Multiple Splice Variants of the Human HIF-3alpha Locus Are Targets of the von Hippel-Lindau E3 Ubiquitin Ligase Complex*

Mindy A. MaynardDagger §, Heng QiDagger §, Jacky ChungDagger , Eric H. L. LeeDagger , Yukihiro Kondo||, Shuntaro Hara**, Ronald C. ConawayDagger Dagger , Joan W. ConawayDagger Dagger , and Michael OhhDagger §§

From the Dagger  Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada, the || Department of Urology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, the ** Department of Public Health, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan, and Dagger Dagger  Stowers Institute for Medical Research, Kansas City, Missouri 64110

Received for publication, August 23, 2002, and in revised form, January 14, 2003

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Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor protein is the cause of familial VHL disease and sporadic kidney cancer. The VHL gene product (pVHL) is a component of an E3 ubiquitin ligase complex that targets the hypoxia-inducible factor (HIF) 1 and 2 alpha  subunits for polyubiquitylation. This process is dependent on the hydroxylation of conserved proline residues on the alpha  subunits of HIF-1/2 in the presence of oxygen. In our effort to identify orphan HIF-like proteins in the data base that are potential targets of the pVHL complex, we report multiple splice variants of the human HIF-3alpha locus as follows: hHIF-3alpha 1, hHIF-3alpha 2 (also referred to as hIPAS; human inhibitory PAS domain protein), hHIF-3alpha 3, hHIF-3alpha 4, hHIF-3alpha 5, and hHIF-3alpha 6. We demonstrate that the common oxygen-dependent degradation domain of hHIF-3alpha 1-3 splice variants is targeted for ubiquitylation by the pVHL complex in vitro and in vivo. This activity is enhanced in the presence of prolyl hydroxylase and is dependent on a proline residue at position 490. Furthermore, the ubiquitin conjugation occurs on lysine residues at position 465 and 568 within the oxygen-dependent degradation domain. These results demonstrate additional targets of the pVHL complex and suggest a growing complexity in the regulation of hypoxia-inducible genes by the HIF family of transcription factors.

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von Hippel-Lindau (VHL)1 disease is a multisystem disorder characterized by the development of hypervascular tumors in numerous organs, including brain, spine, retina, pancreas, adrenal gland, and kidney (1). Clinically, VHL disease displays a dominant pattern of inheritance due to variable but virtually complete degree of penetrance (1). However, VHL-associated tumorigenesis begins with the functional inactivation of both copies of the VHL allele (1). Thus, at a cellular level, VHL disease is recessive and occurs at an approximate frequency of 1 in 36,000 individuals (1). Moreover, a vast majority (60-80%) of sporadic renal clear cell carcinoma also demonstrates a biallelic loss of VHL, suggesting a requirement for pVHL inactivation in most renal carcinogenesis (1).

The VHL gene product (pVHL) is a component of a multiprotein complex called VEC composed of elongins B/C, Cul-2, and Rbx1 (2, 3). The VEC functions as an E3 ubiquitin ligase that binds and targets specifically prolyl-hydroxylated hypoxia-inducible factor (HIF) 1 and 2 alpha  subunits for polyubiquitylation (4-11). Hydroxylation of HIF-1/2alpha occurs on conserved proline residues within the oxygen-dependent degradation domain (ODD) in the presence of oxygen and iron by the newly identified class of prolyl hydroxylases (PHD) (5-7, 12, 13). The polyubiquitin-tagged HIF-1/2alpha subunits are subsequently captured by the 26 S proteasome for degradation. Thus, under reduced oxygen tension or hypoxia, HIF-1/2alpha subunits remain unhydroxylated and consequently escape ubiquitin-mediated proteolysis. The HIF-1/2alpha subunits bind to constitutively stable HIF-beta (also known as ARNT; aryl-hydrocarbon receptor nuclear translocator) subunit forming an active heterodimeric HIF transcription factor that binds to the hypoxia-responsive elements in the promoters of numerous hypoxia-inducible genes, triggering a physiologic response to hypoxia (14, 15).

pVHL contains two major recognizable domains, alpha  and beta  (16). Whereas the alpha  domain is required for binding elongin C, which bridges pVHL with the rest of the VEC complex (i.e. elongin B, Cul-2, and Rbx1), the beta  domain functions as a protein-docking site and is required for binding HIF-1/2alpha subunits via the ODD (9, 16-18). It is now known that all renal tumor-causing pVHL mutants are either defective in binding elongin C or HIF-1/2alpha (4, 9, 19). Concordantly, tumor cells devoid of functional pVHL produce inordinate amounts of hypoxia-inducible genes that are regulated by HIF-1/2, such as vascular endothelial growth factor, platelet derived growth factor-B, glucose transporter-1, and transforming growth factor-alpha , irrespective of ambient oxygen tension (20-23). Expression of these and other hypoxia-inducible angiogenic factors likely explains the vascular phenotype of VHL-associated tumors. These observations, taken together, support the notion that constitutive stabilization of HIF-1/2alpha in the absence of functional pVHL may be causally linked to tumorigenesis in VHL patients.

Recently, Kondo et al. (24) showed that a variant of HIF-2alpha , which retains the ability to activate HIF target genes but escapes degradation via VEC due to an arginine substitution of the conserved proline within the ODD, when overproduced in HIF-1alpha (-/-) renal carcinoma cells reconstituted with functional pVHL restored their ability to form tumors in nude mouse. This result suggests that down-regulation of HIF-2alpha is necessary for tumor suppression by pVHL. However, Maranchie et al. (25) demonstrated that the degradation-resistant variant of HIF-1alpha was not sufficient to reproduce tumorigenesis, indicating that it is not the critical oncogenic substrate of pVHL. Interestingly, competitive inhibition of the pVHL substrate-recognition site with a peptide derived from the ODD of HIF-1alpha recapitulates the tumorigenic phenotype of pVHL-deficient cells (25). These observations suggest that the tumor suppressor function of pVHL, through its interaction with the alpha  subunits of HIF-1/2, is more complicated than once perceived and/or that VEC E3 ubiquitin ligase may have more substrates than HIF-1/2alpha .

Here, we describe the genomic organization of human HIF-3alpha locus and report multiple alternatively spliced variants as follows: hHIF-3alpha 1, hHIF-3alpha 2 (also referred to as hIPAS in the GenBankTM), hHIF-3alpha 3, hHIF-3alpha 4, hHIF-3alpha 5, and hHIF-3alpha 6. We show that hHIF-3alpha 1, -3alpha 2, and -3alpha 3 share a common ODD, which includes the consensus oxygen-dependent prolyl hydroxylation motif, and are targeted for ubiquitylation by the VEC complex in vitro and in vivo. Thus, the growing number of potential oxygen-dependent transcription factors targeted for ubiquitin-mediated destruction via VEC adds to the complexity of not only the mechanisms governing our physiologic response to hypoxia but also to the tumor suppressor function of pVHL.

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Cells-- 786-O renal clear cell carcinoma (RCC) subclones stably transfected to produce wild-type pVHL (WT8) or transfected with empty plasmid (pRC3) were as described previously (26, 27). Osteosarcoma U2OS, colon carcinoma HCT116, and prostate carcinoma PC-3 cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Sigma) at 37 °C in a humidified 5% CO2 atmosphere.

Antibodies-- Monoclonal anti-Gal4 (RK5C1) and anti-HA (12CA5) antibodies were from Santa Cruz Biotechnology and Roche Molecular Biochemicals, respectively. Monoclonal anti-T7 antibody was from Novagen (Madison, WI). Monoclonal anti-pVHL antibody (IG32) was as described (26, 27). Purified polyclonal anti-HIF-3alpha antibody was as described (28).

Plasmids-- Mammalian expression plasmids, pRc-CMV, containing HA-VHL(WT, Y98H, Q164F, 63-154, and 155-213) and T7-VHL(WT) were described previously (9). The cloning and generation of the mammalian expression plasmid, pcDNA3, encoding the full-length human HIF-3alpha (FL) was described previously (28). pcDNA3-HA-hHIF-3alpha 1-3[ODD] was generated by PCR from human fetal brain library using 5'-GCGCGGATCCGCCACCATGGTGCACAGACTCTTCACCT-3' and 5'-GCGCGAATTCTCACTCGTCCTCTGAGCTCTG-3'. The PCR product was then ligated into the BamHI and EcoRI in the multiple cloning sites of pcDNA3-HA and pcDNA3-T7 plasmids. pcDNA3-HA-hHIF-3alpha ODD(K465R), -(K568R), and -(P490A) were generated using Stratagene QuikChange site-directed mutagenesis kit (La Jolla, CA) and the following primer sets: 5'-CTTCACCTCCGGGAGAGACACTGAGG-3'/5'-CCTCAGTGTCTCTCCCGGAGGTGAAG-3', 5'-CTGGGGCTCGGAGAAGGACCCTGGC-3'/5'-GCCAGGGTCCTTCTCCGAGCCCCAG-3', and 5'-GGAGATGCTGGCCGCCTACATCTCCATG-3'/5'-CATGGAGATGTAGGCGGCCAGCATCTCC-3', respectively. All plasmids made by PCR were confirmed by DNA sequencing.

Northern Blot Analysis-- The hHIF-3alpha probe for the human multiple tissue Northern (MTN) blot (Clontech) was prepared by amplifying a fragment (nucleotides 273-761) of hHIF-3alpha 1 by PCR using 5'-GGAGGGCTTCGTCATGGT-3' and 5'-TCGTCACAGTAGGTGAACTTC- ATG-3' primers. The amplified fragment was then labeled with [alpha -32P]dCTP using the DECAprime II random priming kit (Ambion). The MTN blot was incubated in hybridization buffer for 30 min, and the labeled probe was then added (2 × 106 cpm/ml) for ~1 h at 68 °C. The membrane was washed 3 times in 2× SSC (0.3 M NaCl, 0.03 M sodium citrate (pH 7.0)) and 0.05% SDS for 30 min, and 2 times in 0.1× SSC, 0.1% SDS at 50 °C for 40 min. hHIF-3alpha bands were visualized by autoradiography. beta -Actin was subsequently probed as a loading control.

Immunoprecipitation and Immunoblotting-- Immunoprecipitation and Western blotting were performed as described previously (29). In brief, cells were lysed in EBC buffer (50 mM Tris (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40) supplemented with protease and phosphatase inhibitors (Roche Molecular Biochemicals). Immunoprecipitates, immobilized on protein A-Sepharose (Amersham Biosciences AB), were washed 5 times with NETN buffer (20 mM Tris (pH 8.0), 120 mM NaCl, 1 mM ETDA, 0.5% Nonidet P-40), eluted by boiling in SDS-containing sample buffer, and separated by SDS-PAGE.

Purification of HIF PHD-- Extracts containing enriched PHD were purified from rabbit reticulocyte lysate as described previously (30). Briefly, ~1 liter of rabbit reticulocyte lysate (Green Hectares, Oregon, WI) was diluted to 5 liters in 50 mM Tris-HCl (pH 7.4), 0.1 M KCl, and 5% (v/v) glycerol and precipitated with 0.213 g/ml (NH4)2SO4. After centrifugation at 16,000 × g for 45 min at 4 °C, the resulting supernatant was precipitated with an additional 0.153 g/ml (NH4)2SO4. After centrifugation at 16,000 × g for 45 min at 4 °C, the pellet was resuspended in Buffer A (40 mM HEPES-NaOH (pH 7.4) and 5% (v/v) glycerol), dialyzed against Buffer A to a conductivity equivalent to Buffer A containing 0.2 M KCl, and applied at 0.5 liters/h to 0.5 liter of phosphocellulose (Whatman, P11) column equilibrated in Buffer A containing 0.2 M KCl. The phosphocellulose column was eluted stepwise at 1 liter/h with Buffer A containing 0.5 M KCl, and 100-ml fractions were collected. Proteins eluting in the phosphocellulose 0.5 KCl step were pooled and precipitated with 0.4 g/ml (NH4)2SO4. After centrifugation at 16,000 × g for 45 min at 4 °C, the pellet was resuspended in 4 ml of Buffer A. Following centrifugation at 35,000 × g for 30 min at 4 °C, the resulting supernatant was applied at 2 ml/min to a TSK SW3000 high pressure liquid chromatography column (Toso-Haas, Montgomeryville, PA; 21.5 × 600 mm) equilibrated in Buffer A containing 0.15 M KCl. The SW3000 column was eluted at 2 ml/min, and 4-ml fractions containing enriched PHD were collected.

In Vitro Binding Assay-- Assay for binding of hHIF3alpha to pVHL was performed as described previously (9, 31). Reticulocyte lysate translation products were synthesized in the presence (for pcDNA3-T7-pVHL) or absence (for pcDNA3-HA-hHIF-3alpha [FL] and -[ODD]) of [35S]methionine. hHIF3alpha translation products were treated with or without cellular fractions containing enriched prolyl hydroxylase for 30 min at 37 °C. HA-HIF-3alpha (10 µl) and T7-pVHL (10 µl) translation products were incubated with anti-HA antibody and protein A-Sepharose in 750 µl of EBC buffer (50 mM Tris (pH 8), 120 mM NaCl, 0.5% Nonidet P-40). After five washes with NETN buffer (20 mM Tris (pH 8), 100 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA), the bound proteins were resolved on SDS-PAGE and detected by autoradiography.

In Vitro Ubiquitylation Assay-- An in vitro ubiquitylation assay was performed as described previously (9). [35S]Methionine-labeled reticulocyte lysate HA-HIF3alpha [FL] or -[ODD] translation products (4 µl) were incubated in RCC 786-O S100 extracts (100-150 µg), prepared as described previously (9), supplemented with 8 µg/µl ubiquitin (Sigma), 100 ng/µl ubiquitin-aldehyde (BostonBiochem, Inc., Cambridge, MA), and an ATP-regenerating system (20 mM Tris (pH 7.4), 2 mM ATP, 5 mM MgCl2, 40 mM creatine phosphate, 0.5 µg/µl of creatine kinase) in a reaction volume of 20-30 µl for 1.5 h at 30 °C.

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pVHL tumor suppressor complex VEC targets the ODD within hHIF-1/2alpha for oxygen-dependent ubiquitylation (4-6, 9-11). We searched the GenBankTM for other cellular proteins containing ODD-like residues, in particular LAPYIXMD motif, with the aim of identifying additional targets of VEC.

Genomic Organization and Multiple Splicing Variants of hHIF-3alpha -- Fig. 1 and Table I summarize our data base search uncovering multiple alternatively spliced variants of human HIF-3alpha (hHIF-3alpha ). The third alpha -class of HIF subunit was first isolated in mouse in 1998 (32). A dominant-negative regulator of murine mHIF-1 called inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS; GenBankTM accession number AF416641) was later identified to be a splicing variant of mHIF-3alpha (33, 34). The human homologue of the mHIF-3alpha was isolated and revealed high homology to hHIF-1 and -2alpha (28). The search of the GenBankTM data base with hHIF-3alpha cDNA (GenBankTM accession numbers NM_152794 and AB054067) revealed that hHIF-3alpha has at least six alternatively splicing variants. In fact, the genomic organization of hHIF-3alpha was described previously in 1999 when the BAC 82621 genomic clone (GenBankTM accession number AC007193) was completed and mapped to chromosome 19q13.2. Although several GenBankTM entries were proposed to be hHIF-3alpha splicing variants, a detailed intron-exon structure of the hHIF-3alpha locus has not been reported previously.


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Fig. 1.   Multiple spliced variants of the human HIF-3alpha locus. A, genomic organization of the hHIF-3alpha alternatively spliced variants. Alternatively spliced isoforms of the hHIF-3alpha locus are illustrated with the shaded boxes representing the open reading frame. The locations of the conserved domains are indicated with bars. Cen, centromere; tel, telomere; bHLH, basic helix-loop-helix; PAS, Per/Arnt/Sim; PAC, PAS-associated C-terminal domain. B, structural alignment of hHIF-1/2/3alpha subunits. Functional domains are illustrated as open boxes, Dots represent LXXLL motifs. NAD, N-terminal transactivation domain; CAD, C-terminal transactivation domain. C, amino acid alignment of ODD in hHIF-1/2/3alpha and mHIF-3alpha . hHIF-3alpha [ODD] is represented by hHIF-3alpha 1[ODD]. Shaded regions represent conserved amino acids, and asterisk represents the conserved proline residue within the prolyl hydroxylation motif. D, multiple tissue Northern blot of hHIF-3alpha . Human MTN blot was hybridized with 32P-labeled 489-bp region within exons 3-6 of hHIF-3alpha 1, and hybridized bands were detected by autoradiography. The arrows indicate multiple hHIF-3alpha probe-specific RNAs (upper panel). beta -Actin probe was subsequently hybridized to the blot (lower panel) as internal loading control.


                              
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Table I
Exon-intron structure of hHIF-3alpha gene
The exon and intron sequences are described as uppercase and lowercase letters, respectively. hHIF-3alpha 1 (GenBankTM accession numbers NM_152794/AB054067) begins at exon 1c and ends at exon 16. hHIF-3alpha 2/IPAS (accession numbers NM_152795/AF463492) starts from exon 1a and ends at exon 13a. hHIF-3alpha 3 (accession numbers NM_022462/AK021653) starts at exon 1b and ends at exon 17 without exons 15 and 16. hHIF-3alpha 4 (accession numbers BC026308) begins at exon 1a and ends within imperfect intron 8. hHIF-3alpha 5 (accession numbers NM_152796) begins at exon 1b and ends at exon 15 without exons 3, 12, and 13. hHIF-3alpha 6 (accession numbers AC024095) begins at exon 1b and ends within intron 8 without exon 3. The intron donor sequences (in boldface) flanking the 3' and 5' of exons 14b and 17 do not obey the GT-AG rule.

Here we describe the genomic structures of six splicing variants of hHIF-3alpha locus. Similar to mHIF-3alpha , hHIF-3alpha gene consists of 19 exons (Table I), which span about 43 kb on chromosome 19q13.2. Three unique exons, namely exons 1a, -b, and -c, likely contain the transcription start sites for the six splicing variants. As shown in Fig. 1A, exon 2 encodes a basic helix-loop-helix domain, and exons 3-9 encode PASa, PASb, and PAC (PAS-associated C-terminal domain) domains. The 3' portion of exon 11, entire exon 12, and 5' portion of exon 13 encode the ODD domain. Exons 14a and 16 encode a leucine zipper (LZIP).

The first human HIF-3alpha was reported in 2001 (28). Thus, we have designated it hHIF-3alpha 1. As shown in Fig. 1A, hHIF-3alpha 1 begins at exon 1c and ends at exon 16, which is absent in all other splicing variants (Fig. 1A). hHIF-3alpha 1 encodes a 668-amino acid protein that contains a single N-terminal transactivation domain (NAD) like hHIF-1 and -2alpha , but it does not contain a C-terminal transactivation domain (CAD) (Fig. 1B). Interestingly, hHIF-3alpha 1 contains a LZIP domain composed of four-septad leucines not found on hHIF-1/2alpha or on any other hHIF-3alpha splicing variants (Fig. 1B). LZIP domains mediate both DNA-binding and protein-protein interactions (35). Another distinguishing characteristic of hHIF-3alpha 1 is the signature LXXLL protein-protein interaction motif found immediately upstream of the ODD and LZIP domains. The LXXLL motif is found mostly in nuclear receptor co-factors (36). Both LZIP and LXXLL structures are absent in hHIF-1 and -2alpha subunits, suggesting that hHIF-3alpha 1 may bind DNA/promoter sequences or novel interacting protein(s) uncommon to those recognized by hHIF-1/2alpha .

Recently, a dominant-negative regulator of mHIF-1 called mIPAS, which lacks transactivation domains, was identified as an alternatively spliced variant of mHIF-3alpha (see Fig. 1B) (33, 34). mIPAS dimerizes with the mHIF-1alpha subunit and consequently prevents the interaction of mHIF-1alpha to mHIF-beta subunits (33). Furthermore, the mIPAS/mHIF-1alpha complex does not bind to the hypoxia-responsive elements of target genes (33). Thus, mIPAS inhibits hypoxia-mediated transcriptional activation. The cDNA sequence of hIPAS was deposited in the GenBankTM (GenBankTM accession numbers NM_152795 and AF463492). hIPAS starts at exon 1a and ends at exon 13a without exon 1b and -c (Fig. 1A and Table I). Although referred to as hIPAS, its cDNA sequence and 632-amino acid composition are more similar to hHIF-3alpha 1 than mIPAS (see Fig. 1B). For example, mIPAS does not contain a recognizable ODD, and hence it is presumably not subjected to ubiquitylation by VEC, whereas hIPAS contains a well conserved ODD. Unlike mIPAS, hIPAS contains a NAD (Fig. 1B). For these reasons, we refer to hIPAS as hHIF-3alpha 2. hHIF-3alpha 2, due to alternative splicing (see Fig. 1A and Table I), retains one of the two LXXLL motifs and lacks the LZIP motif found on the C terminus of hHIF-3alpha 1 (Fig. 1B).

cDNA entries with GenBankTM accession numbers AK021653 and NM_022462 were also found to be derived from the hHIF-3alpha locus. We refer to it as hHIF-3alpha 3, which begins at exon 1b and ends at exon 17 without exons 1a, 1c, 15, and 16 (Fig. 1A and Table I). hHIF-3alpha 3 utilizes the shorter exon 13b and 14b instead of 13a and 14a. Although hHIF-3alpha 3 is a 648-amino acid protein that contains an ODD and both LXXLL motifs, it does not contain any recognizable DNA-binding sequences, such as bHLH and LZIP domains (Fig. 1B).

Another full-length cDNA (GenBankTM accession number BC026308) that begins at exon 1a and ends within the imperfect intron 8 was derived from the same locus as other hHIF-3alpha splice variants (Fig. 1A and Table I). The intron 7 was not spliced. We refer to it as hHIF-3alpha 4 that encodes a 363-amino acid protein. Amino acid sequence alignment suggests that hHIF-3alpha 4 is most similar to mIPAS (Fig. 1B). Both proteins lack a NAD and CAD, as well as ODD, suggesting that, like mIPAS, hHIF-3alpha 4 could potentially act as a dominant-negative regulator of hHIF activity. Furthermore, with respect to the other hHIF-3alpha variants, hHIF-3alpha 4 does not contain LXXLL or LZIP structures.

Two additional GenBankTM cDNA entries were recently found to be splicing variants of hHIF-3alpha . We have named them hHIF-3alpha 5 (GenBankTM accession number NM_152796) and hHIF-3alpha 6 (GenBankTM accession number AK024095). Both isoforms start at exon 1b and lack exon 3. hHIF-3alpha 5 contains a short exon 14c and ends at exon 15. hHIF-3alpha 5 encodes a protein containing a partial PASa, PASb, and PAC domains. hHIF-3alpha 6, like hHIF-3alpha 4, contains intron 7 and ends at intron 8. hHIF-3alpha 6 contains only a partial C-terminal domain of PASb.

It should be noted that Makino and colleagues (34) described previously several EST sequences that were located on the hHIF-3alpha locus. ESTs with GenBankTM accession numbers BG699633, AL528423, and AL519496 are largely identical to hHIF-3alpha 2, whereas BQ067192 and AL535689 are fragments of hHIF-3alpha 3 and hHIF-3alpha 5, respectively. However, EST entry with accession number BM119659 is derived from mouse.

To discern the expression profile of hHIF-3alpha , we performed human multiple tissue Northern blot analysis. The tissue blot was hybridized with 32P-labeled 489-bp region within exons 3-6 of hHIF-3alpha 1, which also overlaps on hHIF-3alpha 2-6. We observed multiple hybridized RNA species with sizes 7.5, 7.0, 3.0, and 1.5 kb (Fig. 1D). A strong expression pattern was observed in the heart, placenta, and skeletal muscle, whereas a weak expression profile was found in the lung, liver, and kidney (Fig. 1D). In contrast, Northern blot analysis of hHIF-1 or -2alpha revealed single RNA species (37, 38). Based on the primary sequences of hHIF-3alpha isoforms, the predicted sizes of the RNA species vary from 1.5 to 3 kb. The higher 7.5- and 7.0-kb bands could represent RNA species with extended poly(A) tails or yet novel hHIF-3alpha -like transcripts. Hence, the hHIF-3alpha locus likely generates multiple alternatively spliced variants.

pVHL Binds Oxygen-dependent Degradation Domain of hHIF-3alpha -- hHIF-3alpha 1, -3alpha 2, and -3alpha 3 splice variants share a common ODD (Fig. 1B). The alignment of hHIF-1/2/3alpha and mHIF-3alpha ODDs shows that this domain is highly conserved, in particular the LAPYIXMD motif, which has been demonstrated to be critical for pVHL to bind hHIF-1/2alpha (Fig. 1C) (5). Specifically, hydroxylation of the conserved proline residue within this motif, which occurs via the newly identified class of PHDs in the presence of dioxygen, is necessary for binding pVHL (5, 6, 12, 13). To determine whether hHIF-3alpha [ODD] is capable of binding pVHL and whether prolyl hydroxylation is required for pVHL/hHIF-3alpha [ODD] interaction, we generated hHIF-3alpha [ODD; WT] and hHIF-3alpha [ODD; P490A] mutant fused in-frame with an N-terminal hemagglutinin (HA) epitope tag. HA-hHIF-3alpha [ODD; WT and P490A] chimeras were treated with or without a cellular fraction enriched with PHD, mixed with 35S-labeled pVHL in vitro translates, and immunoprecipitated with an anti-HA antibody. pVHL bound to hHIF-3alpha [ODD; WT] and this interaction was moderately enhanced in the presence of PHD (Fig. 2A). It should be noted that even in the absence of PHD-enriched fraction, there is some binding of pVHL and hHIF-3alpha [ODD; WT] (Fig. 2A, lane 2). This is likely due to the presence of endogenous PHD in the reticulocyte extract used to translate in vitro hHIF-3alpha [ODD; WT]. However, hHIF-3alpha [ODD; P490A] bound to pVHL weakly even in the presence of PHD (Fig. 2A). These results were similar to that of pVHL/hHIF-1alpha [ODD; WT and P564A] control interactions (Fig. 2A) (5) and suggest that pVHL binds hHIF-3alpha [ODD] dependent on hydroxylation of the conserved proline residue. Recovered amounts of the hHIF-3alpha [ODD; WT and P490A] and hHIF-1alpha [ODD; WT and P564A] were comparable as determined by anti-HA immunoblotting (Fig. 2B).


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Fig. 2.   Binding of pVHL to hHIF-3alpha [ODD] is enhanced by PHD and is dependent on P490. A, in vitro translates of HA-hHIF-3alpha [ODD; WT], HA-hHIF-3alpha [ODD; P490A], HA-hHIF-1alpha [ODD; WT], and HA-hHIF-1alpha [ODD; P564A] were treated with or without cellular extracts enriched for PHD and mixed with in vitro translated 35S-labeled T7-pVHL. Reaction mixtures were then immunoprecipitated with anti-HA antibody, and pVHL bound to hHIF-1 or 3alpha [ODD] was separated on SDS-PAGE and visualized by autoradiography. B, equal amounts of in vitro translated HA-hHIF-3alpha [ODD; WT or P490A] and Gal4-HA- hHIF-1alpha [ODD; WT or P564A] used in the binding assay were immunoprecipitated and immunoblotted with anti-HA antibody. IP, immunoprecipitation; IB, immunoblot; AR, autoradiography.

pVHL Binds hHIF-3alpha via the beta  Domain-- pVHL is composed of two domains, alpha  and beta  (16). The alpha  domain consists of three C-terminal helices and is required for binding elongin C, which bridges pVHL to elongin B and Cul2 and Rbx1, thereby nucleating the VEC complex (2, 16, 17). The beta  domain is a substrate-docking interface that is required for binding prolyl-hydroxylated hHIF-1/2alpha [ODD] (5, 9, 16). To determine which domain of pVHL was required for binding hHIF-3alpha , 35S-labeled full-length pVHL-(1-213), pVHL-(155-213), which encompasses most of the alpha  domain, and pVHL-(63-154), which encompasses most of the beta  domain, were mixed with either T7-hHIF-3alpha [ODD] or full-length hHIF-3alpha 1 that had been pre-treated with PHD. Both hHIF-3alpha [ODD] and full-length hHIF-3alpha 1 bound to pVHL-(1-213) and pVHL-(63-154) (Fig. 3A). However, pVHL-(155-213) did not associate with hHIF-3alpha [ODD] or full-length hHIF-3alpha 1 (Fig. 3A). Recovered amounts of hHIF-3alpha [ODD] and hHIF-3alpha 1 were comparable as determined by anti-T7 and anti-hHIF-3alpha 1 immunoblotting, respectively (Fig. 3B). We next examined the binding capabilities of two tumor-derived pVHL mutants, a representative beta  domain mutant Y98H and a representative alpha  domain mutant Q164R, to hHIF-3alpha [ODD]. The beta  domain pVHL mutant Y98H bound poorly to hHIF-3alpha [ODD], whereas Q164R effectively bound hHIF-3alpha [ODD] (Fig. 3C). These results taken together demonstrate that pVHL binds hHIF-3alpha [ODD] via its beta  domain. It follows then that tumor-derived mutations affecting the surface residues in the beta  domain effectively abrogate the interaction of pVHL to not only hHIF-1 and -2alpha subunits but also hHIF-3alpha 1-3 variants. Furthermore, binding of hHIF-3alpha [ODD] to the beta  domain of pVHL was much stronger than to the full-length pVHL (Fig. 3A), which suggests a possible inhibitory effect of some portion of pVHL or may reflect a more accessible folding conformation of the beta  domain in the absence of the rest of pVHL residues.


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Fig. 3.   hHIF-3alpha [ODD] and the full-length hHIF-3alpha 1 bind to the beta  domain of pVHL. A, in vitro translates of T7-hHIF-3alpha [ODD] and hHIF-3alpha 1[full-length] were treated with cellular extracts enriched for PHD and mixed with 35S-labeled HA-pVHL-(1-213; WT) or -(155-213) or -(63-154). Reaction mixtures were then immunoprecipitated with anti-T7 (lanes 4-6) or anti-hHIF-3alpha 1 (lanes 7-9) antibodies, and the co-immunoprecipitating pVHL was separated on SDS-PAGE and visualized by autoradiography. 20% input of the radiolabeled pVHL fragments was separated on SDS-PAGE and visualized by autoradiography (lanes 1-3). B, expression of in vitro translated T7-hHIF-3alpha [ODD] and hHIF-3alpha 1 used in the binding assay were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. C, in vitro translates of T7-hHIF-3alpha [ODD] were treated with cellular extracts enriched for PHD and then mixed with 35S-labeled HA-pVHL(WT) or tumor-derived (Y98H) or (Q164R) mutants. Reaction mixtures were immunoprecipitated with anti-T7 antibody (lanes 4-6), and the co-immunoprecipitating pVHL was separated on SDS-PAGE and visualized by autoradiography. 20% input of the radiolabeled pVHL was separated on SDS-PAGE and visualized by autoradiography (lanes 1-3).

Ubiquitylation of hHIF-3alpha Is Dependent on pVHL and Oxygen-- hHIF-1 and -2alpha are degraded via VEC-dependent, oxygen-dependent ubiquitin-proteasome pathway (5-7, 9, 16). To determine whether pVHL ubiquitylates hHIF-3alpha [ODD], we performed an in vitro ubiquitylation assay. There was efficient poly-ubiquitylation of hHIF-3alpha [ODD; WT] in S100 extracts (cellular extracts devoid of 26S proteasome) that contained wild-type pVHL (Fig. 4A). However, S100 extract that lacked functional pVHL was incapable of ubiquitylating hHIF-3alpha [ODD; WT] (Fig. 4A). This suggests that pVHL is critical for targeting hHIF-3alpha [ODD] for ubiquitylation. Furthermore, the addition of PHD moderately enhanced the ubiquitylation of hHIF-3alpha [ODD; WT] (Fig. 4A). This is likely due to the increased prolyl hydroxylation of hHIF-3alpha [ODD; WT] in the presence of dioxygen, which enhances its binding to pVHL (see Fig. 2A). In support, hHIF-3alpha [ODD; P490A] mutant showed dramatically reduced ubiquitylation in the presence of S100 extract containing wild-type pVHL and PHD (Fig. 4A). Interestingly, it should be noted that the intensity of the first band immediately above hHIF-3alpha [ODD; P490A], likely representing mono-ubiquitylated species of hHIF-3alpha [ODD] did not diminish with respect to the wild-type counterpart (Fig. 4A, lanes 3 and 4). This suggests that although Pro-490 is critical for pVHL-dependent polyubiquitylation of hHIF-3alpha [ODD], it does not appear to be required for mono-ubiquitylation.


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Fig. 4.   Ubiquitylation of hHIF-3alpha [ODD] and hHIF-3alpha 1[full-length] is dependent on pVHL and is blocked by hypoxia mimetic. A, in vitro ubiquitylation of 35S-labeled HA-hHIF-3alpha [ODD; WT or P490A], treated with or without PHD, was performed in the presence of S100 786-O renal carcinoma cellular extracts devoid of pVHL (R) or reconstituted with wild-type pVHL (W). Reaction mixtures were immunoprecipitated with anti-HA antibody. Bound proteins were then separated on SDS-PAGE and visualized by autoradiography. B and C, in vitro ubiquitylation of 35S-labeled HA-hHIF-3alpha [ODD] (B) or 35S-labeled hHIF-3alpha 1[full-length] (C) was performed in the presence of S100 (R or W) and PHD, with increasing amounts of CoCl2 (lanes 2-4; 0, 10, and 100 µM, respectively). Reaction mixtures were immunoprecipitated with anti-HA (B) or anti-hHIF-3alpha 1 (C) antibody. Bound proteins were then separated on SDS-PAGE and visualized by autoradiography.

To confirm whether the ubiquitylation of hHIF-3alpha is regulated by oxygen, we performed an in vitro ubiquitylation assay in the presence or absence of hypoxia mimetics, such as CoCl2. Treatment with CoCl2 attenuated the ubiquitylation of hHIF-3alpha [ODD] in a dosage-dependent manner (Fig. 4B). A similar result was also obtained with desferrioxamine (DFO) (data not shown). These results taken together with the aforementioned observations of Fig. 4A strongly suggest that hHIF-3alpha [ODD] is targeted for ubiquitylation dependent on pVHL and oxygen.

We next asked whether the full-length hHIF-3alpha 1 was likewise targeted for pVHL-dependent ubiquitylation. Like hHIF-3alpha [ODD], the polyubiquitylation of hHIF-3alpha 1 occurred only in S100 extracts that contained pVHL (Fig. 4C, compare lanes 1 and 2). However, full-length hHIF-3alpha 1 was ubiquitylated less robustly than hHIF-3alpha [ODD], which raises the possibility that there are structural elements in the full-length protein that inhibit ubiquitylation. We have previously made similar observations with hHIF-1 and -2alpha (data not shown and Ref. 9). Treatment with CoCl2 dramatically inhibited the ubiquitylation of hHIF-3alpha 1 (Fig. 4C). Thus, the targeting of full-length hHIF-3alpha 1 for ubiquitylation is most likely pVHL- and oxygen-dependent. Whether other full-length hHIF-3alpha variants that contain a recognizable ODD, such as hHIF-3alpha 2 and hHIF-3alpha 3, are also targeted for pVHL/oxygen-dependent ubiquitylation or whether all hHIF-3alpha subunits containing the ODD bind to pVHL with equal affinity remains to be resolved.

Ubiquitylation of hHIF-3alpha Occurs on Lysine Residues at Positions 465 and 568-- Conjugation of ubiquitin on targeted substrates invariably occurs on lysine residues (39, 40). There are two lysine residues on hHIF-3alpha [ODD]. To determine which lysine residue(s) within the ODD of hHIF-3alpha was required for ubiquitin conjugation via VEC, we generated hHIF-3alpha [ODD; K465R], hHIF-3alpha [ODD; K568R], and hHIF-3alpha [ODD; K465R/K568R] using PCR-based site-directed mutagenesis and performed an in vitro ubiquitylation assay. Although the single Lys to Arg substitution mutants were still capable of being tagged with ubiquitin comparable with the wild-type hHIF-3alpha [ODD], the double substitution mutant had significantly reduced levels of poly-ubiquitylation (Fig. 5). These results demonstrate that both lysine residues are capable of accepting the activated ubiquitin. It is, however, formally possible that other lysine residues outside the ODD are targeted for ubiquitin conjugation by VEC.


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Fig. 5.   pVHL-dependent ubiquitin conjugation on hHIF-3alpha [ODD] occurs on lysine 465 and lysine 568. In vitro ubiquitylation of 35S-labeled HA-hHIF-3alpha [ODD; WT], HA-hHIF-3alpha [ODD; K465R], HA-hHIF-3alpha [ODD; K568R], and HA-hHIF-3alpha [ODD; K465R/K568R] treated with PHD was performed in the presence of S100 (R, devoid of pVHL, or W, reconstituted with pVHL). Reaction mixtures were immunoprecipitated with anti-HA antibody. Bound proteins were then separated on SDS-PAGE and visualized by autoradiography.

Stability of Endogenous hHIF-3alpha 1 Is Regulated by pVHL in Vivo-- To address whether the stability of endogenous hHIF-3alpha 1 is affected by pVHL in vivo, we first asked whether pVHL binds hHIF-3alpha 1 in the absence of overexpression (Fig. 6A). 786-O RCC cells (VHL -/-) and PC-3 prostate carcinoma cells (VHL +/+) were treated with the proteasome inhibitor MG132 and immunoprecipitated with anti-hHIF3alpha 1 antibody. Bound proteins were separated by SDS-PAGE, and anti-pVHL immunoblot revealed co-immunoprecipitating pVHL. pVHL30 and pVHL19, which are both wild-type forms of pVHL (41, 42), were found to co-immunoprecipitate with hHIF-3alpha 1 (Fig. 6A, lane 2). It should be noted that although the expression level of hHIF-3alpha 1 under the presence of hypoxia mimetics is comparable with the level achieved under the presence of proteasome inhibitor, pVHL failed to co-immunoprecipitate with hHIF-3alpha 1 (data not shown). This is likely due to the lack of prolyl hydroxylation of hHIF-3alpha 1 in the presence of hypoxia mimetics, which renders it unrecognizable by pVHL. It should also be noted that the hHIF-3alpha 1 antiserum was raised against residues 564-583 of hHIF-3alpha 1 (28). This region of amino acids is also present on hHIF-3alpha 2 and -3alpha 2. Thus, in the absence of specific antibodies against each of the hHIF-3alpha splicing variants, it is formally possible that pVHL interacts in vivo with one or more of hHIF-3alpha 1, -3alpha 2, and -3alpha 3.


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Fig. 6.   A, endogenous interaction between pVHL and hHIF-3alpha 1. 786-O RCC cells (lane 1) and PC-3 prostate carcinoma cells (lane 2) were treated with the proteasome inhibitor MG132 (10 µM) for 4 h. Cells were then lysed and immunoprecipitated with anti-hHIF3alpha 1 antibody. Bound proteins were separated by SDS-PAGE, and anti-hHIF3alpha 1 (upper panel) and anti-pVHL (lower panel) immunoblots were performed. Asterisk represents a protein yet unidentified recognized by anti-hHIF-3alpha 1 antibody. B, regulation of endogenous hHIF-3alpha 1 by pVHL and the effect of hypoxia mimetic. 786-O RCC, HCT116 colon carcinoma, PC-3 prostate carcinoma, and U2OS osteosarcoma cells were treated with the hypoxia mimetic CoCl2 for 4 h. Cells were then lysed and immunoprecipitated (IP) with anti-hHIF3alpha 1 antibody. Bound proteins were separated by SDS-PAGE and visualized by anti-hHIF3alpha 1 immunoblot (IB). Endogenous expression of pVHL (- or +) in each of the cell line is indicated on the top. In vitro translated hHIF-3alpha 1 was loaded in lane 1 as a positive control. C, 786-O RCC cells stably transfected with pRc-CMV plasmid alone (RC3) or pRC-CMV-HA-VHL (WT8) were treated with hypoxia mimetics CoCl2 (10 µM) or DFO (10 µM) for 4 h. Cells were then lysed and immunoprecipitated with anti-hHIF3alpha 1 (top and middle panels) or anti-HA (bottom panel) antibody. Bound proteins were separated by SDS-PAGE and detected by anti-hHIF3alpha 1 or anti-HA immunoblot, respectively.

We next asked whether pVHL affected the expression level of endogenous hHIF-3alpha 1 in vivo. HCT116 colon carcinoma, PC-3 prostate carcinoma, and U2OS osteosarcoma cells that express endogenous pVHL showed dramatically reduced levels of hHIF-3alpha 1 (Fig. 6B). However, these cells when treated with the hypoxia mimetic CoCl2 (or DFO; data not shown) markedly accumulated hHIF-3alpha 1 (Fig. 6B). Concordantly, 786-O RCC cells devoid of functional pVHL showed elevated expression of hHIF-3alpha 1 irrespective of hypoxia mimetics (Fig. 6B). These results taken together demonstrate that endogenous pVHL and hHIF-3alpha 1 interact in vivo, and under normal oxygen tension, VEC targets hHIF-3alpha 1 for ubiquitin-mediated proteolysis.

In further support of these observations, 786-O stably reconstituted with wild-type pVHL (WT8) restored the normal profile of hHIF-3alpha 1 (Fig. 6C). Therefore, the presence of pVHL dramatically reduced the level of hHIF-3alpha 1 under normoxia, and accordingly, in the presence of hypoxia mimetics, such as DFO or CoCl2, hHIF-3alpha 1 level was significantly elevated. As a control, 786-O cells stably transfected with empty plasmid alone (RC3) failed to restore the normal profile of hHIF-3alpha 1 (Fig. 6C). Consistent with the in vitro data, these results demonstrate that pVHL binds hHIF-3alpha 1 and destabilizes its expression in vivo.

The hHIF-3alpha locus contains multiple alternatively spliced variants, hHIF-3alpha 1-6. hHIF-3alpha 1-3 contain a common ODD and hence are potential targets of the pVHL tumor suppressor complex VEC for ubiquitin-mediated destruction. This activity requires the beta  domain of pVHL to recognize the ODD that has been post-translationally hydroxylated at the conserved proline residue within the LAPYIXMD motif. This modification requires PHD, which is known to function selectively in the presence of oxygen (5, 6, 12, 13). Thus, pVHL is intricately involved in the regulation of hypoxia-inducible transcription factors hHIF-1, -2 and now -3, whose activities are turned "ON" only under reduced oxygen tension. It will be important to determine whether the splice variants of the hHIF-3alpha locus are involved in the transcriptional regulation of genes not activated by hHIF-1 or -2. Finding LZIP and LXXLL motifs on hHIF-3alpha that are absent on hHIF-1/2alpha supports this notion. The challenge will be to define uncommon DNA/promoter sequences or interacting protein(s) recognized singularly by hHIF-3alpha , which will undoubtedly shed new light into the understanding of mechanisms governing our physiologic response to hypoxia. Moreover, understanding the growing network of interactions between pVHL and hHIF family of transcription factors may help us better understand the genotype-phenotype correlation that exists in VHL disease.

    ACKNOWLEDGEMENTS

We thank the members of the Ohh laboratory for helpful discussions and comments. We also thank Dr. Keiichi Kondo and Sherri K. Leung for technical assistance.

    FOOTNOTES

* This work was supported in part by the National Cancer Institute of Canada and Terry Fox Run Grant 13030.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.

§ Both authors contributed equally to this work.

Recipient of the NSERC scholarship.

§§ Canada Research Chair in Molecular Oncology. To whom correspondence should be addressed: Dept. of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-946-7922; Fax: 416-978-5959; E-mail: michael.ohh@utoronto.ca.

Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M208681200

    ABBREVIATIONS

The abbreviations used are: VHL, von Hippel-Lindau; ODD, oxygen-dependent degradation domain; E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin; LZIP, leucine zipper; PHD, prolyl hydroxylases; HIF, hypoxia-inducible factor; hHIF, human HIF; RCC, renal clear cell carcinoma; MTN, multiple tissue Northern; WT, wild type; IPAS, inhibitory Per/Arnt/Sim; hIPAS, human inhibitory PAS domain protein; mIPAS, murine IPAS; DFO, desferrioxamine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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