Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan...

20
Chen et al. 1 Direct interactions between HIF-1α and Mdm2 modulate p53 function Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College of Physicians & Surgeons, Columbia University 1150 St. Nicholas Ave, New York, NY 10032, USA Running Title: Regulation of p53 function by HIF-1α Keywords: p53, HIF-1α, ubiquitination, Mdm2, nuclear export, stabilization *Corresponding author Wei Gu Berrie Research Pavilion Rm 412C Institute for Cancer Genetics Columbia University 1150 St. Nicholas Avenue New York, NY 10032 Phone: 212-851-5282 (Office) 212-851-5285/5286 (Lab) Fax: 212-851-5284 e-mail: [email protected] Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc. JBC Papers in Press. Published on February 26, 2003 as Manuscript C200694200 by guest on April 5, 2020 http://www.jbc.org/ Downloaded from

Transcript of Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan...

Page 1: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

1

Direct interactions between HIF-1α and Mdm2 modulate p53 function

Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu*

Institute for Cancer Genetics, and Department of Pathology

College of Physicians & Surgeons, Columbia University

1150 St. Nicholas Ave, New York, NY 10032, USA

Running Title: Regulation of p53 function by HIF-1α Keywords: p53, HIF-1α, ubiquitination, Mdm2, nuclear export, stabilization

*Corresponding author Wei Gu Berrie Research Pavilion Rm 412C Institute for Cancer Genetics Columbia University 1150 St. Nicholas Avenue New York, NY 10032 Phone: 212-851-5282 (Office) 212-851-5285/5286 (Lab) Fax: 212-851-5284 e-mail: [email protected]

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on February 26, 2003 as Manuscript C200694200 by guest on A

pril 5, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 2: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

2

Summary

The p53 tumor suppressor is maintained at low levels in normal cells by Mdm2-

mediated degradation, and strongly stabilized in response to various types of stress

including hypoxia. Although hypoxia-inducible factor 1α (HIF-1α) has been

implicated to be involved in p53 stabilization, the precise mechanism by which HIF-

1α regulates p53-mediated function remains unknown. Here, we found that HIF-1α

directly binds Mdm2 both in vitro and in vivo; in contrast, p53 fails to directly

interact with HIF-1α in vitro. Interestingly, Mdm2 expression can significantly

enhance the in vivo association between p53 and HIF-1α, indicating that Mdm2 may

act as a bridge and mediate the indirect interaction between HIF-1α and p53 in

cells. Furthermore, HIF-1α protects p53 degradation mediated by Mdm2, and leads

to activation of p53-mediated transcription in cells. To elucidate the mechanism of

HIF-1α-mediated effect, we also found that HIF-1α can significantly suppress

Mdm2-mediated p53 ubiquitination in vitro, and blocks Mdm2-mediated nuclear

export of p53. These results have significant implications regarding the molecular

mechanism by which p53 is activated by HIF-1α in response to hypoxia.

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 3: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

3

Introduction

Cellular hypoxia is an important phenomenon in developmental biology, normal

physiology, and many pathological conditions, including cancer. Hypoxia triggers a

multifaceted adaptive response that is primarily mediated by the heterodimeric

transcription factor hypoxia-inducible factor (HIF). HIF-1 is a heterodimer composed of

two subunits, the rate-limiting factor HIF-1α and the constitutive expressed HIF-1β (1,2).

HIF-1β has been characterized as an aryl hydrocarbon receptor nuclear translocator

(ARNT), and this family of proteins has previously been shown to heterodimerize with

the aryl hydrocarbon receptor (AHR) (3). On the other hand, HIF-1α specifically

mediates hypoxic responses. In normoxia, HIF-1α is maintained at low and often

undetectable levels. HIF-1α is targeted for degradation by the ubiquitination-proteasome

pathway through directly binding to the von Hippel-Lindau tumour suppressor gene

(pVHL), which forms the recognition component of an E3 ubiquitin-protein ligase

leading to ubiquitination of HIF-1α (4-7). Recent reports demonstrate that HIF-1α

undergoes an iron and oxygen-dependent modification before it can interact with pVHL.

This modification is catalyzed by a specific family of enzymes termed HIF-1α-proline

hydroxylases (8-10). During hypoxia, this specific hydroxylase is inactive since it

requires dioxygen for its activity. As a result, HIF-1�� �������������� ��� ���� �������� �

pVHL to recognize its non-hydroxylated form. HIF-1α then translocates to the nucleus

and dimerizes with the constitutively present HIF-1β (11).

The importance of the HIF-1��response pathway in human tumorigenesis is underscored

by the finding that HIF-1α is overexpressed in multiple human cancers, because tumor

cells, unlike normal cells from the same tissue, are often chronically hypoxic (12). The

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 4: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

4

tumor suppressor protein p53 integrates numerous signals that control cell life and death

(13). Wild-type p53 is expressed at low levels in most cells because of its short half-life

under normal conditions. In contrast, the p53 protein is stabilized, and its level increases

in response to various stresses such as DNA damage, hypoxia, and inappropriate

oncogene signaling (14,15). In its active form, p53 can bind DNA in a sequence-specific

manner and activate transcription of target genes. p53 levels are regulated in large part by

Mdm2, the product of a p53-inducible gene. Mdm2 can interact with the N terminus of

p53, which also contains the major acidic transcriptional activation domain. The

interaction between Mdm2 and p53 can inhibit p53 transcriptional activity by interfering

with the ability of p53 to contact transcriptional coactivators such as p300/CBP (16).

Importantly, Mdm2 binding also promotes the ubiquitination of p53 and its export from

the nucleus to the cytoplasm, where p53 is then degraded by cytoplasmic proteasomes

(17).

Hypoxic induction of p53 requires concomitant induction of HIF-1α, whereby HIF-1α

can then bind to and stabilizes p53 (18-21). However, the molecular mechanism by which

HIF-1α stabilizes p53 remains unknown. Moreover, it is not clear whether HIF-1α

interacts with p53 directly despite previous indications that p53 associated with HIF-1α

in cells. Nevertheless, an array of immobilized peptide assay showed that the core domain

of p53 has an affinity for the ODD domain of HIF-1α (22). As described in this report,

we have found a strong interaction between HIF-1α and Mdm2 while we failed to detect

any direct interaction of p53 with HIF-1α. Our data demonstrate that HIF-1α regulates

p53 activity including stability and nuclear export through interactions with Mdm2. These

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 5: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

5

results provide a potential mechanism for p53 stabilization by HIF-1α in response to

hypoxia.

Experimental procedures

Cell culture and transfection

p53-null H1299 lung carcinoma cells and mouse embryonic fibroblast cells (MEFs)

were cultured in Dulbecco's modified Eagle's medium (Mediatech, VA) supplemented

with penicillin/streptomycin and 10% fetal bovine serum (Mediatech, VA).

1X106 or 2X105 cells were plated in 10 cm or 6-well plates, respectively, and 24 h later

transfections were done by calcium phosphate precipitation procedures. After a 24 h

incubation with 20% O2, the cells were harvested.

Plasmids

Full length HIF-1α DNA was generated by PCR using hemagglutinin (HA)-tagged HIF-

1α from David Livingston (Dana Farber Cancer Institute), and was subcloned into

pcDNA3.1/v5-His-Topo (Invitrogen) or p3xFlag-CMV-14 (Sigma). Green fluorescence

protein (GFP)-tagged wild-type p53 was obtained from Yanping Zhang (M.D. Anderson

Cancer Center). Plasmid DNA for transfections was isolated using Qiagen plasmid maxi

kit (Qiagen).

Recombinant Protein Preparation and GST Pull-down Assay

Glutathione S-transferase (GST) fusions of p53 and Mdm2 were expressed in

Escherichia coli BL-21 (DE3) (Promega), and induced with 0.1 mM isopropyl-1-thio-β-

D-galactopyranoside. Bacterial pellets were lysed in BC500 (25 mM Tris, pH 7.8, 500

mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.2 % NP40, fresh 1 mM PMSF)

with sonication. Levels of expressed GST fusion proteins were estimated by incubation

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 6: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

6

with glutathione-sepharose beads, washing, and quantification by SDS-PAGE followed

by staining with Coomassie Brilliant Blue R-250. Known amounts of bovine serum

albumin were used as standards.

35S-labeled in vitro translated HIF-1α was prepared by using the TNT system

(Promega). Equal amounts (1 µg) of GST, GST-p53 and GST-Mdm2 immobilized on

glutathione-Sepharose beads were incubated with in vitro translated HIF-1α in BC200 for

3 h at 40C. After washing, the bound proteins were eluted with SDS sample buffer and

were separated by SDS-PAGE, followed by autoradiography.

Immunoblotting and Co-immunoprecipitation

Cells were lysed in Flag lysis buffer (50 mM Tris, 137 mM NaCl, 10 mM NaF, 1mM

EDTA, 1% Triton X-100, 0.2% Sarkosyl, 1 mM DTT, 10% glycerol, pH 7.8) with fresh

protease inhibitors (1mM PMSF, protease inhibitor cocktail (Sigma)). Aliquots (30µg) of

cell extracts were resolved in SDS/8% polyacrymide gels and then transferred to

nitrocellulose membranes in 20 mM Tris-HCL, pH 8.0/150 mM glycine/20%(vol/vol)

methanol. Membranes were blocked with 5% (vol/vol) nonfat dry milk/TBST (20 mM

tris⋅HCl, pH 7.6/137 mM NaCl/0.1% Tween 20), incubated with α-p53 (DO-1) antibody

(Santa Cruz), or anti-GFP (Clontech), and detected with ECL reagents (Amersham).

Co-immunoprecipitation assay was performed essentially as described previously (23).

In brief, 50 µl of proteasome inhibitor LLNL was added to the co-transfected culture 6 h

before harvest. 24 h after transfection, the cells were lysed in BC100 (25 mM Tris, pH

7.8, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.2 % NP40, fresh 1 mM

PMSF), and incubated with anti-Flag M2 beads (Sigma) for overnight at 40C. The beads

were washed five times with 1 ml of lysis buffer, after which the associated proteins were

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 7: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

7

eluted with BC100/0.2% NP-40 plus 0.2 mg/ml Flag peptide (Sigma). The eluted proteins

were resolved on 8% SDS PAGE and western blot with the anti-p53 or anti-Mdm2

(SMP14, Santa Cruz) monoclonal antibody.

Luciferase Assay

Luciferase activity was determined using a dual luciferase assay system (Promega)

following the manufacturer's protocol. Cells in six-well were removed by scraping into

100 µl of reporter lysis buffer. Cell lysate was collected by centrifugation for 15 min at

12,000g. Luciferase activity was measured using a Lumat LB 9507 luminometer (EG&G

Wallac, Gaithersburg, MD).

In vitro ubiquitination assays

The in vitro ubiquitination assay was performed as previously described (24) with some

modifications. For a standard reactions, the purified Flag-p53 proteins from H1299 cells

were mixed with other purified components including E1, E2 (GST-UbcH5C), E3 (GST-

Mdm2) and His-ubiquitin in reaction buffer (40 mM Tris, 5 mM MgCl2, 2 mM ATP, 2

mM DTT, pH 7.6). The reaction was stopped after 60 min at 370C by additions of SDS

sample buffer, and subsequently resolved SDS-PAGE gels for western blot analysis with

�-p53 (DO-1).

Nuclear export assay for p53

H1299 cells were plated on 6-well containing glass coverslips and GFP-p53 was

transfected as described. 50 µM of proteasome inhibitor LLNL was added for 6 hr before

fixation. Twenty-four hours after transfection, cells on the coverslips were washed three

times with phosphate-buffered saline (PBS), and then fixed in 4% paraformaldehyde for

10 min at room temperature. After fixation, cells were washed in PBS three times and

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 8: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

8

then permeabilized in ice-cold PBS containing 0.2% triton X-100 for 10 min. Cells were

blocked in PBS containing 1 % bovine serum albumin and 1µg of DAPI (Sigma)/ml at

room temperature for 30 min. Cells were washed three times with PBS and the stained

cells were mounted with mounting medium (Polysciences, Inc. PA) and sealed with nail

polish. Immunofluorescence was recorded using an immunofluorescence microscope.

Results

Interaction between HIF-1α and Mdm2

Although earlier studies indicated that p53 can bind HIF-1α in cells, we repeatedly

failed to detect any direct interaction between these two protein in a GST-pull down assay

(data not shown), suggesting that HIF-1α may interact with p53 through other factors. To

examine the notion that p53 may interact with HIF-1α through Mdm2, we tested whether

HIF-1α directly binds Mdm2 in vitro by GST pull-down assays. Both GST-Mdm2 and

GST were expressed in bacteria and purified to near homogeneity. As shown in Figure 1a,

the 35S-labeled in-vitro translated HIF-1α strongly bound to immobolized GST-Mdm2

(lane 2) but not to immobilized GST (lane 3). However, using the same assay, no

significant interaction was detected between GST-p53 and HIF-1α. Furthermore, we

tested the interaction between HIF-1α and Mdm2 in vivo by coimmunoprecipitation

assays. Flag-tagged full-length HIF-1α was transiently co-transfected with Mdm2 in

H1299 cells. Immunoprecipitations were performed with anti-Flag M2 beads, and the

precipitated proteins were analyzed by western blot with an anti-Mdm2 antibody. As

shown in Figure 1b, the Mdm2-HIF-1α complexes were readily detected in cells co-

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 9: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

9

transfected with Flag-HIF-1α and Mdm2 (lane 4) but not by Mdm2 alone (lane 3),

indicating that there is a specific in vivo interaction between Mdm2 and HIF-1α.

Mdm2 enhances the in vivo binding between p53 and HIF-1α

To examine the possibility that the HIF-1α-p53 interaction is mediated by Mdm2, we

tested whether Mdm2 expression is required for the HIF-1α-p53 interaction in cells. p53

and Flag-HIF-1α were transiently transfected with or without Mdm2 in H1299 cells.

Immunoprecipitations were carried out from cell extracts using anti-Flag M2 beads. As

shown in Figure 2, p53 was barely detectable in the HIF-1α-associated immuno-

complexes in the absence of Mdm2 expression (lane 5), further confirming the idea that

p53 can not directly bind HIF-1α in vivo. In contrast, when Mdm2 was expressed in the

cells, p53 was efficiently co-precipitated with HIF-1α by the same method (lane 6).

These results indicate that Mdm2 acts as a bridge mediating the binding between HIF-1α

and p53.

HIF-1α abrogates Mdm2-mediated p53 degradation and transcription repression

Mdm2 is a key regulator of p53 and can both inhibit p53 transcriptional activity and

target it for degradation (24). We tested the possibility whether HIF-1α could protect p53

from degradation mediated by Mdm2. The p53-null H1299 cells were transfected with

CMV-p53, CMV-Mdm2, CMV-GFP, and pTOPO-HIF-1α. 24 hr after transfection the

cells were lysed in a Flag-lysis buffer and analyzed by western blot. As shown in Figure

3a, HIF-1α effectively rescues p53 from Mdm2-mediated degradation. Overexpression of

Mdm2 significantly induced p53 degradation (lane 2 vs lane 1), whereas this degradation

was inhibited in a dose–dependent manner upon overexpression of HIF-����������������

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 10: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

10

lane 2). Interestingly, HPV E6 protein can induce p53 degradation through the E6/E6 AP

ubiquitin ligase complex. However, overexpression of HIF-1�� ���� ���� �������� ����

degradation mediated by E6 (Fig. 3b). Taken together, these results demonstrated an

effect of HIF-�� directly on Mdm2 mediated degradation of p53. Furthermore, we used

p53 transcriptional activity assay to support the notion. To this end, we co-transfected

mouse embryo fibroblasts (MEFs, p53-/-) with vectors expressing p53, Mdm2 and HIF-

1α, along with a reporter construct containing the minimal p21 promoter (p21min-luc).

As indicated in Figure 3c, co-transfection of Mdm2 with p53 strongly repressed p21

luciferase activity due to p53 degradation. However, HIF-1α significantly abrogated the

inhibitory effect of Mdm2 in a dose-dependent manner. The above data demonstrate that

HIF-1α can strongly stabilize p53 by abrogating Mdm2-mediated effects, which leads to

activation of p53-mediated transcription.

HIF-1α suppresses p53 ubiquitination mediated by Mdm2

To investigate whether HIF-1α can suppress p53 ubiquitination mediated by Mdm2, we

carried out an in vitro ubiquitination assay. As shown in Figure 3d, ubiquitinated p53

was easily detected at the reaction with Mdm2 but no HIF-1��(lane 2 vs lane 1). However,

in the presence of HIF-��, the levels of ubiquitinated p53 decreased dramatically (lane 3

vs lane 2). This result demonstrates that HIF-��� ���� ���� �������� ������ Mdm2-

mediated p53 ubiquitination.

HIF-1α blocks nuclear export mediated by Mdm2

Current models indicate that Mdm2 can also induce nuclear export of p53 (25,26). To

test the possibility that HIF-1α can block this export, we transfected H1299 cells with

vectors expressing GFP-p53, Mdm2 or HIF-1α and examined them by

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 11: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

11

immunofluorescence microscope (Figure 4a). The distribution of GFP-p53 was almost

entirely nuclear when expressed alone, but it localized to the cytoplasm to varying extents

when co-expressed with Mdm2. We scored the extent by which p53 localized to the

cytoplasm when expressed alone or when expressed together with Mdm2 (Figure 4b).

Cell co-transfected with p53 and Mdm2 had a 76% cytoplasmic expression vs 24% when

p53 was transfected alone, indicating that nuclear Mdm2 can promote the cytoplasmic

localization of p53 (25, 26). Interestingly, only 26% of cytoplasmic expression occurred

in cells co-transfected with HIF-1α in addition to p53 and Mdm2 (Figure 4a, b). These

results indicate that HIF-1α can strongly block Mdm2-mediated p53 nuclear export.

Discussion

How cells sense changes in ambient oxygen is a central question in biology. In

mammalian cells, lack of oxygen, or hypoxia, leads to stabilization of a sequence-specific

DNA binding transcriptional factor called HIF. Downstream genes of HIF are linked to

processes such as angiogenesis and glucose metabolism (20). On the other hand, hypoxia

induces accumulation of the tumor suppressor p53. Earlier studies indicated that HIF-1α

interacts with p53 in vivo (18), however the nature of this interaction has not been

elucidated. In this study, we demonstrate for the first time that HIF-1α directly binds to

Mdm2 both in vitro and in vivo. In the absence of Mdm2, the binding between HIF-1α

and p53 is almost undetectable. Furthermore, Mdm2 expression significantly induces the

indirect interaction between p53 and HIF-1α in cells, indicating that Mdm2 may act as a

bridge mediating the p53-HIF-1α interaction. In this regard, we failed to detect any

significant interaction between p53 and HIF-1α in MEF Mdm2-/- cells, and stabilization

of p53 induced by HIF-1α expression was also severely abrogated (data not shown). Our

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 12: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

12

findings seem at odds with the recent report of tight binding between the ODD domain of

HIF-���������������������� ��������an array of immobilized peptide assay (22). It is

likely that the experiment was based on p53 pepetide fragments, which may not be

equivalent to the native, folded protein. However, the existence of Mdm2 somehow might

cause the conformational change of p53 native protein, thereby favoring the exposure of

binding sites of p53 to HIF-1α.

In addition, our results demonstrate that HIF-1α protects p53 from degradation

mediated by Mdm2 and can abrogate p53 transcriptional repression by Mdm2.

Considering that p53 degradation is mainly induced by Mdm2 in normal cells, we also

found that Mdm2-mediated ubiqutination of p53 is significantly inhibited by HIF-1α.

Since Mdm2-mediated p53 ubiquitination promotes its nuclear export (25, 26), we further

demonstrate that HIF-1α expression can block Mdm-2-mediated nuclear export of p53.

Thus, these results have significant implications regarding the molecular mechanism by

which HIF-1α modulates p53 function in vivo.

Our results are also consistent with published results indicating HIF-1α interacts with

the wild-type p53 protein but not the tumor-derived p53 mutant form in cells (18). Since

wild-type p53 protein is capable of inducing Mdm2 expression in cells, the observed

interaction between p53 and HIF-1α is most likely mediated by endogenous Mdm2. In

contrast, because the tumor-derived p53 mutant is completely inactive in transcriptional

activation of endogenous Mdm2, HIF-1α failed to interact with p53 since there is no (or

very low levels) Mdm2 in cells expressing mutated p53. Recent studies also indicate that

Mdm2 is involved in modulating HIF-1α stability under hypoxic conditions (21), further

supporting the notion that HIF-1α directly interacts with Mdm2 but not p53. Mdm2 is a

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 13: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

13

potent E3 ubiquitin ligase and induces both p53 degradation and nuclear export of p53

through ubiquitination. Therefore, it is possible that Mdm2 also directly mediates

degradation and nuclear export of HIF-1α. Our study, together with several other studies

(15,18,21), strongly implicates the important role of HIF-1α in the regulation of the p53-

Mdm2 pathway in response to hypoxia.

Acknowledgements We thank C. Brooks for carefully reading the manuscript and other members of W. Gu laboratory for sharing unpublished data and critical comments. We thank David Livingston and Yanping Zhang for providing plasmids. This work was supported in part by grants from the Leukemia & Lymphoma Society, Avon Foundation, the Stewart Trust, the Irma T. Hirschl Trust, and NIH/NCI to W.G.. W.G is a Leukemia & Lymphoma Society Scholar.

References 1. Wang, G.L. and Semenza, G.L. (1995) J. Biol. Chem. 270, 1230-1237. 2. Semenza, G.L. (2001) Curr. Opin. Cell Biol. 13, 167-171. 3. Hoffman, E.C., Reyes, H., Chu, F.F., Sander, F., Conley, L.H., Brooks, B.A. and Hankinson, O. (1991) Science 252, 954-958. 4. Maxwell, P.H., Wiesener, M.S., Chang, G.W., Clifford, S.C., Vaux, E.C., Cockman, M.E., Wykoff, C.C., Pugh, C.W., Maher, E.R., and Ratcliffe, PJ. (1999) Nature 399, 271-275. 5. Kamura T, Sato S, Iwai K, Czyzyk-Krzeska M, Conaway RC, Conaway JW. (2000) Proc Natl Acad Sci U S A 97, 10430-10435. 6. Blagosklonny, M.V. (2001) Oncogene 20, 395-398. 7. Ohh, M., Park, C.W., Ivan, M., Hoffman, M.A., Kim, T.Y., Huang, L.E., Pavletich, N., Chau, V., and Kaelin, W.G. (2000) Nature Cell Biol. 2, 423-427. 8. Jaakkola, P., Mole, D.R., Tian, Y.M., Wilson, M.I., Gielbert, J., Gaskell, S.J., Kriegsheim, Av., Hebestreit, H.F., Mukherji, M., Schofield, C.J., Maxwell, P.H., Pugh, C.W., and Ratcliffe, P.J. (2001) Science 292, 468-472. 9. Epstein, A.C., Gleadle, J.M., McNeill, L.A., Hewitson, K.S., O'Rourke, J., Mole, D.R., Mukherji, M., Metzen, E., Wilson, M.I., Dhanda, A., Tian, Y.M., Masson, N., Hamilton, D.L., Jaakkola, P., Barstead, R., Hodgkin, J., Maxwell, P.H., Pugh, C.W., Schofield, C.J., and Ratcliffe, P.J.. (2001) Cell 107, 43-54. 10. Bruick, R.K., and McKnight, S.L. (2001) Science 294, 1337-1340. 11. Semenza, G.L. (1999) Annu. Rev. Cell Dev. Biol. 15, 551-578. 12. Harries, A. L. (2002) Nature Reviews Cancer 2, 38-47. 13. Vogelstein, B., Lane, D., and Levine, A.J. (2000) Nature 408, 307 - 310. 14. Giaccia, A.J., and Kastan, M.B. (1998) Genes Dev. 12, 2973-2983.

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 14: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

14

15. Koumenis, C., Alarcon, R., Hammond, E., Sutphin, P., Hoffman, W., Murphy, M., Derr, J., Taya, Y., Lowe, S.W., and Giaccia, K.M., (2001) Mol Cell Biol 21, 1297-1310. 16. Prives, C., and Hall, P.A. (1999) J. Pathol. 187, 112-126. 17. Ryan, K.M., Phillips, A.C. and Vousden, K.H. (2001) Curr. Opin. Cell Biol. 13, 332-337. 18. An, W.G., Kanekal, M., Simon, M.C., Maltepe, E., Blagosklonny, M.V., and Neckers, L.M. (1998) Nature 392, 405-408. 19. Blagosklonny, M.V., An, W.G., Romanova, L.Y., Trepel, J. Fojo, T., and Neckers, L. (1998) J. Biol. Chem. 273, 11995-11998. 20. Semenza, G.L., (2000) Genes Dev. 14, 1983-1991. 21. Ravi, R., Mookerjee, B., Bhujwalla, Z.M., Sutter, C.H., Artemov, D., Zeng, Q., Dillehay, L.E., Madan, A., Semenza, G.L., and Redi, A. (2000) Genes Dev. 14, 33-44. 22. Hansson, L.O., Friedler, A., Freund, S., Rudiger, S. and Fersht, A. R. (2002) Proc Natl Acad Sci U S A 99, 10305-10309. 23. Gu, W., Shi, X.L., and Roeder R.G. (1997) Nature 387, 819-823. 24. Li, M., Luo, J., Brooks, C.L., and Gu, W. (2002) J. Biol. Chem. 277, 50607-50611. 25. Geyer, R.K., Yu, Z.K., and Maki, C.G. (2000) Nature Cell Biol. 2, 569-573. 26. Boyd, S.D., Tsai, K.Y., and Jacks, T. (2000) Nature Cell Biol. 2, 563-568.

Figure legends

Figure 1. HIF-1α interacts with Mdm2 in vitro and in vivo. (a) The in vitro interaction of

Mdm2 and HIF-1α. The GST (lane 3) and GST-Mdm2 (lane 2) fusion protein were used

in a GST pull down assay with in vitro translated 35S-labeled full length HIF-1α. (b)

Mdm2 interacts with HIF-1α in vivo. Western blot analysis of whole cell extracts (lanes

1,2) or immunoprecipiates with the anti-flag M2 beads (IP/M2) (lanes 3,4) from cells co-

transfected with Flag-HIF-1α (10 µg) and Mdm2 (10 µg) (lanes 2,4) or Mdm2 alone (lane

1,3) by anti-Mdm2 antibody.

Figure 2. Mdm2 enhances the binding between p53 and HIF-1α. Western blot analysis

of whole cell extracts (lanes 1,2,3) or immunoprecipites with the anti-flag M2 beads

(IP/M2) (lanes 4,5,6) from cells co-transfected with Flag-HIF-1α (10 µg) and p53 (5 µg)

(lanes 2,5), plus Mdm2 (5 µg) (lanes 3,6) or p53 alone (lanes 1,4) by anti-p53

monoclonal antibody (DO-1).

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 15: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Chen et al.

15

Figure 3. (a) Protection of p53 from Mdm2-mediated degradation by HIF-1α. Western

blot analysis of extracts from cells transfected with p53 (lane 1), or co-transfected with

p53 (1µg) and Mdm2 (2µg) (lane 2), or in combination with different amounts of HIF-

1α (lanes 3, 4), by the anti-p53 monoclonal antibody (DO-1). The CMV-GFP expression

vector was included in each transfection as a transfection efficiency control, and the

levels of GFP were detected with the anti-GFP monoclonal antibody (JL-8, Clontech). (b)

Failure to protect E6-mediated p53 degradation. Western blot analysis of extracts from

cells transfected with p53 (lane 1), or co-transfected with p53 (1µg) and E6 (0.5 µg)

(lane 2), or in combination with different amounts of HIF-1α (lanes 3, 4). (c) Abrogation

of Mdm2-mediated repression of p53-dependent transcription activation. MEF (p53-/-)

cells were transfected with p53 alone (0.25µg), or co-transfected with p53 (0.25µg) and

Mdm2 (0.5µg), or in combination with indicated amount of HIF-1α expression vector

����� together with the p21-Luc reporter construct (2µg). (d) HIF-1α suppresses Mdm2-

mediated p53 ubiquitination. The ubiquitination reactions were performed in the absence

of Mdm2 as control (lane 1), with Mdm2 (lane 2), and Mdm2 plus HIF-1α (lane 3).

Figure 4. HIF-1α blocks p53 nuclear export mediated by Mdm2. (a) Subcellular

localization of GFP-p53 in H1299 cells transfected with GFP-p53 alone (top row), GFP-

p53+Mdm2 (mid), and GFP-p53+Mdm2+HIF-1α (bottom). GFP-p53 images are shown

in left column, co-staining with DAPI in mid, and merge of GFP-p53 and DAPI in right.

(b) The proportion of cells expressing cytoplasmic p53 following transfection of GFP-

p53, Mdm2 or HIF-1α in H1299 cells. 150 cells were scored, and data from 3

independent experiments were averaged.

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 16: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

20%

Inpu

t

GS

T1 2 3

a

HIF-1αG

ST-

Mdm

2

Figure 1

Mdm2Flag-HIF-1α

Input IP/M2b

Mdm2

1 2 3 4

+ + + +_ + _ +

16

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 17: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Flag-HIF-1α

Input IP

p53

1 2 3 4 5 6

+ - - +

- + + - + +

- -Mdm2

Figure 2

/M2

17

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 18: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Figure 3

p53

GFP

Mdm2HIF-1α

+ + ++ +

10

20

30

p53

Mdm2HIF-1α

_ + + + + +_ _ + + + +_ _ _ 2 5 15

c

1 2 3 4

Rel

ativ

elu

cife

rase

activ

ity(fo

ld)

a

p53

p53-ub

Mdm2HIF-1α

+++

1

---

3

d

---

2

18

p53

GFP

E6HIF-1α

-- -

+++

++

b

1 2 3 4

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 19: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Figure 4

p53 DAPI

p53Mdm2HIF-1α

+ + +_ + +_ _ +

20

40

60

80

a

b

Mdm2

Mdm2+HIF-1α

%

Merge

19

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Page 20: Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* 1150 St ... · Delin Chen, Muyang Li, Jianyuan Luo, and Wei Gu* Institute for Cancer Genetics, and Department of Pathology College

Delin Chen, Muyang Li, Jianyuan Luo and Wei GuDirect interactions between HIF-1 alpha and Mdm2 modulate p53 function

published online February 26, 2003J. Biol. Chem. 

  10.1074/jbc.C200694200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from