From the Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden
Received for publication, September 11, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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Under normoxic conditions
the hypoxia-inducible factor-1 Mammalian organisms are able to adapt to low oxygen levels by
activating a network of genes encoding erythropoietin, vascular endothelial growth factor, and glycolytic enzymes (1).
Hypoxia-dependent activation of transcription is mediated
by the heterodimeric complex of the hypoxia-inducible factor-1 HIF-1 Plasmid Constructs--
pFLAG-GAL4/mHIF-1 Cell Culture and Transient Transfection Experiments--
Human
embryonic kidney (HEK) 293 cells were maintained in Dulbecco's
modified Eagle's medium (DMEM) and Ham's F-12 medium in a 1:1 ratio
containing 10% fetal calf serum, 50 IU/ml penicillin, and 50 mg/ml
streptomycin sulfate. Medium and other products for cell culture were
purchased from Invitrogen. HEK 293 cells were transfected using
LipofectAMINE (Invitrogen) following the instructions of the
manufacturer. Cells were cultured 24 h before transfection in 6-well plates, and cells incubated after transfection were cultured
at normoxia (21% O2) or hypoxia (1% O2) for
different times, as detailed in the figure legends. For reporter gene
assays the cells were harvested 36 h after transfection, and the
luciferase activity was determined. Total protein concentration was
analyzed in whole cell extracts using the Bradford method
(Bio-Rad).
Immunoprecipitation Assays--
In vitro
immunoprecipitation assays were performed with proteins translated in
rabbit reticulocyte lysate (Promega) either in the presence or absence
of [35S]methionine. Translated proteins were incubated
for 1 h at room temperature in lysis buffer in a volume of 100 µl (17) and added to 20 µl of protein G-Sepharose (Amersham
Biosciences) preincubated with anti-FLAG-M2 (Sigma) antibodies. After a
further incubation for 1.5 h at room temperature under rotation,
the Sepharose pellet was washed three times with lysis buffer, and the
precipitated proteins were analyzed by SDS-PAGE followed by autoradiography.
In in vivo immunoprecipitation assays transfected cells were
used to prepare whole cell extracts by sonication of cells for 5 s
(performed twice) in lysis buffer (17). Whole cell extracts (800 µg
to 1 mg of total protein) were incubated at 4 °C for 16 h with
anti-FLAG-M2 antibodies bound to protein G-Sepharose as described
previously (36). The Sepharose pellet was washed three times with TBS
buffer (150 mM NaCl and 50 mM Tris-HCl, pH
4.0), and precipitated proteins were eluted from Sepharose by
incubation under rotation with 0.5 mg/ml FLAG peptide (Sigma) in
TBS for 1.5 h at room temperature.
Immunoblotting Assays--
Immunoprecipitated proteins or 50 µg of whole cell extract protein were separated by SDS-PAGE and
blotted onto nitrocellulose filters. Blocking was performed in TBS with
5% non-fat milk and was followed by incubation for 1 h at room
temperature with anti-FLAG or anti-VHL (Pharmingen) antibodies diluted
1:500 and 1:250, respectively, in TBS with 5 and 1% non-fat milk,
respectively. After several washes with TBS containing 0.5% Tween 20, the filters were incubated with 1:1000 dilutions of anti-mouse
IgG-horseradish peroxidase conjugate (Amersham Biosciences) in TBS with
1% non-fat milk and washed several times with TBS containing 0.5%
Tween 20. Proteins were visualized using enhanced chemiluminescence
(Amersham Biosciences).
In Vitro Binding of pVHL to the N-TAD Degradation Box Depends on
the Integrity of the PYI Motif and on Hydrophobic and Acidic Residues
Surrounding the PYI Sequence--
We have previously identified the
N-TAD as an interaction interface with pVHL and have identified the PYI
motif (residues 563-565 in mHIF-1 pVHL Interacts with N-TAD under Conditions of
Reoxygenation--
We and others (17, 19, 23) have observed previously
that pVHL is able to interact with hHIF-1 Identification of Critical Residues for in Vivo Interaction between
N-TAD and pVHL, Binding of pVHL Depends on the PYI Motif and the
Integrity of Residues
Asp568-Asp569-Asp570 and
Phe571-Leu573--
In order to know if the
mutants that disrupt the interaction between the N-TAD and pVHL
in vitro are also critical for the binding in
vivo, we performed immunoprecipitation assays using transfected
cells expressing FLAG-GAL4-(531-584) mutants and pVHL. Abrogation of
the interaction between the N-TAD and pVHL was observed in the case of
the mutant P563D/Y564D/I565G (lanes 5 and 6) and the corresponding point mutants P563A (lanes 23 and
24) and Y564G (lanes 13 and 14) (Fig.
1D). Notably, the hydrophobicity of Tyr564 plays
an essential role in the binding to pVHL. Mutation of tyrosine to
glycine completely disrupted binding of pVHL (Fig. 1D). On the other hand, phosphorylation of Tyr564 does not seem to
be relevant for pVHL interaction because the tyrosine residue could be
replaced by phenylalanine without affecting the pVHL binding activity
(data not shown). Mutation of residue Ile565 drastically
reduced the interaction with pVHL but did not completely inhibit
binding activity (lanes 15 and 16). Moreover, the
two HIF-1 N-TAD Mutants That Fail to Bind pVHL Are Resistant to pVHL-mediated
Protein Degradation--
To evaluate the effect of different point
mutations introduced into the N-TAD on the ability of pVHL to mediate
protein degradation at normoxia (21% O2), we transiently
expressed in HEK 293 cells pFLAG-GAL4-mHIF-1 Reoxygenation-dependent Degradation of the N-TAD Is
Mediated by pVHL--
Because we performed our in vivo
binding assays with cells exposed to normoxia or to conditions of
reoxygenation, we also compared the efficiency of pVHL-mediated
degradation under these two conditions (Fig. 2C).
Degradation of the wild type N-TAD motif was observed both at normoxia
and in reoxygenated cells in a manner that was strictly dependent on
the concentration of pVHL. In contrast, the N-TAD P563A mutant did not
show any differences in protein levels between cells exposed to 21 or
1% O2 in the presence of increasing concentrations of pVHL
(Fig. 2C). In the case of the wild type N-TAD motif, pVHL
was significantly more potent in mediating protein degradation under
conditions of reoxygenation, as compared with normoxic cells,
consistent with the notion that pVHL may have a higher affinity for the
N-TAD in the reoxygenated cells (25).
Identification of Residues Important for pVHL Binding Located at
the C Terminus of the PYI Motif--
The mutants D568A/D569A/D570A and
F571A/L573A failed to interact with pVHL in vitro and
in vivo and were resistant to pVHL-mediated degradation at
normoxia (Fig. 1, B and C, and Fig.
2A). Because these are triple and double mutants, we also
generated single amino acid point mutants of all these residues in a
shorter protein fragment of the NTAD spanning residues 546-574
(pFLAG-GAL4-mHIF-1 The P563A Mutant of N-TAD Functions as a Constitutively Active
Transactivation Domain--
The HIF-1
The mutants P563D/Y564D/I565D, Y564G, I565G, D570A, and F571A/573A all
showed similarly low values of transactivation both at normoxia and
hypoxia (P563D/Y564D/I565D, 4.8- and 5-fold activation; Y564G, 3- and
3.5-fold activation; I565G, 6.9- and 6.4-fold activation; D570A, 5.6- and 6.8-fold activation; F571A/L573A, 4.3- and 4.1-fold activation at
normoxia and hypoxia, respectively). In contrast to the Y564G mutant,
mutation of Tyr564 to phenylalanine within the N-TAD
resulted in transactivation levels similar to those produced by the
wild type HIF-1 Activation of Transcription Mediated by P563A Is Enhanced by
Coexpression of CBP and Correlates with the Ability of P563A to Bind
CBP--
The mutation of Pro563 to alanine generated a
constitutive transactivation domain that was significantly more potent
in activation of transcription than the wild type N-TAD in hypoxia. In
addition, in contrast to the wild type N-TAD fragment, this mutant
showed constitutive functional activity. We therefore investigated if the transactivation mediated by the N-TAD P563A mutant could still be
potentiated by coexpression of CBP in a manner similar to that of the
wild type N-TAD. As shown in Fig. 4B, transient
cotransfection of CBP moderately (about 1.7-fold) potentiated
transactivation mediated by the N-TAD both at normoxia and hypoxia. CBP
enhanced to a similar degree transcription activation mediated by the
P563A mutant of N-TAD, indicating that CBP is able to interact
functionally with both the N-TAD and P563A mutant.
Mutations That Negatively Affect Activation of Transcription
Mediated by the N-TAD Decrease Transactivation Mediated by Full-length
HIF-1
The three mutants that showed reduced transactivation activity are
expressed at levels similar to the wild type protein. Mutation of
residue Tyr564 to glycine generates a much weaker
transactivator with a 60% reduction of transcription activation at
normoxia and hypoxia, whereas mHIF-1 In the present study we have characterized by mutation analysis
the bifunctional N-TAD which mediates both transactivation and
conditionally regulated protein degradation. We have identified P563A,
Y564G, I565G, D568A/D569A/D570A, F571A, and L573A as mutations that
interfere with the in vivo binding of pVHL and pVHL-mediated degradation of the N-TAD at normoxia. Furthermore, we have identified the residues Met556-Leu558 and
Asp570 as critical for in vitro binding of pVHL
using proteins expressed in rabbit reticulocyte lysate. Binding of pVHL
to the N-TAD has been shown to be dependent on the hydroxylation status
of Pro564 (21-23). This modification is
oxygen-dependent and is mediated by a number of recently
identified prolyl 4-hydroxylases (25, 26). Two independently elucidated
crystal structures of the pVHL-BC complex with a peptide spanning the
PYI core of the N-TAD (34, 35) have recently demonstrated that
hydroxyproline 563 of the N-TAD is the residue that establishes more
contacts with pVHL in a site that is a hotspot for tumorigenic
mutations. As expected (21-23), mutation of Pro563 to
alanine completely disrupted binding of pVHL both in vitro and in vivo. In addition, our results demonstrated that the
P563A mutation generated a constitutively active and more potent
transactivation domain as compared with the wild type N-TAD. Thus, in
contrast to the C-TAD (13), there is no requirement of any additional hypoxic signal in order for the constitutively stabilized N-TAD to
transactivate. All the other N-TAD mutations that abrogate pVHL binding
negatively affected the transactivation function mediated by the N-TAD
demonstrating overlapping structural requirements for both pVHL binding
and functional interaction with the transcription machinery, and
establishing that only the P563A mutation conferred a conformation
favorable for the transactivation function.
We have demonstrated previously (36) that inactivation of the C-TAD in
the full-length HIF-1 In our studies we have identified two mutants L556A/L558A and
M560A/L561A that impaired transactivation mediated by N-TAD without
affecting pVHL binding in vivo. The residue
Leu559 in hHIF-1 In addition to the P563A mutation, two other point mutants of the PYI
motif, Y564G and I565G, also proved to be critical for pVHL binding and
pVHL-mediated degradation. In transactivation assays these two mutants
generate a weak and constitutively active transactivation response.
Interestingly, the introduction of the Y564G mutation into the
full-length mHIF-1 We also mutated several residues located in the C terminus of the PYI
motif, and we observed that mutants D568A/D569A/D570A, F571A, and L573A
were critical for the in vivo interaction with the pVHL. The
D568A/D569A/D570A triple mutant was resistant to pVHL-mediated
degradation. However, in the subsequent analysis of single amino acid
point mutants only the mutation D570A resulted in disruption of the
binding in vitro, whereas it was still degraded in
vivo at normoxia. This residue has been shown in the structural analysis to interact with Arg107 of pVHL through hydrogen
bonds (34). However, our experiments suggest that this interaction may
not be critical for function in vivo. In contrast, the
mutations F571A and L573A stabilized the N-TAD against degradation by
pVHL at normoxia. In the crystal structure of the N-TAD-peptide-pVHL-BC
complex, these residues were shown to interact with pVHL by van der
Waals contacts (34). Phe572 in hHIF-1 (HIF-1
) protein is targeted for
degradation by the von Hippel-Lindau (pVHL) tumor suppressor protein
acting as an E3 ubiquitin ligase. Binding of pVHL to HIF-1
is
dependent on hydroxylation of specific proline residues by
O2-dependent prolyl 4-hydroxylases. Upon
exposure to hypoxia the hydroxylase activity is inhibited, resulting in stabilization of HIF-1
protein levels and activation of
transcription of target genes. One of the two critical proline
residues, Pro563 in mouse HIF-1
, is located within a
bifunctional domain, the N-terminal transactivation domain
(N-TAD), which mediates both pVHL-dependent degradation at
normoxia and transcriptional activation at hypoxia. Here we have
identified two N-TAD residues, Tyr564 and
Ile565, which, in addition to Pro563, were
critical for pVHL-mediated degradation at normoxia. We have also
identified D568A/D569A/D570A, F571A, and L573A as mutations of the
N-TAD that abrogated binding to pVHL both in vitro and in vivo, and constitutively stabilized N-TAD against
degradation. Moreover, the mutations Y564G, L556A/L558A, and
F571A/L573A drastically reduced the transactivation function of either
the isolated N-TAD or full-length HIF-1
in hypoxic cells.
Interestingly, the P563A mutant exhibited a constitutively active and
potent transactivation function that was enhanced by functional
interaction with the transcriptional coactivator protein CREB-binding
protein. In conclusion, we have identified by mutation analysis several
residues that are critical for either one or both of the interdigitated
and conditionally regulated degradation and transactivation functions of the N-TAD of HIF-1
.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(HIF-1
)1 with the
structurally related partner factor Arnt. In contrast to Arnt, HIF-1
protein expression is regulated in response to alterations in cellular
oxygen levels. At normoxia, HIF-1
is degraded by the
ubiquitin-proteosomal pathway (2-5), whereas upon hypoxic treatment
the protein is stabilized, translocates to the nucleus, and activates
transcription of target genes (6). HIF-1
contains two distinct
transactivation domains that mediate hypoxia-dependent
activation of transcription, the N-TAD (residues 531-584 and 532-585
in mHIF-1
and hHIF-1
, respectively) and the C-TAD (residues
772-822 of mouse HIF-1
corresponding to residues 776-826 in
hHIF-1
) (7-11). The transactivation function mediated by the N- and
C-TAD motifs has been shown to be enhanced by cofactors such as CBP,
SRC-1, and Ref-1 (9, 10). However, the mechanism of regulation of these
two TADs is quite distinct because N-TAD protein stability is strictly
regulated by oxygen levels, whereas the C-TAD is constitutively stable
(9, 12). C-TAD has been recently shown to be hydroxylated at
Asn803 (of hHIF-1
) by a
Fe(II)-2-oxoglutarate-dependent dioxygenase (13-15), and
this modification has been proposed to inhibit the binding to the
cysteine/histidine-rich domain 1 of CBP at normoxia.
is targeted for normoxia-dependent ubiquitylation
by the von Hippel-Lindau tumor suppressor gene product (pVHL) (16-20). The binding of pVHL to HIF-1
is regulated by hydroxylation of Pro402 and Pro564 which is mediated by members
of the Fe(II)-2-oxoglutarate-dependent dioxygenase family
of enzymes (21-26). The VHL tumor suppressor gene was first identified
as the gene responsible for a rare inherited autosomal dominant cancer
syndrome characterized by the development of clear cell renal
carcinoma, hemangioblastoma, and pheochromocytoma (27). In addition,
the VHL gene is also inactivated in sporadic clear cell renal
carcinoma. VHL-negative neoplasms are characterized by being
hypervascularized and by expressing constitutively hypoxia-inducible mRNAs such as vascular endothelial growth factor (28, 29). pVHL has
been shown to harbor an E3 ubiquitin ligase activity in
vitro (30, 31) and shows structural similarity to the
SKP1-CUL-1-F-box E3 ubiquitin ligase complex (32). HIF-1
and
its paralogues HIF-2
and HIF-3
are so far the only known
substrates recognized by the pVHL E3 ubiquitin ligase complex
(19). The
domain of pVHL has been shown to interact with the N-TAD
of HIF-1
, resulting in ubiquitylation and
proteosome-dependent degradation of HIF-1
at normoxia
(17-20, 33). The crystal structure of a N-TAD peptide with the
pVHL-ElonginB-ElonginC (pVHL-BC) complex has been solved by two
independent groups (34, 35) that observed that the hydroxyproline is
buried in a hydrophobic pocket of pVHL and has a central role in the
complex formation. We have identified previously a central
563PYI565 motif within the N-TAD that is
critical for binding of pVHL and conditional degradation of the N-TAD.
In the present work we have identified further residues within the
N-TAD that are important for binding of pVHL. Amino acid mutations that
inhibit interaction with pVHL and subsequent degradation at normoxia
include P563A, Y564G, I565G, D568A/D569A/D570A, F571A, and L573A.
Moreover, reporter gene assays demonstrated that the P563A mutation
generates a more potent and constitutively active transactivation
domain, as compared with the wild type N-TAD. We have also shown that
mutations that decrease N-TAD transactivation affect the activation of
transcription mediated by the full-length HIF-1
.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(531-584) and
pFLAG-GAL4/mHIF-1
-(546-574) were constructed by using PCR fragments
generated with primer pairs carrying EcoRI and
BamHI ends. The PCR fragments were inserted in-frame into
EcoRI-BamHI-restricted pFLAG-GAL4 (36).
pSP72-FLAG-GAL4/mHIF-1
-(531-584) and
pSP72-FLAG-GAL4/mHIF-1
-(546-574) were generated by inserting SacI-SmaI fragments from the corresponding
pFLAG-GAL4 construct into SacI-EcoRV-digested
pSP72 (Promega). pRc/RSV-CBP-HA (expressing full-length mouse CBP) was
obtained from R. H. Goodman (Vollum Institute, Portland, OR).
pT81/HRE-luc, GAL4-driven luciferase reporter gene plasmid, pCMX-VHL,
pFLAG-mHIF1
, pFLAG-GAL4, and pFLAG-GAL4-mHIF1
-(772-822) have
been described previously (10, 17, 36). The inserts generated by PCR
were completely sequenced using the Dynamic sequencing kit (Amersham
Biosciences). Amino acid mutations were introduced using the
QuickChange site-directed mutagenesis kit (Stratagene) according to the
instructions of the manufacturer, and positive mutants were screened by sequencing.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) as an essential motif for this
interaction (17). In the present study we have generated point mutants
of each single residue of the PYI motif and constructed double and triple mutants of the residues located N- or C-terminally of this motif
(schematically represented in Fig.
1A). Interaction of N-TAD (FLAG-GAL4/mHIF-1
-(531-584)) and mutants with pVHL was investigated using proteins in vitro translated in rabbit reticulocyte
lysate. In these assays pVHL was [35S]methionine-labeled
and precipitated by the FLAG-GAL4/mHIF-1
fusion proteins. As shown
in Fig. 1B two of the N-TAD mutants, L556A/L558A (lane
4) and Q572A/R574A (lane 13), maintained wild type
levels of pVHL binding activity, whereas P566E/M567E (lane 10) demonstrated reduced but detectable levels of pVHL binding activity. In contrast, all the point mutants of the PYI motif, P463A
(lane 7), Y564G (lane 8), and I565G (lane
9) did not show any in vitro interaction with pVHL.
Three additional N-TAD mutations, M560A/L561A (lane 5),
D568A/D569A/D570A (lane 11), and F571A/L573A (lane
12) also completely abrogated binding of the N-TAD to pVHL.
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Fig. 1.
Identification of mHIF-1
N-TAD residues critical for pVHL interaction in vitro
and in vivo. A, schematic
representation of the functional architecture of mHIF-1
and
generated N-TAD mutants. The asterisks indicate mutated
residues. B, in vitro interaction of N-TAD mutants with
pVHL. pSP72-FLAG-GAL4-(531-584) (N-TAD) and mutants thereof
were expressed using in vitro translation in rabbit
reticulocyte lysate. Equal concentrations of in vitro
translated proteins were incubated with in vitro translated
35S-labeled pVHL. Immunoprecipitation assays were performed
using anti-FLAG antibodies (
-FLAG-IP) and analyzed by
SDS-PAGE and autoradiography. C, analysis of the
binding of pVHL to the N-TAD at normoxia (N) or under
conditions of reoxygenation (R). HEK 293 cells were
transfected with 800 ng of pFLAG-GAL4/mHIF-1
-(531-584) and 200 ng
of pCMX-VHL. After transfection the cells were allowed to grow for
24 h and were exposed to normoxia or hypoxia for 12 h before
reoxygenation. Cells exposed to hypoxia were harvested after different
times of reoxygenation (1 min, R1; 10 min; R10;
60 min, R60). In indicated cases the cells were treated with
1 mM MG132 (MG) during the last 12 h of
incubation. Immunoprecipitation assays were performed using the
anti-FLAG antibodies (
-FLAG-IP), and precipitated
proteins were detected by immunoblotting with anti-FLAG or anti-VHL
antibodies (
-FLAG and
-VHL). Input material
was analyzed by SDS-PAGE of 50 µg of whole cell extract protein and
immunoblotting. D, in vivo interaction of wild
type or mutant N-TAD proteins with pVHL. HEK 293 cells were transfected
with pVHL expression plasmid, and pFLAG-GAL4/mHIF-1
-(531-584)
containing the wild type sequence (N-TAD) or the indicated
mutations, pFLAG-GAL4/mHIF-1
-(546-574) (546-574), or
pFLAG-GAL4/mHIF-1
-(772-822) (C-TAD). Transfections and
immunoprecipitations were performed as described in the legend of
C. The cells were exposed to normoxia (N) or
hypoxia for 12 h and 10 min of reoxygenation (R) before
harvesting. LL-A, L556A/L558A; ML-A, M559A/L570A;
PYI-D, P563D/Y564D/I565D; PM-E, P566E/M567E;
DDD-A, D568A/D569A/D570A; FL-A, F571A/L573A;
QR-A, Q572A/R574A.
in assays using whole cell
extracts from cells expressing both proteins (hereafter referred as
in vivo interaction) under conditions where
proteosome-mediated degradation has been inhibited. This interaction is
inhibited when cells are exposed to hypoxia or hypoxia-mimicking agents (21-23). In the present report we have examined if binding of pVHL to
the N-TAD can be observed without the use of proteosome inhibitors. To
this end we used experimental conditions where overexpression of N-TAD
will saturate the endogenous degradation machinery (17) (Fig.
1C). HEK 293 cells were transfected with
pFLAG-GAL4/mHIF-1
-(531-584) and VHL expression plasmid, and the
cells were exposed to normoxia (21% O2) or hypoxia (1%
O2) for 16 h followed by different times of
reoxygenation. As shown in Fig. 1C strong binding of VHL to the N-TAD can be observed in the absence of MG132 treatment after 1 (lane 2) and 10 min (lane 3) of reoxygenation. In
contrast, much lower levels of N-TAD- pVHL interaction were observed
either at normoxia (lane 1) or following 1 h
(lane 4) of reoxygenation. Treatment of the cells with the
proteosome inhibitor MG132 increased the levels of expressed pVHL
(lanes 5-8) and inhibited the decrease in pVHL binding
observed after 1 h of reoxygenation (lane 8). Interestingly, binding of VHL to the N-TAD was increased also in the
cells treated with MG132 under hypoxic conditions, suggesting that some
modification occurs during hypoxia that allows a more efficient
recruitment of pVHL when the cells return to normoxia. Possibly, these
observations could be explained by an increase in prolyl 4-hydroxylase
activity during reoxygenation because some of these prolyl
4-hydroxylase enzymes have been shown to be induced at the mRNA
levels by hypoxia (25). Given these results we performed the subsequent
in vivo VHL binding assays using cells not treated with
MG132 and exposed to short periods of reoxygenation.
N-TAD mutants, D568A/D569A/D570A and F571A/L573A, which did not interact with pVHL, also failed to bind pVHL in vivo
(Fig. 1D, lanes 9-12). In contrast to the
in vitro results (Fig. 1B), mutation of residues
Met560 and Leu561 did not inhibit pVHL binding
in vivo (Fig. 1D, lanes 3 and
4), possibly because of the absence in reticulocyte lysate
of appropriate modifying enzymes (i.e. prolyl
4-hydroxylases) required for binding of pVHL to these mutants.
Interestingly, the mutant P566E/M567E demonstrated significantly
stronger pVHL binding activity than the wild type N-TAD (Fig.
1D, lanes 7 and 8). Interaction of
pVHL was also observed with a shorter protein fragment of the N-TAD spanning residues 546-574 (lanes 17 and 18),
whereas no binding was observed with the C-TAD (Fig. 1D,
lanes 19 and 20) that has been shown previously
to be a constitutively stable but hypoxia-regulated transactivation
domain of HIF-1
(9).
-(531-584) in the
absence or presence of increasing concentrations of pVHL. As shown in
Fig. 2A, N-TAD-(531-584)
and N-TAD-(546-574) as well as the mutants L556A/L558A, M560A/L561A, and P565E/M566E were degraded by pVHL in a dose-dependent
manner at normoxia. In these experiments the mutant M560A/L561A was
more resistant to degradation than the other expressed GAL4 fusion proteins. In contrast, mutants P563D/Y564D/I565D, P563A, Y564G, I565G,
D568A/D569A/D570A, FL571A/L573A, and the C-TAD (Fig. 2, A
and B) were not degraded even following exposure to the
highest tested levels of pVHL tested. In conclusion, these results
demonstrate that in vivo binding of pVHL to the various
N-TAD mutants correlated with its ability to mediate degradation of
these protein fragments.
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Fig. 2.
Normoxia-dependent degradation of
wild type or mutant forms of N-TAD by pVHL. A and
B, degradation of mHIF-1 N-TAD mutants under normoxic
conditions in the presence of increasing levels of pVHL. HEK 293 cells
were transfected with pFLAG-GAL4/mHIF-1
-(531-584) encoding either
the wild type (N-TAD) or point-mutated forms of the N-TAD
motif, pFLAG-GAL4/mHIF-1
-(546-574)
(546-574), or pFLAG- GAL4/mHIF-1
-(772-822)
(C-TAD) at concentrations (750 ng) allowing detection of the
protein at normoxia. In indicated cases the cells were also transfected
with increasing concentrations of pCMX-VHL (+, 500 ng; ++,
1000 ng). Following transfection the cells were incubated for 36 h
before harvest. Whole cell extract proteins (25 µg) were analyzed by
SDS-PAGE and immunoblotting using anti-FLAG or anti-VHL antibodies
(
-FLAG,
-VHL). LL-A, L556A/L558A;
ML-A, M559A/L570A; PYI-D, P563D/Y564D/I565D;
PM-E, P566E/M567E; DDD-A, D568A/D569A/D570A;
FL-A, F571A/L573A. C, degradation of
the N-TAD motif under conditions of reoxygenation is mediated by
pVHL. HEK 293 cells were transfected with 750 ng of
pFLAG-GAL4/mHIF-1
-(531-584) (N-TAD) or
pFLAG-GAL4/mHIF-1
-(531-584) (P563A) in the absence or
presence of 250 ng (+), 500 ng (++), or 1000 ng
(+++) pCMX-VHL. Following transfection the cells were
incubated for 24 h at normoxia (N), for 12 h at
hypoxia (H), or for 12 h of hypoxia followed by 2 h of reoxygenation (R). 25 µg of whole cell extract
proteins was analyzed by SDS-PAGE and immunoblotting using anti-FLAG
(
-FLAG) or anti-VHL (
-VHL)
antibodies.
-(546-574)). The mutants D568A, D569A, and Q572A
were able to interact with pVHL in an in vitro binding assay
(Fig. 3A, lanes
5, 6, and 9, respectively). In the case of
the mutant D568A (lane 5) we consistently observed weaker
binding to pVHL. In contrast to these results, mutation of residues
Asp570, Phe571, and Leu572
completely abrogate pVHL-N-TAD interaction (Fig. 3A,
lanes 7, 8, and 10, respectively). In
the in vivo pVHL-mediated degradation assay, the shorter
fragment of the N-TAD spanning residues 546-574 and the mutant D570A
were degraded by pVHL in a dose-dependent manner. The D570A
was more resistant to pVHL-mediated degradation than the wild type.
These results indicate the presence in HEK 293 cells of enzymes
(notably one or several prolyl 4-hydroxylases) that facilitate binding
of pVHL in vivo. The other two mutants F571A and L573A,
which failed to bind pVHL in vitro, were resistant to
pVHL-mediated degradation even at the highest doses of pVHL tested.
These data demonstrated that, in addition to the PYI motif, residues
Phe571 and Leu573 were critical for the
physical interaction with pVHL and pVHL-mediated degradation of the
N-TAD.
View larger version (41K):
[in a new window]
Fig. 3.
Identification of critical residues for pVHL
interaction located in the C-terminal region of the N-TAD.
A, in vitro binding of pVHL to residues 546-574
of wild type or point-mutated forms of N-TAD. Immunoprecipitations
(IP) were performed with in vitro translated
pSP72-FLAG-GAL4/mHIF-1 -(546-574) (546-574) and mutants
as described in Fig. 1B. B, pVHL-dependent
degradation at normoxia of mutants of the residue 546-574-fragment of
the N-TAD. Transfections were performed with
pFLAG-GAL4/mHIF-1
-(546-574) and mutants as described in Fig. 2,
A and B.
N-TAD motif has been shown
previously to function as a bifunctional domain both constituting a
hypoxia-regulated degradation box as well as a
hypoxia-dependent transactivation domain that can be
potentiated by coactivators such as p300/CBP (7-10). We therefore
investigated the effect of the different mutations within the N-TAD on
the ability of this domain to mediate the hypoxia-dependent
transactivation response. To this end we performed transient
transfection experiments in HEK 293 cells using a GAL4-driven
luciferase reporter gene and pFLAG-GAL4-mHIF-1
-(531-584) or
different mutants of this motif. At normoxia the wild type N-TAD
construct mediated about 2.4-fold activation of transcription when
compared with the activity observed following expression of the GAL4
DNA binding domain alone. However, in cells treated for 24 h with
hypoxia 9-fold activation of transcription was observed over the values
produced by the GAL4 DNA binding domain at normoxia, thus resulting in
about a 4-fold hypoxia-dependent activation response (Fig.
4A). In contrast to the wild
type N-TAD, most of the tested N-TAD mutants did not mediate any
significant levels of hypoxia-inducible transactivation. For instance,
the mutants L556A/L558A and M560A/L561A produced only very modest
transactivation responses as compared with the wild type chimeric
protein. Considering the fact that these mutants (L556A/L558A and
M560A/L561A) both show interaction with pVHL, and
pVHL-dependent degradation, a possible explanation for the
low transactivation capacity could be that residues Leu556,
Leu558, Met560, and Leu561 are
important for the interaction with factors involved in the transactivation response.
View larger version (18K):
[in a new window]
Fig. 4.
Analysis of the transactivation function of
N-TAD mutant proteins. A, mutation of residue
Pro563 to alanine generates a constitutively active and
potent transactivation domain. HEK 293 cells were transfected with 500 ng of a GAL4-responsive reporter gene plasmid, 20 ng of wild type N-TAD
(pFLAG-GAL4/mHIF-1 -(531-584)), or mutant expression plasmids and
carrier DNA pFLAG-CMV-2 to keep a constant DNA concentration of 1 µg.
The cells were cultured for 12 h after transfection and exposed to
24 h of normoxia or hypoxia. Data are presented as luciferase
activity relative to cells transfected with pFLAG-GAL4 alone at
normoxia. Values represent the mean ± S.E. of three independent
experiments performed in duplicate. LL-A, L556A/L558A;
ML-A, M559A/L570A; PYI-D, P563D/Y564D/I565D;
FL-A, F571A/L573A. B, CBP potentiates
transactivation activity mediated by the N-TAD or the P563A mutant. HEK
293 cells were transfected as described in A in the absence
or presence of 400 ng of CBP expression plasmid.
N-TAD motif (2.7- and 9.2-fold activation over
background values at normoxia and hypoxia, respectively). Also
P566E/M567E mutant transactivates as efficiently as the wild type N-TAD
(results not shown). The short protein fragment of the N-TAD spanning
residues 546-576 was able to mediate a 2.6-fold
hypoxia-dependent transactivation response, indicating a
reduced ability to transactivate although it maintained all properties
of conditionally regulated protein degradation observed with the larger
N-TAD fragment. The transactivation results obtained with the Y564G and
Y564F mutants are in excellent agreement with the pVHL binding
experiments (Fig. 2B). Mutation of Tyr564 to
phenylalanine affected neither pVHL binding nor the transactivation function of the N-TAD, suggesting that phosphorylation of the Tyr
residue may not be required for interaction with pVHL or the ability to
transactivate. On the other hand, hydrophobicity of this residue plays
an important role in regulation of N-TAD function because the
substitution of the tyrosine by glycine abrogated pVHL binding (Fig. 1)
and generated a weak, constitutively active transactivation domain
(Fig. 4). Interestingly, mutation of Pro563 to alanine also
generated a constitutively active transactivation domain that was much
more potent than the wild type N-TAD. N-TAD P563A was able to mediate
28- and 25-fold activation responses over the background values at
normoxia or hypoxia, respectively (Fig. 4A). Although all
the three mutants of the PYI motif abrogated pVHL-mediated degradation
of the N-TAD, only the mutation of the proline to alanine generated a
more potent, constitutively active transactivation function. We
therefore conclude that the P563A mutation not only inhibited binding
of pVHL and subsequent degradation of the N-TAD but also conferred a
conformational change onto the N-TAD that improved the transactivation
potency of this domain.
--
In addition to the N-TAD, HIF-1
contains a second
hypoxia-responsive transactivation domain, the C-TAD. In our previous
experiments (Fig. 4A), we identified mutations that affected
the activation of transcription mediated by the N-TAD. We therefore
examined if these mutations interfere with the transactivation function of the full-length HIF-1
. We inserted several of the relevant N-TAD
mutations into the context of the full-length mHIF-1
(pFLAG-mHIF-1
). These mutants were tested in transactivation assays
by transfecting HEK 293 cells together with an HRE-driven luciferase
reporter gene. We also monitored the expression levels of the different mHIF-1
mutants (Fig. 5B)
using the same DNA preparations tested in the luciferase reporter
assays. Activation of transcription mediated by wild type HIF-1
produced 8.8- and 27.4-fold activation at normoxia and hypoxia,
respectively, over the value of the expression of pFLAG at normoxia
(Fig. 5A). Three of the tested mutants, mHIF-1
(P563A), mHIF-1
(I565G), and mHIF-1
(P402A/P563A), generated a
transactivation response similar to that of the wild type protein,
whereas the other mutants showed a significant reduction of the
transactivation function both at normoxia and hypoxia (Fig.
5A). The mutant mHIF-1
(P563A) transactivated as
efficiently as the wild type protein but was expressed at lower levels.
It is possible that this mutation alone increased the transactivation
efficiency of mHIF-1
. Mutation of both
Pro402-Pro563 generated a mutant that showed
elevated levels of expression and transactivated 2-fold more potently
at normoxia than the wild type mHIF-1
but nevertheless was still
responsive to hypoxia (2-fold hypoxia-dependent activation
response). The results obtained with mHIF-1
(P563A) and
mHIF-1
(P402A/P563A) are in contrast with a previous study using RCC4
cells stably transfected with pVHL (24) where mutation of
Pro564 in hHIF-1
increased transactivation in normoxia
and protein expression in non-treated cells. In our study the mutant
mHIF-1
(P563A) is as well degraded as the wild type protein. The
double mutant P402A/P564G in hHIF-1
is presented as a constitutive
transactivator in this report (24), whereas in the present study this
protein presents a higher transactivation in normoxia but is still
responsive to hypoxia.
View larger version (37K):
[in a new window]
Fig. 5.
Mutation of N-TAD residues decrease the
transactivation activity of full-length mHIF-1 .
A, relative luciferase activity of mHIF-1
mutants. HEK
293 cells were transfected with 500 ng of HRE-driven reporter gene, 200 ng of pFLAG-mHIF-1
, or mutants thereof and 300 ng of pFLAG-CMV-2.
After transfection the cells were kept at normoxia or exposed to
hypoxia for 36 h before harvesting. Data are presented as
luciferase activity relative to cells transfected with pFLAG-CMV-2 at
normoxia. Values represent the mean ± S.E. of three independent
experiments performed in duplicate. B, expression levels of
wild type or mutated forms of mHIF-1
. HEK 293 cells were transfected
with 750 ng of expression plasmids of pFLAG-mHIF-1
or mutants
thereof and 250 ng of pFLAG-CMV-2. After transfection the cells were
allowed to grow for 24 h and after that were kept at normoxia
(N) or at hypoxia (H) for 12 h. Whole cell
extract proteins were analyzed by SDS-PAGE and immunoblotting with
anti-FLAG antibodies (
-FLAG). HIF-1
(LL-A),
HIF-1
(L556A/L558A); HIF-1
(FL-A),
HIF-1
(F571A/L573A); HIF-1
(PP-A),
HIF-1
(P402A/P563A).
(L556A/L558A) and
mHIF-1
(F571A/L573A) demonstrated a transactivation capacity that was
reduced at hypoxia to 40 and 50%, respectively, of the wild type
levels. Given the fact that these mutants show wild type,
hypoxia-regulated expression levels, these latter results show that
some of the mutations that decreased the transcription potential of the
N-TAD in GAL4 fusion protein experiments also decrease the total
transactivation activity of full-length mHIF-1
.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
by deletion or point mutation does not
completely abrogate functional interaction with CBP, as assessed in
gene reporter assays or assays monitoring nuclear colocalization of
HIF-1
and CBP in accumulation foci. Furthermore, N-TAD-mediated
activation of transcription has been shown to be potentiated by
coactivators such as CBP/p300 (9, 10). In good agreement with this
observation, our results showed that the transcription mediated by the
constitutively active P563A mutant could be further enhanced by CBP
indicating that this coactivator participates in this transcriptional complex.
(corresponding to Leu558 in
mHIF-1
) has previously been mutated alone (37) or in the context of
a double mutant together with a mutation of Asp558 (23) and
shown not to affect the binding of pVHL. Moreover, the mutations
L556A/L558A in the N-TAD did not affect pVHL binding in
vitro or in vivo. However, in contrast to mutants such
as Y564F and P566E/M567E that bind pVHL and transactivate as well as
the wild type N-TAD, the L556A/L558A mutations impaired the
transactivation activity of the N-TAD and significantly reduced the
transactivation potency of full-length mHIF-1
. Residue
Leu562 in hHIF-1
(Leu561 in mHIF-1
) was
also previously mutated in several reports with apparently
contradictory results (22, 23, 37). In our assays mutation of
M560A/L561A resulted in abrogation of the binding of pVHL in
vitro but not in vivo. The use of rabbit reticulocyte lysate or cell extracts could explain the different results described in the previous reports (22, 23, 37). In the co-crystals of the N-TAD
peptide and pVHL-BC complex, both Met561 and
Leu562 have direct contacts with pVHL residues (34, 35).
However, our results demonstrated that even when the two residues are
mutated pVHL can bind and degrade N-TAD at normoxia. The major effect of the M561A/L562A mutation was observed in gene reporter assays where
a drastic reduction in transactivation activity was measured, indicating a critical role of these residues for transcriptional activation.
resulted in a drastic reduction in
transactivation potential, suggesting an important role of this residue
in the N-TAD-mediated transactivation function. Previous studies using
in vitro assays of the effect of the mutation Y565A in
hHIF-1
(corresponding to Tyr564 in mHIF-1
) have
yielded contradictory results with regard to the importance of this
residue for binding of pVHL (21, 22). On the other hand, structural
studies (34, 35) have shown that residue Tyr565 interacts
with His110 of pVHL, and the integrity of this tyrosine has
been suggested to be important for the hydroxylation of
Pro564 by two of the prolyl 4-hydroxylases (26). The
present results obtained with Y564G mutation demonstrated that the
tyrosine residue is critical both for degradation function mediated by
pVHL at normoxia and the transactivation function of the N-TAD. The
residue Ile566 in hHIF-1
(corresponding to
Ile565 in mHIF-1
) has been mutated previously to alanine
and shown not to affect the interaction with pVHL (22, 23). However, following mutation of Ile565 to glycine, both in
vitro and in vivo assays indicated that this residue is
also critical for binding of pVHL. In excellent agreement with the
data, one of the studies of the co-crystal of an N-TAD peptide and the
pVHL-BC complex showed Ileu565 to interact through hydrogen
bonds with two residues, Pro99 and Ile109, of
pVHL (34). Thus, Ile565 constitutes the residue in the
vicinity of Pro564 that makes most contacts with pVHL.
(corresponding to
Phe571 in mHIF-1
) contacts with Ile75 and
Gly106 of pVHL, whereas Leu574 in hHIF-1
(Leu573 in mHIF-1
) contacts with Cys77 and
Arg79 (34). Two of these residues Gly106 and
Arg79 are frequent tumor-derived pVHL missense mutations
(32). Thus, our data suggest that the integrity of both
Phe571 and Leu573 is critical for interaction
between the N-TAD and pVHL. In conclusion, we have identified several
residues in the N-TAD that are important for degradation mediated by
pVHL and the transactivation function of HIF-1
.
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FOOTNOTES |
---|
* This work was supported in part by the Swedish Medical Research Council.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.
Supported by a Post-doctoral Fellowship PRAXIS XXI/BPD/11843/97
from the Ministry of Science/Fundação para a Ciência e
a Tecnologia of Portugal.
§ Supported by a fellowship from the World Health Organization.
¶ Supported by Ph.D. Student Fellowship PRAXIS XXI/BD/19994/99 from the Ministry of Science/Fundação para a Ciência e a Tecnologia of Portugal.
Present address: Dept. of Biochemistry and Biophysics,
Institute of Radiation Biology and Medicine, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan.
** To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden. Tel.: 46-8-728-7330; Fax: 46-8-34-88-19; E-mail: lorenz.poellinger@cmb.ki.se.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M209297200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HIF, hypoxia-inducible factor; pVHL, von Hippel-Lindau tumor suppressor gene product; pVHL-BC, pVHL-ElonginB-ElonginC; HRE, hypoxia-response element; TAD, transactivation domain; N-TAD, N-terminal TAD; C-TAD, C-terminal TAD; CBP, CREB-binding protein; TBS, Tris-buffered saline; HEK, human embryonic kidney.
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