From the 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
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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ABSTRACT |
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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 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 pVHL contains two major recognizable domains, Recently, Kondo et al. (24) showed that a variant of
HIF-2 Here, we describe the genomic organization of human HIF-3 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-3 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-3 Northern Blot Analysis--
The hHIF-3 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 hHIF3 In Vitro Ubiquitylation Assay--
An in vitro
ubiquitylation assay was performed as described previously (9).
[35S]Methionine-labeled reticulocyte lysate
HA-HIF3 pVHL tumor suppressor complex VEC targets the ODD within
hHIF-1/2 Genomic Organization and Multiple
Splicing Variants of hHIF-3
Here we describe the genomic structures of six splicing variants of
hHIF-3
The first human HIF-3
Recently, a dominant-negative regulator of mHIF-1 called mIPAS, which
lacks transactivation domains, was identified as an alternatively
spliced variant of mHIF-3
cDNA entries with GenBankTM accession numbers AK021653
and NM_022462 were also found to be derived from the hHIF-3
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-3
Two additional GenBankTM cDNA entries were recently
found to be splicing variants of hHIF-3
It should be noted that Makino and colleagues (34) described previously
several EST sequences that were located on the hHIF-3
To discern the expression profile of hHIF-3 pVHL Binds Oxygen-dependent Degradation Domain of
hHIF-3 pVHL Binds hHIF-3 Ubiquitylation of hHIF-3
To confirm whether the ubiquitylation of hHIF-3
We next asked whether the full-length hHIF-3 Ubiquitylation of hHIF-3 Stability of Endogenous hHIF-3
We next asked whether pVHL affected the expression level of endogenous
hHIF-3
In further support of these observations, 786-O stably reconstituted
with wild-type pVHL (WT8) restored the normal profile of hHIF-3
The hHIF-3 subunits for
polyubiquitylation. This process is dependent on the hydroxylation of
conserved proline residues on the
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-3
locus as follows:
hHIF-3
1, hHIF-3
2 (also referred to as hIPAS; human inhibitory PAS
domain protein), hHIF-3
3, hHIF-3
4, hHIF-3
5, and hHIF-3
6. We
demonstrate that the common oxygen-dependent degradation
domain of hHIF-3
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
subunits for
polyubiquitylation (4-11). Hydroxylation of HIF-1/2
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-1/2
subunits are subsequently captured by
the 26 S proteasome for degradation. Thus, under reduced oxygen
tension or hypoxia, HIF-1/2
subunits remain unhydroxylated and
consequently escape ubiquitin-mediated proteolysis. The HIF-1/2
subunits bind to constitutively stable HIF-
(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).
and
(16). Whereas
the
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
domain functions as a protein-docking site and is
required for binding HIF-1/2
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-1/2
(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-
, 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-1/2
in the
absence of functional pVHL may be causally linked to tumorigenesis in
VHL patients.
, 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-1
(
/
) renal carcinoma cells reconstituted with functional pVHL restored their
ability to form tumors in nude mouse. This result suggests that
down-regulation of HIF-2
is necessary for tumor suppression by pVHL.
However, Maranchie et al. (25) demonstrated that the degradation-resistant variant of HIF-1
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-1
recapitulates the tumorigenic phenotype of pVHL-deficient
cells (25). These observations suggest that the tumor suppressor
function of pVHL, through its interaction with the
subunits of
HIF-1/2, is more complicated than once perceived and/or that VEC E3
ubiquitin ligase may have more substrates than HIF-1/2
.
locus and
report multiple alternatively spliced variants as follows: hHIF-3
1,
hHIF-3
2 (also referred to as hIPAS in the GenBankTM),
hHIF-3
3, hHIF-3
4, hHIF-3
5, and hHIF-3
6. We show that
hHIF-3
1, -3
2, and -3
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.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
antibody was as
described (28).
(FL) was described previously (28). pcDNA3-HA-hHIF-3
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-3
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.
probe for the human
multiple tissue Northern (MTN) blot (Clontech) was
prepared by amplifying a fragment (nucleotides 273-761) of
hHIF-3
1 by PCR using 5'-GGAGGGCTTCGTCATGGT-3' and 5'-TCGTCACAGTAGGTGAACTTC- ATG-3' primers. The amplified
fragment was then labeled with [
-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-3
bands
were visualized by autoradiography.
-Actin was subsequently probed
as a loading control.
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-3
[FL] and
-[ODD]) of [35S]methionine. hHIF3
translation
products were treated with or without cellular fractions containing
enriched prolyl hydroxylase for 30 min at 37 °C. HA-HIF-3
(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.
[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.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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.
--
Fig. 1 and Table
I summarize our data base search
uncovering multiple alternatively spliced variants of human HIF-3
(hHIF-3
). The third
-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-3
(33, 34). The human
homologue of the mHIF-3
was isolated and revealed high homology to
hHIF-1 and -2
(28). The search of the GenBankTM data
base with hHIF-3
cDNA (GenBankTM accession numbers
NM_152794 and AB054067) revealed that hHIF-3
has at least six
alternatively splicing variants. In fact, the genomic
organization of hHIF-3
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-3
splicing
variants, a detailed intron-exon structure of the hHIF-3
locus has
not been reported previously.
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Fig. 1.
Multiple spliced variants of the human
HIF-3 locus. A, genomic
organization of the hHIF-3
alternatively spliced variants.
Alternatively spliced isoforms of the hHIF-3
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-1/2/3
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-1/2/3
and
mHIF-3
. hHIF-3
[ODD] is represented by hHIF-3
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-3
. Human MTN blot was hybridized with
32P-labeled 489-bp region within exons 3-6 of hHIF-3
1,
and hybridized bands were detected by autoradiography. The
arrows indicate multiple hHIF-3
probe-specific RNAs
(upper panel).
-Actin probe was subsequently hybridized
to the blot (lower panel) as internal loading control.
Exon-intron structure of hHIF-3 gene
1 (GenBankTM accession numbers
NM_152794/AB054067) begins at exon 1c and ends at exon 16. hHIF-3
2/IPAS (accession numbers NM_152795/AF463492) starts from exon
1a and ends at exon 13a. hHIF-3
3 (accession numbers
NM_022462/AK021653) starts at exon 1b and ends at exon 17 without exons
15 and 16. hHIF-3
4 (accession numbers BC026308) begins at exon 1a
and ends within imperfect intron 8. hHIF-3
5 (accession numbers
NM_152796) begins at exon 1b and ends at exon 15 without exons 3, 12, and 13. hHIF-3
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.
locus. Similar to mHIF-3
, hHIF-3
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).
was reported in 2001 (28). Thus, we have
designated it hHIF-3
1. As shown in Fig. 1A, hHIF-3
1
begins at exon 1c and ends at exon 16, which is absent in all other
splicing variants (Fig. 1A). hHIF-3
1 encodes a 668-amino
acid protein that contains a single N-terminal transactivation domain
(NAD) like hHIF-1 and -2
, but it does not contain a C-terminal
transactivation domain (CAD) (Fig. 1B). Interestingly,
hHIF-3
1 contains a LZIP domain composed of four-septad leucines not
found on hHIF-1/2
or on any other hHIF-3
splicing variants (Fig.
1B). LZIP domains mediate both DNA-binding and
protein-protein interactions (35). Another distinguishing
characteristic of hHIF-3
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 -2
subunits, suggesting that hHIF-3
1 may
bind DNA/promoter sequences or novel interacting protein(s) uncommon to
those recognized by hHIF-1/2
.
(see Fig. 1B) (33, 34). mIPAS
dimerizes with the mHIF-1
subunit and consequently prevents the
interaction of mHIF-1
to mHIF-
subunits (33). Furthermore, the
mIPAS/mHIF-1
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-3
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-3
2. hHIF-3
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-3
1
(Fig. 1B).
locus. We refer to it as hHIF-3
3, which begins at exon 1b and ends at exon
17 without exons 1a, 1c, 15, and 16 (Fig. 1A and Table I). hHIF-3
3 utilizes the shorter exon 13b and 14b instead of 13a and
14a. Although hHIF-3
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).
splice variants (Fig. 1A and Table I). The intron 7 was not spliced. We
refer to it as hHIF-3
4 that encodes a 363-amino acid protein. Amino acid sequence alignment suggests that hHIF-3
4 is most similar to
mIPAS (Fig. 1B). Both proteins lack a NAD and CAD, as well as ODD, suggesting that, like mIPAS, hHIF-3
4 could potentially act
as a dominant-negative regulator of hHIF activity. Furthermore, with
respect to the other hHIF-3
variants, hHIF-3
4 does not contain
LXXLL or LZIP structures.
. We have named them
hHIF-3
5 (GenBankTM accession number NM_152796) and
hHIF-3
6 (GenBankTM accession number AK024095). Both
isoforms start at exon 1b and lack exon 3. hHIF-3
5 contains a short
exon 14c and ends at exon 15. hHIF-3
5 encodes a protein containing a
partial PASa, PASb, and PAC domains. hHIF-3
6, like hHIF-3
4,
contains intron 7 and ends at intron 8. hHIF-3
6 contains only a
partial C-terminal domain of PASb.
locus. ESTs
with GenBankTM accession numbers BG699633, AL528423, and
AL519496 are largely identical to hHIF-3
2, whereas BQ067192 and
AL535689 are fragments of hHIF-3
3 and hHIF-3
5, respectively.
However, EST entry with accession number BM119659 is derived from mouse.
, 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-3
1, which also overlaps on hHIF-3
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 -2
revealed single RNA species
(37, 38). Based on the primary sequences of hHIF-3
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-3
-like transcripts. Hence, the hHIF-3
locus likely generates multiple alternatively spliced variants.
--
hHIF-3
1, -3
2, and -3
3 splice variants share a
common ODD (Fig. 1B). The alignment of hHIF-1/2/3
and
mHIF-3
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-1/2
(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-3
[ODD] is capable of binding pVHL and
whether prolyl hydroxylation is required for pVHL/hHIF-3
[ODD]
interaction, we generated hHIF-3
[ODD; WT] and hHIF-3
[ODD;
P490A] mutant fused in-frame with an N-terminal hemagglutinin (HA)
epitope tag. HA-hHIF-3
[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-3
[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-3
[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-3
[ODD; WT]. However, hHIF-3
[ODD; P490A] bound to pVHL
weakly even in the presence of PHD (Fig. 2A). These results
were similar to that of pVHL/hHIF-1
[ODD; WT and P564A] control
interactions (Fig. 2A) (5) and suggest that pVHL binds
hHIF-3
[ODD] dependent on hydroxylation of the conserved proline
residue. Recovered amounts of the hHIF-3
[ODD; WT and P490A] and
hHIF-1
[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-3 [ODD] is enhanced by PHD and is
dependent on P490. A, in vitro
translates of HA-hHIF-3
[ODD; WT], HA-hHIF-3
[ODD;
P490A], HA-hHIF-1
[ODD; WT], and HA-hHIF-1
[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 3
[ODD] was separated on SDS-PAGE and
visualized by autoradiography. B, equal amounts of in
vitro translated HA-hHIF-3
[ODD; WT or P490A] and Gal4-HA-
hHIF-1
[ODD; WT or P564A] used in the binding assay were
immunoprecipitated and immunoblotted with anti-HA antibody.
IP, immunoprecipitation; IB, immunoblot;
AR, autoradiography.
via the
Domain--
pVHL is composed of
two domains,
and
(16). The
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
domain is a substrate-docking interface that is
required for binding prolyl-hydroxylated hHIF-1/2
[ODD] (5, 9, 16).
To determine which domain of pVHL was required for binding hHIF-3
,
35S-labeled full-length pVHL-(1-213),
pVHL-(155-213), which encompasses most of the
domain, and
pVHL-(63-154), which encompasses most of the
domain, were mixed
with either T7-hHIF-3
[ODD] or full-length hHIF-3
1 that had been
pre-treated with PHD. Both hHIF-3
[ODD] and full-length hHIF-3
1
bound to pVHL-(1-213) and pVHL-(63-154) (Fig.
3A). However, pVHL-(155-213)
did not associate with hHIF-3
[ODD] or full-length hHIF-3
1 (Fig.
3A). Recovered amounts of hHIF-3
[ODD] and hHIF-3
1
were comparable as determined by anti-T7 and anti-hHIF-3
1 immunoblotting, respectively (Fig. 3B). We next examined the
binding capabilities of two tumor-derived pVHL mutants, a
representative
domain mutant Y98H and a representative
domain
mutant Q164R, to hHIF-3
[ODD]. The
domain pVHL mutant Y98H
bound poorly to hHIF-3
[ODD], whereas Q164R effectively bound
hHIF-3
[ODD] (Fig. 3C). These results taken together
demonstrate that pVHL binds hHIF-3
[ODD] via its
domain. It
follows then that tumor-derived mutations affecting the surface
residues in the
domain effectively abrogate the interaction of pVHL
to not only hHIF-1 and -2
subunits but also hHIF-3
1-3 variants.
Furthermore, binding of hHIF-3
[ODD] to the
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
domain in the
absence of the rest of pVHL residues.
View larger version (29K):
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Fig. 3.
hHIF-3 [ODD] and
the full-length hHIF-3
1 bind to the
domain of pVHL. A, in
vitro translates of T7-hHIF-3
[ODD] and
hHIF-3
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-3
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-3
[ODD] and hHIF-3
1 used in the binding assay were
immunoprecipitated (IP) and immunoblotted (IB)
with the indicated antibodies. C, in vitro
translates of T7-hHIF-3
[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).
Is Dependent on pVHL and
Oxygen--
hHIF-1 and -2
are degraded via
VEC-dependent, oxygen-dependent
ubiquitin-proteasome pathway (5-7, 9, 16). To determine whether pVHL
ubiquitylates hHIF-3
[ODD], we performed an in vitro ubiquitylation assay. There was efficient poly-ubiquitylation of
hHIF-3
[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-3
[ODD; WT] (Fig. 4A). This suggests that pVHL is
critical for targeting hHIF-3
[ODD] for ubiquitylation.
Furthermore, the addition of PHD moderately enhanced the ubiquitylation
of hHIF-3
[ODD; WT] (Fig. 4A). This is likely due to the
increased prolyl hydroxylation of hHIF-3
[ODD; WT] in the presence
of dioxygen, which enhances its binding to pVHL (see Fig.
2A). In support, hHIF-3
[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-3
[ODD; P490A], likely representing mono-ubiquitylated species of hHIF-3
[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-3
[ODD], it
does not appear to be required for mono-ubiquitylation.
View larger version (38K):
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Fig. 4.
Ubiquitylation of
hHIF-3 [ODD] and
hHIF-3
1[full-length] is dependent on pVHL
and is blocked by hypoxia mimetic. A, in
vitro ubiquitylation of 35S-labeled HA-hHIF-3
[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-3
[ODD]
(B) or 35S-labeled hHIF-3
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-3
1 (C) antibody. Bound proteins were then
separated on SDS-PAGE and visualized by autoradiography.
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-3
[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-3
[ODD] is targeted for ubiquitylation dependent on pVHL and oxygen.
1 was likewise targeted
for pVHL-dependent ubiquitylation. Like hHIF-3
[ODD], the polyubiquitylation of hHIF-3
1 occurred only in S100 extracts that contained pVHL (Fig. 4C, compare lanes 1 and
2). However, full-length hHIF-3
1 was ubiquitylated less
robustly than hHIF-3
[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 -2
(data not shown and Ref. 9). Treatment with
CoCl2 dramatically inhibited the ubiquitylation of
hHIF-3
1 (Fig. 4C). Thus, the targeting of full-length
hHIF-3
1 for ubiquitylation is most likely pVHL- and
oxygen-dependent. Whether other full-length hHIF-3
variants that contain a recognizable ODD, such as hHIF-3
2 and
hHIF-3
3, are also targeted for pVHL/oxygen-dependent
ubiquitylation or whether all hHIF-3
subunits containing the ODD
bind to pVHL with equal affinity remains to be resolved.
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-3
[ODD]. To determine which lysine residue(s)
within the ODD of hHIF-3
was required for ubiquitin conjugation via
VEC, we generated hHIF-3
[ODD; K465R], hHIF-3
[ODD; K568R], and
hHIF-3
[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-3
[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.
View larger version (23K):
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Fig. 5.
pVHL-dependent ubiquitin
conjugation on hHIF-3 [ODD] occurs on lysine
465 and lysine 568. In vitro ubiquitylation of
35S-labeled HA-hHIF-3
[ODD; WT], HA-hHIF-3
[ODD;
K465R], HA-hHIF-3
[ODD; K568R], and HA-hHIF-3
[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.
1 Is Regulated by pVHL in
Vivo--
To address whether the stability of endogenous hHIF-3
1 is
affected by pVHL in vivo, we first asked whether pVHL binds
hHIF-3
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-hHIF3
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-3
1 (Fig. 6A, lane 2). It
should be noted that although the expression level of hHIF-3
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-3
1 (data not shown). This is likely
due to the lack of prolyl hydroxylation of hHIF-3
1 in the presence
of hypoxia mimetics, which renders it unrecognizable by pVHL. It should
also be noted that the hHIF-3
1 antiserum was raised against residues
564-583 of hHIF-3
1 (28). This region of amino acids is also
present on hHIF-3
2 and -3
2. Thus, in the absence of specific
antibodies against each of the hHIF-3
splicing variants, it is
formally possible that pVHL interacts in vivo with one or
more of hHIF-3
1, -3
2, and -3
3.
View larger version (22K):
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Fig. 6.
A, endogenous interaction
between pVHL and hHIF-3 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-hHIF3
1 antibody.
Bound proteins were separated by SDS-PAGE, and anti-hHIF3
1
(upper panel) and anti-pVHL (lower panel)
immunoblots were performed. Asterisk represents a protein
yet unidentified recognized by anti-hHIF-3
1 antibody. B,
regulation of endogenous hHIF-3
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-hHIF3
1 antibody. Bound
proteins were separated by SDS-PAGE and visualized by anti-hHIF3
1
immunoblot (IB). Endogenous expression of pVHL (
or +) in
each of the cell line is indicated on the top. In
vitro translated hHIF-3
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-hHIF3
1
(top and middle panels) or anti-HA (bottom
panel) antibody. Bound proteins were separated by SDS-PAGE and
detected by anti-hHIF3
1 or anti-HA immunoblot, respectively.
1 in vivo. HCT116 colon carcinoma, PC-3 prostate carcinoma, and U2OS osteosarcoma cells that express endogenous pVHL
showed dramatically reduced levels of hHIF-3
1 (Fig. 6B). However, these cells when treated with the hypoxia mimetic
CoCl2 (or DFO; data not shown) markedly accumulated
hHIF-3
1 (Fig. 6B). Concordantly, 786-O RCC cells devoid
of functional pVHL showed elevated expression of hHIF-3
1
irrespective of hypoxia mimetics (Fig. 6B). These results
taken together demonstrate that endogenous pVHL and hHIF-3
1 interact
in vivo, and under normal oxygen tension, VEC targets
hHIF-3
1 for ubiquitin-mediated proteolysis.
1
(Fig. 6C). Therefore, the presence of pVHL dramatically reduced the level of hHIF-3
1 under normoxia, and accordingly, in the
presence of hypoxia mimetics, such as DFO or CoCl2,
hHIF-3
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-3
1 (Fig. 6C). Consistent with the
in vitro data, these results demonstrate that pVHL binds
hHIF-3
1 and destabilizes its expression in vivo.
locus contains multiple alternatively spliced variants,
hHIF-3
1-6. hHIF-3
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
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-3
locus are involved in the transcriptional regulation of genes not activated by hHIF-1 or -2. Finding LZIP and LXXLL motifs
on hHIF-3
that are absent on hHIF-1/2
supports this notion. The challenge will be to define uncommon DNA/promoter sequences or interacting protein(s) recognized singularly by hHIF-3
, 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.
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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
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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.
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