. et al., Mol. Cell. Biol., 20:402-415, 2000). Thus, the functional architecture of the N-TAD encompasses overlapping structures which have two different functions. In fact the functions are opposing, i.e. protein degradation versus activation of gene transcription. This creates an important “switch” in regulation of HIF-1 alpha protein function. As outlined in the model in FIG. 25 , the hypoxia-dependent intranuclear mechanism of protection may involve dimerization with Arnt, recruitment of coactivators, and/or recruitment of Ref-1. Importantly, the present data indicate that protein stabilization per se does not provide the sole basis for rendering HIF-1 alpha transcriptionally active. Even if the N-TAD protein was made constitutively stable and resistant to VHL by point mutagenesis, it still required hypoxic inducibility with regard to its transactivation function. It is possible that covalent modification of the C-terminus of HIF-1 alpha may play a role in determining this regulatory effect. In analogy to the steroid receptor system (Xu, L. et al., Curr. Opin. Genet. Dev., 9:140-147, 1999), it is also an attractive scenario that the hypoxic signal may determine a conformational change in HIF-1 alpha which facilitates recruitment of the coactivators and inactivates VHL function. The foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed broadly to include all variations falling within the scope of the appended claims and equivalents thereof.