MOLECULAR AND CELLULAR BIOLOGY, Jan. 2003, p. 26–37
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 23, No. 1
The Transformation Suppressor Pdcd4 Is a Novel Eukaryotic
Translation Initiation Factor 4A Binding Protein
That Inhibits Translation
Hsin-Sheng Yang,1* Aaron P. Jansen,1Anton A. Komar,2Xiaojing Zheng,2
William C. Merrick,2Sylvain Costes,3Stephen J. Lockett,3
Nahum Sonenberg,4and Nancy H. Colburn1
Gene Regulation Section, Center for Cancer Research,1and Image Analysis Laboratory, Science Applications International
Corporation,3National Cancer Institute, Frederick, Maryland 21702; Department of Biochemistry, School of Medicine,
Case Western Reserve University, Cleveland, Ohio 44106-49352; and Department of Biochemistry and
McGill Cancer Research Centre, University of McGill, Montreal, Quebec H3G 1Y6, Canada4
Received 26 August 2002/Returned for modification 19 September 2002/Accepted 2 October 2002
Pdcd4 is a novel transformation suppressor that inhibits tumor promoter-induced neoplastic transformation
and the activation of AP-1-dependent transcription required for transformation. A yeast two-hybrid analysis
revealed that Pdcd4 associates with the eukaryotic translation initiation factors eIF4AI and eIF4AII. Immu-
nofluorescent confocal microscopy showed that Pdcd4 colocalizes with eIF4A in the cytoplasm. eIF4A is an
ATP-dependent RNA helicase needed to unwind 5? mRNA secondary structure. Recombinant Pdcd4 specifically
inhibited the helicase activity of eIF4A and eIF4F. In vivo translation assays showed that Pdcd4 inhibited
cap-dependent but not internal ribosome entry site (IRES)-dependent translation. In contrast, Pdcd4D418A, a
mutant inactivated for binding to eIF4A, failed to inhibit cap-dependent or IRES-dependent translation or
AP-1 transactivation. Recombinant Pdcd4 prevented eIF4A from binding to the C-terminal region of eIF4G
(amino acids 1040 to 1560) but not to the middle region of eIF4G(amino acids 635 to 1039). In addition, both
Pdcd4 and Pdcd4D418Abound to the middle region of eIF4G. The mechanism by which Pdcd4 inhibits
translation thus appears to involve inhibition of eIF4A helicase, interference with eIF4A association-dissoci-
ation from eIF4G, and inhibition of eIF4A binding to the C-terminal domain of eIF4G. Pdcd4 binding to eIF4A
is linked to its transformation-suppressing activity, as Pdcd4-eIF4A binding and consequent inhibition of
translation are required for Pdcd4 transrepression of AP-1.
Initiation of protein synthesis in eukaryotic cells is a multi-
step process leading to the assembly of ribosomes and Met-
tRNAiat the initiation codon of an mRNA (12, 14). The rate-
limiting step of this process is the binding of the 40S ribosomal
subunit to mRNA. Several eukaryotic translation initiation
factors (eIFs), including the eIF4F complex, participate in this
process. Translation initiation factor eIF4F is a multiple-sub-
unit complex comprising eIF4A, eIF4E, and eIF4G.
eIF4A is an ATP-dependent RNA helicase belonging to the
DEAD box protein family (25) that has nine highly conserved
motifs shared with other DEAD box proteins. The RNA heli-
case activity of eIF4A is further increased by eIF4B, eIF4H, or
as a subunit of eIF4F (1, 41, 43, 45). Mutations in the nine
motifs greatly reduce the RNA binding ability, ATPase ac-
tivity, or helicase activity of eIF4A and produce inhibition of
translation (37). eIF4A is thought to catalyze the unwinding of
mRNA secondary structure at the 5? untranslated region, al-
lowing the 40S ribosomal subunit to bind the mRNA and scan
in a 5?-to-3? direction, searching for the initiation codon (14).
In mammals, three eIF4A isoforms have been identified.
eIF4AI and eIF4AII encoded by two different genes are highly
(91%) identical in amino acid sequence (33). eIF4AI and
eIF4AII are functionally indistinguishable and exchangeable,
performing similar kinetics of incorporation into eIF4F (50).
The third factor, eIF4AIII, is less identical to eIF4AI (?65%)
and functions as a translation inhibitor (23).
eIF4G functions as a scaffold containing several translation
initiation factor binding sites, including the sites for cap-bind-
ing protein eIF4E (27) and for eIF4A (16). eIF4E is required
for cap-dependent translation and binds to the N-terminal
one-third of eIF4G (amino acids 1 to 634). Cleavage of this
domain from eIF4G results in inhibition of cap-dependent
translation (13). Two eIF4A binding sites in eIF4G are located
within the middle one-third (amino acids 635 to1039) and the
C-terminal one-third (amino acids 1040 to 1560) (32). The
middle one-third of eIF4G is sufficient for cap-independent
5?-end-dependent translation (8) and internal ribosome entry
site (IRES)-mediated translation (26). The C-terminal one-
third of eIF4G has been reported to serve as a regulatory
domain for translation (32).
Pdcd4 was found in a differential display analysis of mouse
epidermal JB6 variants to be highly expressed in transforma-
tion-resistant (P?) but not in transformation-susceptible (P?)
cells (5). Expression of the pdcd4 gene is upregulated during
apoptosis in response to several inducers (46) and downregu-
lated by topoisomerase inhibitor treatment (34). No causal
relationship to apoptosis or to topoisomerase inhibitor-in-
duced cytotoxicity has been reported. The reduction of Pdcd4
in P? cells by overexpression of antisense pdcd4 is accompa-
* Corresponding author. Mailing address: Gene Regulation Section,
Bldg. 567, Rm. 180, Center for Cancer Research, National Cancer
Institute, Frederick, MD 21702. Phone: (301) 846-6564. Fax: (301)
846-6907. E-mail: firstname.lastname@example.org.
nied by acquisition of a transformation-susceptible phenotype
(5). Conversely, overexpression of sense pdcd4 in stably trans-
fected P? cells renders them resistant to tumor promoter-
induced transformation, indicating that elevated expression of
Pdcd4 protein is sufficient to inhibit transformation (49).
In order to elucidate the molecular target(s) of Pdcd4, we
performed a yeast two-hybrid analysis with pdcd4 cDNA as
bait. Translation initiation factors eIF4AI and eIF4AII were
identified as binding partners of Pdcd4. Pdcd4 inhibits eIF4A
helicase activity and inhibits translation in vivo. Interestingly,
Pdcd4 prevents eIF4A from binding to the C-terminal region
of eIF4G, and Pdcd4 itself binds to the middle region of
eIF4G, suggesting that Pdcd4 functions as a novel regulatory
factor for translation initiation.
MATERIALS AND METHODS
Construction of plasmids. pdcd4 cDNA was excised from pcDNA-Pdcd4 (49)
and ligated into the EcoRI and SmaI sites of the pGBKT7 DNA binding domain
vector (Clontech), and named pGBKT7-Pdcd4. This plasmid was the bait con-
struct used in the yeast two-hybrid assay. Plasmids pAcHLT-A-Pdcd4 and
pAcGHLT-A-Pdcd4 were used for generating recombinant His-Pdcd4 and glu-
tathione S-transferase (GST)-Pdcd4, respectively, in Sf-9 cells. pdcd4 cDNA was
inserted into the EcoRI and PstI sites of the pAcHLT-A or pAcGHLT-A vector
(Pharmingen). The plasmid Xpress-eIF4A was used to generate an Xpress-
tagged eIF4A. eIF4A cDNA was inserted into the BamHI and XhoI sites of
pcDNA4/Max (Invitrogen). Plasmids pCMV-BD-Pdcd4 and pCMVAD-eIF4A
were used for mammalian two-hybrid analysis with pdcd4 cDNA and eIF4A
cDNA inserted into the EcoRI and XbaI sites of the pCMV-DB and the BamHI
and XhoI sites of the pCMV-AD vectors (Stratagene), respectively. All con-
structs were subsequently sequenced to confirm in-frame fusion of pdcd4.
Yeast two-hybrid screen and assay. pGBKT7-Pdcd4 was transformed into
yeast strain PJ69-2A and mated with the pretransformed mouse brain Match-
maker cDNA library (Clontech) according to the manufacturer’s protocol. The
yeast cells were selected by their growth on Leu-, Try-, His-, and Ade-free plates
for 7 days. The colonies from these plates were subsequently assayed for ?-ga-
lactosidase expression three times with a ?-galactosidase liftover assay according
to the manufacturer’s protocol (Clontech). The library plasmids from Leu?/
Trp?/His?/Ade??-galactosidase-positive clones were isolated and sequenced.
Recombinant protein expression and purification. His-Pdcd4 or GST-Pdcd4
was expressed in insect Sf-9 cells. Sf-9 cells were infected with recombinant virus
at a multiplicity of infection of 5 and cultured for 72 h at 27°C. Cells were then
lysed in lysis buffer [25 mM sodium phosphate (pH 8), 300 mM NaCl, 1% Triton
X-100], and expressed proteins were purified by glutathione-Sepharose resins
(Pharmingen) or Ni-agarose affinity resins (Qiagen). The protein concentrations
were determined by comparison of band intensity with bovine serum albumin as
the standard on sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) following staining with Coomassie blue.
His-eIF4A, GST-eIF4G(672-1065), and GST-eIF4G(1201-1445) were ex-
pressed in Escherichia coli BL21(DE3) and purified on Ni-agarose resin (Qiagen)
as described previously (32, 38).
Immunoprecipitation and pull-down assays. JB6 P? cells were transfected
with plasmid pcDNA-Pdcd4 (49) or Xpress-eIF4A plasmid with Lipofectamine
(Invitrogen). After 48 h, cells were harvested and lysed in lysis buffer [20 mM
HEPES-KOH (pH 7.6), 100 mM KCl, 0.5 mM EDTA, 20% glycerol, 0.5% Triton
X-100, 50 ?g of RNase A per ml, and 1? protease inhibitor cocktail (Boehringer
Mannheim)]. For immunoprecipitation, eIF4A antibody (0.1 volume of super-
natant) was added to cell lysates from cells transfected with plasmid pcDNA-
Pdcd4 and incubated for 1 h at 4°C. Ten microliters of protein A-Sepharose
beads (CL-4B; Phamacia) were washed with 250 ?l of lysis buffer twice, added to
the cell lysates, and rotated for 2 h at 4°C.
For the GST pull-down assay, 50 ?g of recombinant GST-Pdcd4 was added to
lysates of JB6 P? cells that had been transiently transfected with Xpress-eIF4A
plasmid and incubated for 1 h at 4°C. Ten microliters of glutathione-Sepharose
beads (Pharmingen) were washed with 250 ?l of lysis buffer twice, added to the
cell lysates, and rotated for 2 h at 4°C. The beads were washed three times with
250 ?l of lysis buffer and boiled in SDS sample buffer. The bound proteins were
resolved by SDS-PAGE and detected by Pdcd4 or Xpress antibodies.
Mammalian two-hybrid assay of protein-protein binding. In the mammalian
two-hybrid assay, a luciferase reporter becomes activated when a DNA binding
domain (BD) fusion protein binds to an activation domain (AD) fusion protein.
pdcd4 and eIF4A cDNAs were inserted into pCMV-BD (Stratagene) and
pCMV-AD (Stratagene), respectively. Then 104JB6 P? cells were seeded in
24-well plates in Eagle’s minimal essential medium (EMEM) with 4% fetal
bovine serum. Cells were transfected with pCMV-BD-Pdcd4 (5 to 50 ng),
pCMV-AD-eIF4A (5 to 50 ng), Gal4-luciferase reporter gene (25 ng), and
thymidine kinase (TK)-Renilla luciferase gene (10 ng) with 2 ?l of Lipofectamine
(Invitrogen) for 4 h, and cells were then incubated with fresh EMEM with 4%
fetal bovine serum. After 48 h, cells were lysed in 1? passive lysis buffer (Pro-
mega), and luciferase activity was measured as previously described (49).
Immunohistochemistry analysis of Pdcd4 and eIF4A localization. JB6 P?
cells were grown on no. 1 glass coverslips in EMEM containing 4% fetal bovine
serum, washed twice in phosphate-buffered saline, fixed in 4% paraformalde-
hyde, and permeabilized in 0.5% Triton X-100. Cells were incubated with a 1:200
dilution of a rabbit polyclonal anti-eIF4A antibody. After being washed three
times with phosphate-buffered saline, the slides were incubated with goat anti-
rabbit immunoglobulin G coupled to fluorescein isothiocyanate (Molecular
Probes). After being washed three more times, the cells were incubated with
anti-Pdcd4 antibody previously labeled with Alexa Fluor 568 (Molecular Probes)
at a dilution of 1:100. The cells were washed twice with phosphate-buffered
saline, and nuclei were stained with 4?,6?-diamidino-2-phenylindole (1:10,000).
The cells were washed twice with phosphate-buffered saline and then mounted
with antifading mounting fluid (Prolong; Molecular Probes). Fluorescence im-
ages were obtained with the 63? objective of a Zeiss LSM 410 confocal micro-
scope at the National Cancer Institute Image Analysis Lab.
For quantification, we used image analysis to detect and quantify the colocal-
ization. The analysis consisted of first calculating the probability that real colo-
calization of the two proteins existed and, if so, then calculating the amount of
colocalization of each protein relative to the other. The first step in the analysis
was to crop the image to the region where both proteins were allowed to exist.
In this case the region was the cytoplasm of a cell. In the following text, “image”
refers only to this region.
The probability that real colocalization existed, in addition to the apparent
random colocalization seen when the signals from two proteins overlapped in the
color image, was calculated as follows. First, the Pearson correlation coefficient
(r) of the two images, which measures the degree of similarity of the patterns of
the proteins in the two images, was calculated (30). The r value ranges from 0 to
1; 1 indicates a high similarity of the patterns, suggesting real colocalization of
the two proteins, whereas 0 indicates only random colocalization. Next, the
spatial arrangement of the protein pattern in one of the images was randomized
in order to destroy any real (nonrandom) colocalization between the two pro-
teins, followed by calculating r again between the randomized region and the
region in the image of the other protein (rran). This process was repeated
hundreds of times in order to obtain a distribution of rranvalues. In the final step,
the initial r from the pair of images (that were not randomized) was compared to
the distribution of rranvalues. If r was significantly higher than the rranvalues, it
can be concluded that there is a significant probability that real colocalization
was present. The probability for each cell that the colocalization was the result of
a random overlap was estimated to be less than 0.1%.
Because it was concluded that real colocalization was present, the amount and
locations of real colocalization were estimated based on the assumption that
positions where the proteins were colocalized most likely corresponded to loca-
tions in the images where the same point in both images had high intensity. (A
point in an image is called a pixel, and in fluorescence images the intensity at a
given pixel is approximately proportional to the concentration of protein at the
equivalent position in the specimen.) The estimation was performed by succes-
sively removing pixel pairs from the images, starting with the pair with the highest
intensity and stopping when r calculated for the remaining pixels was 0 (implying
that only random colocalization remained). The pixels removed were marked as
those corresponding to real protein colocalization. Summing the intensities of
the colocalized pixels in its image and dividing the sum by the intensities of all the
pixels in its image calculated the degree of colocalization for each protein.
Helicase activity assay. The unwinding of duplex RNA was performed as
described previously (44). In brief, eIF4A was incubated with or without Pdcd4
in a 20-?l reaction which contained 2 nM RNA duplex, 1 mM ATP, 1 mM
MgCl2, 20 mM HEPES-KOH (pH 7.5), 70 mM KCl, 2 mM dithiothreitol, and
1 mg of bovine serum albumin per ml. The sequence of the long RNA strand was
C-3?, and the sequence of the short,32P-radiolabeled strand was 5?-GCUUUA
CGGUGC-3? or 5?-GCUUUACGGUGCU-3?. The duplex contained 12 bp or
Reaction mixes were incubated for 15 min at 35°C, and the reactions were
stopped with 5 ?l of a solution containing 50% glycerol, 2% SDS, 20 mM EDTA,
VOL. 23, 2003NOVEL eIF4A BINDING PROTEIN Pdcd4 27
and 0.05% each bromophenol blue and xylene cyanol dyes. Duplex and single
strands were resolved by gel electrophoresis on 15% native polyacrylamide gels
at 4°C for about 2 h at 200 V in 1? TBE (Tris-borate-EDTA) buffer. Radioac-
tivity was determined with an Ambis radioanalytic scanner, and the resulting data
were quantified as previously described (44). eIF4A, eIF4B, and eIF4F were
purified from rabbit reticulocyte lysate. Yeast Ded1p was provided by Eckhard
Jankowsky (Department of Biochemistry, Case Western Reserve University).
Analysis of Pdcd4 alone indicated that there was no nuclease or phosphatase
contamination (data not shown).
In vitro translation. Ten microliters of nuclease-treated rabbit reticulocyte
lysate (Promega) were mixed with 1 ?l of SUPERase In (40 U, Ambion), 1 ?l of
an amino acid mixture lacking methionine (1 mM each of the other amino acids),
0.2 ?g of bicistronic CAT/EMCV/LUC (47) mRNA, 15 ?Ci of [35S]methionine,
and His-Pdcd4 protein (0 to 4.8 ?g). The bicistronic CAT/EMCV/LUC mRNA
contains chloramphenicol acetyltransferase (CAT) and luciferase reporter genes.
Translation of the CAT open reading frame is cap dependent, whereas the
luciferase open reading frame is encephalomyocarditis virus (EMCV) IRES
The reaction mixture was added to buffer A [20 mM HEPES-KOH (pH 7.6),
100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol] to a final
volume of 20 ?l and incubated at 30°C for 1 h. The products of translation were
resolved by SDS-PAGE, fixed with 40% methanol–7% acetic acid, and treated
with Amplify (Amersham). The intensity of bands was determined by a Storm
850 Phosphorimager (Molecular Dynamics).
Transient-transfection assays of in vivo translation and of AP-1-dependent
transcription. An in vivo translation assay of the bicistronic reporter system was
based on that described previously (28). Briefly, 5 ? 104JB6 RT101 cells were
seeded in a six-well plate in EMEM with 4% fetal bovine serum. After transfec-
tion with 0 to 2 ?g of pcDNA-Pdcd4 (or pcDNA-Pdcd4D418A), 0.2 ?g of the
bicistronic reporter system, pcDNA-CAT/EMCV/LUC, and 10 ng of the TK-
Renilla luciferase gene, cells were allowed to recover for 12 to 18 h in EMEM
with 4% fetal bovine serum. Cells were then serum starved with 0.2% fetal
bovine serum in EMEM for 24 h and incubated in EMEM with 4% fetal bovine
serum for an additional 24 h. Cells were harvested for CAT assay and luciferase
assay. CAT activity was measured with the Quan-T-CAT kit (Amersham). The
radioactivity was determined with a Beckman LS 3801. Luciferase activity was
measured as described above. Transfection efficiency was normalized to Renilla
Transient transfection of the 4? AP-1–luciferase reporter and assay of tumor
promoter-induced activation of AP-1-dependent transcription were done as
previously described (49). Transfection efficiency was normalized to Renilla lu-
ciferase activity. In this assay, nonresponsiveness of other promoter-luciferase
constructs establishes that transcription, not luciferase translation, is being mea-
In vitro binding assay. Fifty micrograms of GST-eIF4G(672-1065) or GST-
eIF4G(1201-1445) recombinant protein immobilized on a 10-?l bed volume of
glutathione-Sepharose resin was incubated with 5 ?g of His-eIF4A in the pres-
ence or absence of 5 ?g of His-Pdcd4 in 10 ?l of binding buffer [20 mM Tris-HCl
(pH 7.5), 100 mM KCl, 2.5 mM Mg Cl2, 0.1 mM EDTA, 10% glycerol, 0.4%
Triton X-100] on ice for 20 min. The beads were washed three times with 250 ?l
of binding buffer and boiled in SDS sample buffer. The bound proteins were
resolved by SDS-PAGE and detected by immunoblot with penta-His antibody
(Qiagen), GST antibody (Santa Cruz Biotechnology), or Pdcd4 antibody, as
indicated. The Pdcd4 peptide antibody recognizes a single band of 64 kDa. This
Pdcd4 antibody has high specificity, since this 64-kDa band was erased by the
peptide that was used to generate this antibody. (49).
Identification of eIF4A as a Pdcd4-interacting protein in a
yeast two-hybrid screen. Overexpression of Pdcd4 in JB6 P?
cells suppresses tetradecanoyl phorbol acetate-induced trans-
formation and inhibits the AP-1 activation required for trans-
formation (49). However, ectopic introduction of AP-1 pro-
teins (Fra-1, JunD, or c-Jun) does not relieve inhibition of
AP-1-dependent transcription by Pdcd4. In addition, recombi-
nant GST-Pdcd4 did not pull down Fra-1 or c-Fos from tetra-
decanoyl phorbol acetate-treated P? cell lysates (data not
shown), indicating that Pdcd4 does not physically interact with
In order to identify the binding partner(s) of Pdcd4, we per-
formed a yeast two-hybrid screen of a mouse brain MATCH-
MAKER cDNA library with full-length Pdcd4 as the bait.
After several iterations of ?-galactosidase liftover assays, the
DNA sequences of clones 1 and 19 of 31 positive clones were
identical to translation initiation factor eIF4AI and eIF4AII,
respectively. Since eIF4AII is preferentially expressed in the
brain (33), the higher number of eIF4AII-positive clones ob-
served was expected. To further confirm this interaction, we
performed a coimmunoprecipitation with eIF4A antibody. As
shown in Fig. 1A, eIF4A antibody but not preimmune serum
coprecipitated Pdcd4 from lysates of (low-Pdcd4) JB6 P? cells
transfected with the Pdcd4 expression plasmid. We generated
recombinant GST-Pdcd4 from baculovirus-infected Sf-9 cells
and purified it with glutathione-Sepharose (Fig. 1B). GST-
Pdcd4 was able to pull down eIF4A from lysates of JB6 P?
cells transfected with the Xpress-tagged eIF4A expression
plasmid (Fig. 1C).
To demonstrate that the association of Pdcd4 and eIF4A
occurred in vivo, we used a mammalian two-hybrid system. The
pdcd4 cDNA and eIF4A cDNA were fused with the Gal4
DNA-binding domain (pCMV-BD) and NF-?B activation
domain (pCMV-AD), respectively. Since both plasmids
pCMV-BD and pCMV-AD contain nuclear localization sig-
nals, Gal4-Pdcd4 and NF-?B–eIF4A fusion proteins are able
to translocate into nuclei. As shown in Fig. 1D, the level of
luciferase expression was greatly increased when pCMV-BD-
Pdcd4 (bait) and pCMV-AD-eIF4A (prey) were cotransfected.
Transfection of pCMV-BD-Pdcd4 along with pCMV-AD
(empty vector) or of pCMV-BD (empty vector) with pCMV-
AD-eIF4A showed only the background level of luciferase
activity. These results indicate that Pdcd4 and eIF4A physically
interact in vivo and in vitro.
Pdcd4 colocalizes with eIF4A in the cytoplasm. Human
H731 is a homolog of Pdcd4 reported to be expressed in the
cytoplasm, the nucleus, or both (51, 52). However, based on
the data shown above, we would expect Pdcd4 to associate with
eIF4A in the cytoplasm. In order to address this question and
to further confirm the Pdcd4-eIF4A interaction in vivo, we
used multicolor confocal immunofluorescence microscopy to
ascertain the subcellular localizations of Pdcd4 and eIF4A in
JB6 P? cells. Both red-fluorescent Pdcd4 (Fig. 2A) and green-
fluorescent eIF4A (Fig. 2B) displayed diffuse cytoplasmic ex-
pression with concentrated perinuclear distribution. Although
all cells displayed colocalization in the cytoplasm, the perinu-
clear region appeared to have the most concentrated colocal-
ization (Fig. 2C). The antibody binding to Pdcd4 and eIF4A
was successfully erased by recombinant His-Pdcd4 protein and
native eIF4A protein purified from rabbit reticulocyte lysate,
respectively (data not shown).
Based on the analysis of seven randomly selected cells, the
average Pearson correlation in the cytoplasm between the two
proteins was 55% ? 11% (30). The Pearson correlation was
used as a cutoff value to select which pixels in the cytoplasm
were colocalized. Within the cytoplasm, 63% ? 6% of all
Pdcd4 proteins and 65% ? 6% of all eIF4A proteins were
determined to be compartmentalized within the perinuclear
region. Upon selection of the colocalized pixels in the perinu-
clear region, the approximate percent colocalization for each
protein was determined by dividing the sum of all colocalized
28YANG ET AL.MOL. CELL. BIOL.
pixel intensities by the total intensity of each component within
the region. By doing so, 54 ? 20% of all Pdcd4 proteins within
the perinuclear region were determined to be colocalized with
eIF4A. A similar analysis of all eIF4A within the perinuclear
region revealed that 58% ? 21% of all eIF4A proteins were
colocalized with Pdcd4. Thus, the ratio of Pdcd4-bound eIF4A
to free eIF4A was about 1:1. Small shifts away from this Pdcd4
bound to unbound ratio could be functionally significant.
Pdcd4 inhibits the helicase activity of eIF4A. To address
whether Pdcd4 alters the eIF4A helicase activity, we per-
formed a helicase activity assay with recombinant His-Pdcd4,
native eIF4A, and a synthetic duplex RNA. The helicase ac-
tivity of eIF4A was determined by measuring the unwinding of
a 12- or 13-bp RNA duplex (42, 44). As shown in Fig. 3A,
recombinant His-Pdcd4 inhibited eIF4A helicase activity in a
concentration-dependent manner. Helicase activity of eIF4A
was enhanced by eIF4B or as a subunit of eIF4F (1, 41, 43).
However, in the presence of recombinant His-Pdcd4, eIF4B
was not able to stimulate the eIF4A helicase activity (Fig. 3B
and 3C). Pdcd4 inhibited not only the free eIF4A helicase
activity, but also the helicase activity when eIF4A was a subunit
of eIF4F (Fig. 3B and 3C). Recombinant His-Pdcd4 inhibited
eIF4F helicase activity in a concentration-dependent manner
(Fig. 3C). These results indicate that Pdcd4 functions as a
dominant eIF4A inhibitor.
In the absence of eIF4A, recombinant His-Pdcd4 was not
able to unwind the RNA duplex (in either the presence or
absence of ATP), indicating that Pdcd4 does not have helicase
activity (data not shown). Recombinant His-Pdcd4 did not
inhibit the helicase activity of RNA helicase Ded1p up to a 4:1
Pdcd4-Ded1p molar ratio (Fig. 3D). Ded1p is a DEAD box
family helicase that is essential for translation initiation in
Saccharomyces cerevisiae (17). In contrast, Pdcd4 appeared to
inhibit eIF4A at about a 1:1 molar ratio of eIF4A to His-
Pdcd4. At a 4:1 His-Pdcd4 to eIF4A molar ratio, Pdcd4 inhib-
ited the helicase activities of eIF4A and eIF4F by approxi-
mately 70% (Fig. 3C, lanes 6 and 10). The inhibition noted in
Fig. 3A is similar to that observed with more stable duplexed
RNA (i.e., 18 to 20 bp), which was also inhibited at a less than
a 1:1 ratio (42). Thus, Pdcd4 specifically suppresses eIF4A
Pdcd4 inhibits translation. The helicase activity of eIF4A is
critical for translation. Mutational inactivation of the helicase
activity of eIF4A inhibits protein translation in vitro (36). Since
Pdcd4 inhibits the helicase activity of eIF4A (Fig. 3), Pdcd4
would be predicted to inhibit protein translation. A test of this
hypothesis measured cap- and IRES-dependent translation in
vitro with a capped bicistronic CAT/EMCV/LUC mRNA in a
rabbit reticulocyte lysate assay to which recombinant His-
Pdcd4 was added. Translation of the CAT open reading frame
is cap dependent, whereas translation of the luciferase open
reading frame is EMCV IRES-dependent. Since nickel resin-
bound proteins purified from uninfected Sf-9 cells did not
affect in vitro translation, the proteins copurified with His-
Pdcd4 from nickel resin were assumed not to inhibit transla-
tion (data not shown).
Addition of native eIF4A to the reticulocyte lysate did not
affect either cap- or IRES-dependent translation (Fig. 4A,
compare lanes 1 and 2), in agreement with previous observa-
tions (36) that addition of eIF4A did not alter the rate of
translation. Addition of recombinant His-Pdcd4 (0.15 to 2.4
?g) to the reticulocyte lysate inhibited both cap-dependent and
IRES-dependent translation in a concentration-dependent
manner (Fig. 4A, lanes 2 to 7). Addition of eIF4F (0.75 and 1.5
?g) purified from rabbit reticulocyte lysates produced a partial
recovery of cap-dependent translation but not of IRES-depen-
FIG. 1. Identification of Pdcd4 binding to eIF4A. (A) Coimmuno-
precipitation of Pdcd4 with eIF4A. JB6 P? cell lysates isolated fol-
lowing transient transfection with Pdcd4 expression plasmid were im-
munoprecipitated (IP) with goat serum (lane 2) or eIF4A antibody
(Ab) (lane 3). The immunoprecipitates were resolved by SDS–10%
PAGE followed by immunoblotting with Pdcd4 antibody. Lane 1
shows one-tenth of the cell lysates. (B) Coomassie blue staining of
GST-Pdcd4. One microgram of GST-Pdcd4 expressed in SF-9 cells and
purified from a glutathione column as described in Materials and
Methods was loaded onto SDS-PAGE (10%) and stained with Simple-
Blue (Invitrogen). Lane M, protein molecular size markers. (C) GST
pull-down of eIF4A with Pdcd4. JB6 P? cell lysates isolated following
transient transfection with Xpress-tagged eIF4A expression plasmid
were pulled down with GST (lane 2) or GST-Pdcd4 (lane 3). The
bound proteins were resolved by SDS–10% PAGE followed by immu-
noblotting with Xpress antibody. Lane 1 shows one-tenth of the cell
lysates. (D) Mammalian two-hybrid assay of Pdcd4 binding to eIF4A.
Various amounts (5, 10, and 50 ng) of plasmid pCMV-BD-Pdcd4 (or
its empty vector, pCMV-DB) and pCMV-AD-eIF4A (or its empty
vector, pCMV-AD) along with the Gal4-luciferase reporter gene were
cotransfected into JB6 P? cells. After 48 h, cells were lysed, and the
luciferase activity was measured. The luciferase activity from the cells
with 5 ng of pCMV-BD-Pdcd4 and 5 ng of pCMV-AD-eIF4A was
designated as 1. These experiments were repeated three times, each
with five independent transfections, and representative data are
shown. Results are expressed as the mean ? standard deviation. RLU,
relative luciferase units.
VOL. 23, 2003 NOVEL eIF4A BINDING PROTEIN Pdcd429
dent translation (Fig. 4B). However, addition of native eIF4A
(1.5 ?g and 3.0 ?g) purified from rabbit reticulocyte lysates did
not relieve the inhibition of either CAT or luciferase transla-
tion by recombinant His-Pdcd4 (0.6 ?g) (Fig. 4A, lanes 8 to
10), suggesting that the mechanism of inhibition of translation
by Pdcd4 is not simply to quench or sequester eIF4A activity.
To investigate whether Pdcd4 inhibits translation in vivo, the
pcDNA-Pdcd4 expression plasmid and the cytomegalovirus-
driven bicistronic CAT/IRES/LUC reporter system, pcDNA-
CAT/EMCV/LUC, were transiently cotransfected into (low-
Pdcd4) JB6 RT101 cells. The effect of Pdcd4 on translation was
measured by CAT and luciferase activity. Transfection with
increasing concentrations of pcDNA-Pdcd4 DNA produced a
concentration-dependent decrease in translation of the CAT
reporter (Fig. 5A). Transfection of 2 ?g of pcDNA-Pdcd4
DNA inhibited CAT expression (cap-dependent translation)
by 40 to 45%. It is noteworthy that inhibiting protein transla-
tion by 50% is sufficient to alter a cell’s physiological function
(48). For example, tumstatin inhibits cap-dependent transla-
tion in endothelial cells by 25 to 45%, with the result that
activation of phosphatidylinositol 3-kinase and Akt kinase was
inhibited and apoptosis was stimulated (28, 29). In contrast,
Pdcd4 did not inhibit luciferase expression (IRES-dependent
translation) at a concentration of up to 2 ?g of pcDNA-Pdcd4
DNA, suggesting that Pdcd4 preferentially inhibits cap-depen-
dent translation in vivo. Greater selectivity was seen in vivo
than in vitro, a not uncommon observation. Since the CAT and
luciferase reporter genes are located on the same bicistronic
mRNA, and since luciferase expression was unaffected, it fol-
lows that CAT transcription was also unaffected by Pdcd4.
Thus, the inhibition of cap-dependent CAT expression by
Pdcd4 is occurring at the level of translation. A Pdcd4 mutant,
Pdcd4D418A, in which glutamine 418 was changed to alanine
showed a dramatic decrease in binding to eIF4A when ex-
pressed in JB6 RT101 cells (Fig. 5B). Transfection of pcDNA-
Pdcd4D418ADNA with the bicistronic reporter system into
RT101 cells produced no inhibition of CAT or luciferase ex-
pression (cap-dependent and IRES-dependent translation, re-
spectively) (Fig. 5C). These results indicate that binding of
Pdcd4 to eIF4A is required for Pdcd4 to inhibit translation
Pdcd4D418does not suppress AP-1-dependent transcription
in JB6 P? cells. To determine whether Pdcd4 binding to
eIF4A is physiologically significant, we asked whether the
Pdcd4D418Amutant is defective in inhibiting AP-1-dependent
transcription. Wild-type Pdcd4 inhibits AP-1-dependent tran-
scriptional activation but not NF-?B or ornithine decarboxyl-
ase activation in JB6 P? cells (49). AP-1 transactivation is one
of the few molecular events known to be required for tumor
promoter-induced neoplastic transformation in JB6 P? cells
(15) and in mouse skin tumor promotion in vivo (53), and AP-1
activation is the only transformation-relevant molecular event
known to be targeted by Pdcd4 (49). Inhibition of AP-1-de-
pendent transcription by dominant negative c-jun suppresses
tumor promoter-induced transformation in JB6 cells (9) and
tumorigenesis in mouse skin (53).
As shown in Fig. 6A, transient cotransfection of wild-type
Pdcd4 expression plasmid with the 4? AP-1-LUC reporter
into JB6 P? cells inhibited both basal and tetradecanoyl phor-
bol acetate-induced AP-1-dependent luciferase expression in a
concentration-dependent manner, in agreement with previous
observations (49). In contrast, cotransfection of Pdcd4D418A
expression plasmid with the 4? AP-1 reporter did not inhibit
basal or tetradecanoyl phorbol acetate-induced AP-1-depen-
dent luciferase expression (Fig. 6B). Wild-type Pdcd4 inhibits
the luciferase expression driven by the 4? AP-1 promoter but
not that driven by the NF-?B or serum response element
promoter (49). Since these three luciferase reporter plasmids
have identical sequences in their 5?untranslated regions, there
is no basis for differential Pdcd4 effects at the level of luciferase
translation. Thus, Pdcd4 is acting, albeit indirectly, to inhibit
AP-1-dependent transcription. Because Pdcd4D418Adoes not
FIG. 2. Immunofluorescent detection of colocalization of Pdcd4 and eIF4a in JB6 P? cells. P? cells were immunostained for Pdcd4 (A) and
eIF4A (B) and viewed by confocal microscopy. (C) The merged images of panels A and B display a yellow color indicative of colocalization of the
two proteins to the perinuclear region of the cytoplasm. The cell shown is a representative example of multiple P? cells. Of seven randomly
selected cells, 63% ? 5% of total Pdcd4 was colocalized with the eIF4A, and 65% ? 6% of total eIF4A was colocalized with Pdcd4 in the
cytoplasm. The nucleus, which did not show either Pdcd4 or eIF4A staining, was masked for the purpose of limiting the area and therefore the
stringency by which the spatial statistical algorithm could test whether the colocalization was the result of random overlap. The estimated
probability that the colocalization was due to random overlap was 0.1%. Bar, 15 ?m.
30YANG ET AL.MOL. CELL. BIOL.
bind to eIF4A (Fig. 5B), the results shown in Fig. 6 suggest that
Pdcd4 binding to eIF4A is required for suppression of AP-1-
Pdcd4 prevents eIF4A association with the C-terminal one-
third but not the middle one-third of eIF4G. Analysis of the
Pdcd4 protein sequence reveals a recognizably conserved fea-
ture, two ?-helical MA-3 domains (also designated MI do-
mains) (2, 39). The function of the MA-3 domain is not well
understood. This domain is also found in the C-terminal one-
third of eIF4G and located within the second eIF4A binding
region (32) (Fig. 7A). This location suggests that the MA-3
domain may play an important role in the binding of eIF4A. If
this hypothesis is correct, Pdcd4 should be able to compete
with the C-terminal one-third of eIF4G for binding to eIF4A.
To address this question, we performed an in vitro binding
assay that used recombinant His-Pdcd4, recombinant His-
eIF4A (32), GST-eIF4G(672 to 1065) (middle region) (32),
and GST-eIF4G(1201 to 1445) (C-terminal region) (32) (Fig.
7B). GST-eIF4G(672-1065) and GST-eIF4G(1201-1445) were
immobilized on glutathione-Sepharose beads and incubated
with His-eIF4A in the presence and absence of His-Pdcd4. The
bound protein was resolved by SDS-PAGE and analyzed by
immunoblotting with GST, penta-His, and Pdcd4 antibodies
(Fig. 7C). His-eIF4A bound to GST-eIF4G(1201-1445) (Fig.
7C, lane 3), but His-Pdcd4 did not (Fig. 7C, lane 2). Addition
of His-Pdcd4 along with eIF4A to the binding reaction mixture
that included eIF4G(1201-1445)-bound beads (ratio of His-
eIF4A to His-Pdcd4 of 1:1) dramatically decreased (five- to
sixfold, as determined by densitometry) the association of His-
eIF4A with GST-eIF4G(1201-1445) (Fig. 7C, compare lanes 3
FIG. 3. Inhibition of eIF4A RNA helicase activity by Pdcd4. (A) Pdcd4 inhibits eIF4A helicase activity. Unwinding of a 2 nM RNA duplex (12
bp; ?G ? ?21.4 kcal/mol) by 1.5 ?M eIF4A (open circles) was performed as described in Materials and Methods. As a control (solid squares),
eIF4A was incubated with the duplex in the absence of ATP. Separate controls indicated that there was no unwinding by Pdcd4 in the presence
of 1 mM ATP (not shown). After the 15-min incubation at 35°C, unwinding was quantitated by gel electrophoresis and subsequent analysis with
an Ambis radioanalytic scanner. (B) Pdcd4 inhibits eIF4F helicase activity and eIF4B does not stimulate eIF4A helicase activity in the presence
of Pdcd4. A 2 nM RNA duplex (13 bp; ?G ? ?23.1 kcal/mol) was incubated with the following proteins: 13.5 pmol of eIF4A (lane 4), 13.5 pmol
of eIF4A plus 27 pmol of Pdcd4 (lane 5), 6.75 pmol of eIF4A (lane 6), 6.75 pmol of eIF4A plus 6.75 pmol of eIF4B (lane 7), 6.75 pmol of eIF4A
plus 6.75 pmol of eIF4B plus 13.5 pmol of Pdcd4 (lane 8), 6.75 pmol of eIF4F (lane 9), or 6.75 pmol of eIF4F plus 13.5 pmol of Pdcd4 (lane 10)
for 15 min at 35°C. Samples were separated and quantitated as described in Materials and Methods. Lane 1, duplex RNA incubated under
unwinding conditions without protein for 15 min at 0°C; lane 2, duplex RNA incubated under the same conditions for 5 min at 95°C; lane 3, duplex
RNA incubated under the same conditions without ATP and proteins for 15 min at 35°C. (C) Pdcd4 inhibits helicase activities of eIF4A plus eIF4B
and eIF4F in a concentration-dependent manner. Unwinding of a 2 nM RNA duplex (13 bp; ?G ? ?23.1 kcal/mol) by eIF4A (6.75 pmol) plus
eIF4B (6.75 pmol) without (lane 3) or with increasing concentrations of Pdcd4 (3.4 to 13.5 pmol, lanes 4 to 6) or by eIF4F (6.75 pmol) without
(lane 7) or with increasing concentrations of Pdcd4 (3.4 pmol to 13.5 pmol, lanes 8 to 10) was performed as described in Materials and Methods.
Lane 1, duplex RNA incubated under unwinding conditions without protein for 15 min at 0°C; lane 2, duplex RNA incubated under the same
conditions for 5 min at 95°C. (D) Pdcd4 does not inhibit Ded1p helicase activity. Unwinding of a 2 nM RNA duplex (13 bp; ?G ? ?23.1 kcal/mol)
by Ded1p helicase (0.5 pmol) in the absence (lane 4) or presence (lanes 5 and 6, 1 and 2 pmol, respectively) of Pdcd4 was performed as described
in Materials and Methods. Lane 1, duplex RNA incubated under unwinding conditions without protein for 15 min at 0°C; lane 2, duplex RNA
incubated under the same conditions for 5 min at 95°C.
VOL. 23, 2003 NOVEL eIF4A BINDING PROTEIN Pdcd4 31
and 4). However, addition of His-Pdcd4 with eIF4A to the
reaction mixture that included GST-eIF4G(672-1065)-immo-
bilized beads showed an unchanged level of association of
His-eIF4A with GST-eIF4G(672-1065) (Fig. 7C, compare
lanes 7 and 8). These results indicate that Pdcd4 prevents
eIF4A from binding to the C-terminal one-third of eIF4G but
does not prevent eIF4A from binding to the middle one-third
Pdcd4 Binds to the middle one-third of eIF4G. Figure 7C
shows that His-Pdcd4 also associates with eIF4G(672-1065) in
the absence or presence of eIF4A (lanes 6 and 8, anti-His- and
anti-Pdcd4 immunoblots). The independence of Pdcd4 and
eIF4A binding to the middle domain of eIF4G suggests that
the Pdcd4 binding site on eIF4G(672-1065) is different from
the site on eIF4G(672-1065) that binds to eIF4A.
To seek independent verification that Pdcd4 is able to bind
to the middle domain of eIF4G, we performed a GST pull-
down. As shown in Fig. 6D, GST-eIF4G(672-1065) was able to
pull down eIF4A from lysates of JB6 P? cells transiently trans-
fected with pcDNA3.1 (vector), pcDNA-Pdcd4 (Pdcd4) or
pcDNA-Pdcd4D418A(Pdcd4D418A) (Fig. 6D, lanes 3, 6, and 9;
anti-eIF4A immunoblot). GST-eIF4G(672-1065) also pulled
down Pdcd4 and Pdcd4D418from lysates of cells transfected
with pcDNA-Pdcd4 and pcDNA-Pdcd4D418A, respectively
(Fig. 6D, lanes 6 and 9, anti-Pdcd4 immunoblot). Since
Pdcd4D418Adoes not bind to eIF4A (Fig. 5B), the results
shown in Fig. 7D indicate that Pdcd4 binding to the eIF4G
middle domain occurs independently of eIF4A-Pdcd4 binding.
We also used GST-Pdcd4 to test whether Pdcd4 binds to full-
length endogenous eIF4G. As expected, GST-Pdcd4 pulled
down endogenous eIF4G from JB6 P? cell lysates (Fig. 7E,
lane 3) but GST did not (Fig. 7E, lane 2). Binding of the
human Pdcd4 homolog H731 to eIF4G has also been reported
The GST-eIF4G(672-1065) and GST-eIF4G(1201-1445) im-
FIG. 4. Inhibition of in vitro translation by Pdcd4. (A) Rabbit re-
ticulocyte lysate was preincubated with eIF4A alone (lane 1), with
increasing amounts of Pdcd4 (0.15 to 4.8 ?g, lanes 2 to 7), or with
Pdcd4 and increasing amounts of eIF4A (lanes 8 to 10) for 5 min at
30°C prior to the addition of the bicistronic CAT/EMCV/LUC mRNA
(0.2 ?g). Translation was performed in a total volume of 20 ?l as
described in Materials and Methods. The band intensity was deter-
mined by Phosphorimager. The value obtained for both cap- and
IRES-dependent translation in the absence of added Pdcd4 and eIF4A
was designated as 100%. (B) Rabbit reticulocyte lysate was incubated
with eIF4F (lanes 1 to 3) or eIF4F and Pdcd4 (lanes 4 to 6) for 5 min
at 30°C prior to the addition of the bicistronic CAT/EMCV/LUC
mRNA (0.2 ?g). Translation was performed in a total volume of 20 ?l
as described in Materials and Methods. The band intensity was deter-
mined by Phosphorimager. The value obtained for both cap- and
IRES-dependent translation in the absence of added Pdcd4 and eIF4F
was designated as 100%.
FIG. 5. Pdcd4 inhibits translation in vivo. (A and C) The plasmid pcDNA-CAT/EMCV/LUC reporter system (0.2 ?g) was transiently
transfected with (A) pcDNA-Pdcd4 (0 to 2 ?g) or (C) pcDNA-Pdcd4D418A(0 to 2 ?g) into JB6 RT101 cells. Total DNA was maintained at 2.2
?g by adding pcDNA3.1? vector DNA. After transfection, cells were serum starved (0.2% fetal bovine serum) for 24 h and then incubated with
normal medium (4% fetal bovine serum) for an additional 24 h. The CAT and luciferase activities from the cells with 0 ?g of pcDNA-Pdcd4 (A) or
pcDNA-Pdcd4D418A(C) transfection was designated as 100%. These experiments were repeated three times in triplicate, and representative data
are shown. Results are expressed as mean ? standard deviation. * and ** indicate significant differences compared with the control as determined
by Student’s t test (*, ?0.005; **, ?0.0001). (B) Pdcd4D418Amutant does not bind to eIF4A. Plasmid pCMV-BD-Pdcd4 (50 ng) (wild type [WT])
or pCMV-BD-Pdcd4D418A(50 ng) (D418A) was transiently transfected with pCMV-AD-eIF4A (50 ng) and Gal4-luciferase reporter DNA (25 ng)
into JB6 RT101 cells. After 48 h, cells were lysed and luciferase activity was measured. The luciferase activity of wild-type Pdcd4 was designated
as 100%. These experiments were repeated three times with five independent transfections each, and representative data are shown. Results are
expressed as mean ? standard deviation. The inset shows an immunoblot of RT101 cells transiently transfected with pcDNA3.1? (lane 1),
pcDNA-Pdcd4 (lane 2), or pcDNA-Pdcd4D418A(lane 3) and detected with Pdcd4 antibody.
32 YANG ET AL.MOL. CELL. BIOL.
mobilized on glutathione-Sepharose beads used in the above
experiments were analyzed by immunoblotting with GST an-
tibody to indicate that similar amounts were bound (Fig. 7C).
In summary, Pdcd4 interferes with eIF4A binding to the C-
terminal but not to the middle-domain binding site on eIF4G,
and Pdcd4 itself binds to the middle domain of eIF4G. The
domains on Pdcd4 that bind to eIF4A and to eIF4G appear to
This study demonstrates that the transformation suppressor
Pdcd4 physically associates with the translation initiation fac-
tor eIF4A, resulting in inhibition of helicase activity (Fig. 3)
and translation (Fig. 4 and 5). The inhibition of translation
requires Pdcd4 binding to eIF4A, as Pdcd4D418A, a mutant
inactivated for binding to eIF4A, has no effect on translation
(Fig. 5C). These findings are in agreement with previous ob-
servations that mutational inhibition of the helicase and/or
ATPase activity of eIF4A inhibits translation (35–37, 47).
Therefore, Pdcd4 is not only a novel eIF4A binding protein, it
is the first example of a protein that inhibits translation
through inactivation of eIF4A activity.
Wild-type Pdcd4 but not the Pdcd4D418Amutant inhibited
AP-1-dependent transcription (Fig. 6). Inhibition of AP-1 is
sufficient to inhibit tumor promotion, both in the JB6 cell
model and in mouse skin carcinogenesis in vivo (9, 53). The
lack of eIF4A binding by the mutant Pdcd4 appears to disable
the inhibition of translation and consequently to disable the
transrepression of AP-1 that contributes to Pdcd4’s suppres-
sion of transformation. Pdcd4D418Ais not inactivated for all its
activities, as it retains the ability to bind the middle domain of
eIF4G (Fig. 7D). Taken together, these results suggest that the
loss of binding to eIF4A results in the loss of a transformation-
How does Pdcd4 inhibit translation? A model to explain the
mechanism underlying the translational inhibition by Pdcd4
must take into account the findings that (i) Pdcd4 inhibits the
helicase activity of eIF4A, (ii) Pdcd4 blocks eIF4A binding to
the C-terminal one-third of eIF4G, (iii) Pdcd4 binds to the
middle one-third of eIF4G independently of eIF4A binding,
and (iv) the Pdcd4 domains for binding to eIF4A and the
middle domain of eIF4G are distinct (Fig. 8). The C-terminal
domain of eIF4G is a regulatory domain for translation, and its
binding to eIF4A has been shown to greatly enhance transla-
tion (32). Therefore, prevention of eIF4A binding to the C-
terminal domain by Pdcd4 (Fig. 7C) contributes to inhibition
eIF4A exchanges between free eIF4A and eIF4A bound in
the eIF4F complex (36). Biochemical and kinetic studies have
shown that eIF4A may proceed through two to three associa-
tion-dissociation cycles at the beginning of translation initia-
tion to unwind double-stranded RNA (42, 43). Blocking this
process inhibits translation. Pdcd4 binding to the middle one-
third of eIF4G independent of eIF4A (Fig. 7C and 7D) sug-
gests that eIF4A may be trapped by Pdcd4 on the middle
domain of eIF4G, thus blocking the association-dissociation
cycle of eIF4A through eIF4F. The observation (Fig. 4A) that
addition of eIF4A to the rabbit reticulocyte lysate does not
relieve inhibition of translation by Pdcd4 further supports this
model. eIF4A, when trapped in a Pdcd4-inactivated form
bound to eIF4G, would not be displaced by added free eIF4A.
Further testing of this model will be important.
Our data do not indicate the stoichiometry for the ratio of
eIF4G and eIF4A. One molecule of eIF4A may associate with
one molecule of eIF4G to form a “sandwich,” as proposed by
Morino et al. (32) (Fig. 8A), or two molecules of eIF4A may
associate with one molecule of eIF4G. Recently, two groups
have proposed a stoichiometry for eIF4G to eIF4A. Korneeva
et al. (19), using surface plasmon resonance techniques and
recombinant eIF4G proteins, showed a 1:2 ratio for eIF4G to
eIF4A. On the other hand, Li et al. (24), using immunopre-
cipitation of endogenous and tagged eIF4A, concluded that 1:1
was the ratio for eIF4G to eIF4A.
Two human Pdcd4 homologs, H731-L and H731, are 96%
and 93% identical, respectively, in amino acid sequence to
mouse Pdcd4. H731-L and H731 are alternative transcripts of
the same gene. H731 lacks 11 amino acids in the N-terminal
region that are present in H731-L and Pdcd4. H731 was iden-
tified and isolated with the Pr-28 antibody, which recognizes a
nuclear antigen in proliferating cells (31). Recent studies by
Yoshinaga et al. (52) used the human H731 antibody to deter-
mine the expression and localization of H731 in several cell
lines and tissues. H731 was abundantly expressed and localized
in the cytoplasm of cancer cells. In normal cells, however,
FIG. 6. Pdcd4D418Adoes not inhibit AP-1-dependent transcription.
JB6 P? cells were transfected with 0.2 ?g of the 4? AP-1 luciferase
reporter gene and increasing amounts (0 to 0.8 ?g) of pcDNA-Pdcd4
(A) or pcDNA-Pdcd4D418A(B). Total DNA was maintained at 1.0 ?g
by adding pcDNA3.1? vector DNA. The luciferase activity of cells
treated with tetradecanoyl phorbol acetate (TPA) and without
pcDNA-Pdcd4 or pcDNA-Pdcd4D418Awas designated as 100%. These
experiments were repeated three times in triplicate, and representative
data are shown. Results are expressed as mean ? standard deviation.
* and ** indicate significant differences compared with the control
(tetradecanoyl phorbol acetate or dimethyl sulfoxide [DMSO] treat-
ment following transfection with 0 ?g of pcDNA-Pdcd4) as deter-
mined by Student’s t test (*, ?0.005; **, ?0.0001).
VOL. 23, 2003NOVEL eIF4A BINDING PROTEIN Pdcd4 33
FIG. 7. Prevention of eIF4A binding to the C-terminal but not to the middle one-third of eIF4G by Pdcd4: Pdcd4 binds to the middle one-third
of eIF4G. (A) Structures of eIF4G1 and Pdcd4. The numbers refer to the size (in amino acids) of eIF4G1 and Pdcd4 and to the locations of the
eIF4A binding domain and MA-3 domain (2, 32, 40). eIF4A binding domains (open box and arrows) in eIF4G1 are indicated schematically. The
MA-3 domains (grey box) in eIF4G1 and Pdcd4 are indicated schematically. (B) Coomassie blue staining of recombinant GST-eIF4G(672-1065),
GST-eIF4G(1201-1445), His-eIF4A, and His-Pdcd4. Three micrograms of each recombinant GST-eIF4G(672-1065) (lane 2), GST-eIF4G(1201-
1445) (lane 3), His-Pdcd4 (lane 4), and His-eIF4A (lane 5) was resolved by SDS-PAGE and stained with SimpleBlue (Invitrogen). Lane 1, protein
34 YANG ET AL.MOL. CELL. BIOL.
H731 was localized in the nuclei. These observations are in
disagreement with our observations of Pdcd4 localization and
differential expression. First, the level of Pdcd4 expression in
the (less progressed) JB6 P? cells was about 8- to 10-fold
higher than that in JB6 P? cells (49), and transformed JB6
cells (unpublished data). Second, the results of immunofluo-
rescent confocal microscopy analysis indicate that Pdcd4 is
colocalized with eIF4A in the cytoplasm (Fig. 2). In addition,
the immunoprecipitation and GST pulldowns showing that
Pdcd4 physically interacts with eIF4A (Fig. 1) and eIF4G (Fig.
7) provide further support for cytoplasmic localization of
It is unknown whether the two highly identical proteins,
Pdcd4 and H731L, localize differently. The nuclear localization
of H731 might be attributed to differential tissue specificity or
to the use of different antibodies. It is noteworthy that the
Pdcd4 antibody used in the present studies shows high speci-
ficity (49), whereas the H731 antibody recognizes several pro-
teins ranging in molecular mass from 51 to 64 kDa (5).
Comparison of the Pdcd4 protein sequence with proteins in
the GenBank database reveals two ?-helical MA-3 domains
that are located from amino acids 163 to 284 and amino acids
326 to 449 (Fig. 7A). The MA-3 domain extends over approx-
imately 120 amino acids with 80 to 85% consensus secondary
structure; although there are many conserved amino acids in
the MA-3 domain, no specific consensus sequence has been
reported (2, 39). In human and mouse eIF4G, the MA-3 do-
main is located within the second eIF4A binding domain (ami-
no acids 1201 to 1441) (32) (Fig. 7A), implying that the MA-3
domain may play an essential role in binding eIF4A. The
finding that Pdcd4 prevents eIF4A from binding to the C-
terminal one-third of eIF4G (Fig. 7C) supports this hypothesis.
Indeed, deletion or mutation of either MA-3 domain in the
Pdcd4 protein dramatically inactivated the binding to eIF4A in
a mammalian two-hybrid assay (Fig. 5B and unpublished data),
indicating that the MA-3 domain is required for binding
Several tumors and tumor cell lines show elevated levels of
translation initiation factors such as eIF4E (7), eIF4A (10),
and eIF4G (3). Overexpression of eIF4E (22) or eIF4G (11)
FIG. 8. Model of how Pdcd4 inhibits translation. (A) A sandwich model of eIF4A binding to eIF4G is shown (32). The eIF4A molecule binds
to the middle and C-terminal one-third of eIF4G. (B) Model for Pdcd4 inhibition of translation. Pdcd4 inhibits the helicase activity of eIF4A. This
inactivated eIF4A molecule is further trapped by Pdcd4 on the middle one-third of eIF4G. This process will block eIF4A association-dissociation
cycling and keep the eIF4F complex inactivated. In addition, Pdcd4 also prevents from eIF4A binding to the C-terminal one-third of eIF4G. This
will prevent eIF4A from stimulating the activity of eIF4F.
molecular size markers. (C) In vitro binding assay. Bovine liver GST (lanes 1 and 5), recombinant GST-eIF4G(1201-1445) (lanes 2 to 4), or
GST-eIF4G(672-1065) (lanes 6 to 8) was immobilized on glutathione-Sepharose beads and incubated with 5 ?g of His-Pdcd4 only (lanes 2 and
6), 5 ?g of His-eIF4A only (lanes 3 and 7), or 5 ?g of both His-Pdcd4 and His-eIF4A (lanes 1, 4, 5, and 8) on ice for 10 min. After being washed
with binding buffer, the bound proteins were resolved by SDS-PAGE and analyzed by immunoblotting with GST antibody (first panel), penta-His
antibody (second panel), or Pdcd4 antibody (third panel). Ten percent of input His-Pdcd4 and His-eIF4A proteins were subjected to SDS-PAGE
followed by immunoblotting with penta-His antibody (fourth panel). GST-eIF4G(672-1065) and GST-eIF4G(1201-1445) immobilized on gluta-
thione-Sepharose beads were shown as similar amounts. (D) Pulldown of Pdcd4 and Pdcd4D418Awith GST-eIF4G(627-1065). JB6 P? cell lysates
isolated following transient transfection with pcDNA3.1? (lanes 1 to 3), pcDNA-Pdcd4 (lanes 4 to 6), or pcDNA-Pdcd4D418A(lanes 7 to 9) were
pulled down with GST (lanes 2, 5, and 8) or GST-eIF4G(627-1065) (lanes 3, 6, and 9). The bound proteins were resolved by SDS–10% PAGE
followed by immunoblotting with Pdcd4 or eIF4A antibodies. (E) GST pulldown of endogenous eIF4G with Pdcd4. JB6 P? cell lysate was pulled
down with GST (lane 2) or GST-Pdcd4 (lane 3). The bound proteins were resolved by SDS–10% PAGE followed by immunoblotting with eIF4G
antibody. Lane 1 shows one-tenth of the cell lysate.
VOL. 23, 2003NOVEL eIF4A BINDING PROTEIN Pdcd4 35
resulted in transformation of NIH 3T3 cells, suggesting that
translation factors may function as oncogenes. Therefore,
downregulation or inactivation of translation factors may sup-
press transformation. How does Pdcd4 suppress tetradecanoyl
phorbol acetate-induced neoplastic transformation in JB6
cells? A small number of molecular events are known to be
required for tumor promoter-induced transformation of JB6
P? cells and tumorigenesis in vivo. Among these required
molecular events are activation of transcription factors AP-1,
NF-?B, and serum response element as well as ornithine de-
carboxylase activation (15). Of these events, wild-type Pdcd4
inhibits only AP-1 activation (49).
The mRNAs that are translational targets of Pdcd4 are un-
known. One possibility is that Pdcd4 inhibits the translation of
AP-1 proteins or of enzymes or coactivators required for their
activation. Mitogen treatment of cells greatly stimulates the
translation of a group of so-called “translationally repressed”
mRNAs (4, 40). This group of mRNAs are often involved in
cell proliferation. Included are mRNAs for growth factors,
growth promotion genes, and proto-oncogenes (7) that contain
long GC-rich 5? untranslated regions having the potential to
form stable secondary structure(s) at the 5? end. Translation of
this group of mRNAs may be inefficient and highly dependent
on the eIF4A helicase activity (20, 21). Inhibiting or decreasing
eIF4A helicase activity would be expected to limit translation
of the translationally repressed mRNAs resulting in the sup-
pression of cell growth or transformation. For instance, muta-
tion of eIF4A in Schizosaccharomyces pombe inhibited trans-
lation of cdc25 but not of cdc2 and arrested cells in the G2
phase. Deletion of the 5? untranslated region of cdc25 restored
cdc25 translation (6).
In a related study, we found an inverse relationship between
Pdcd4 expression and proliferation within a number of tissues,
but especially the cervical epithelia of mice during estrus,
which includes a cyclical period of actively proliferating cervi-
cal epithelium (A. Jansen, unpublished data). Recent studies
by Svitkin et al. (47), with mRNAs varying in stability of sec-
ondary structure and eIF4A mutants, showed that the more
stable the secondary structure within the 5? untranslated region
of mRNA, the lower the efficiency of translation. These results
further support the idea that the requirement for eIF4A in
translation is proportional to the stability of the secondary
structure within the 5? untranslated region.
In summary, suppression of eIF4A helicase activity and/or
interference with eIF4A binding to eIF4G by Pdcd4 may sup-
press the translation of a set of mRNAs that limits the activa-
tion of AP-1 or other molecular events required for transfor-
mation in JB6 cells. Identification of the genes that are most
sensitive to translational inhibition by Pdcd4 will be important.
We thank Ed Cho for technical assistance for microscopy, Eckhard
Jankowsky and Wen Wang (Department of Biochemistry, Case West-
ern Reserve University) for providing purified yeast Ded1p as a His6-
tagged protein expressed from a plasmid provided by Patrick Linder,
and Terry Copeland for advice and synthesis of Pdcd4 peptides.
1. Abramson, R. D., T. E. Dever, T. G. Lawson, B. K. Ray, R. E. Thach, and
W. C. Merrick. 1987. The ATP?dependent interaction of eukaryotic initia-
tion factors with mRNA. J. Biol. Chem. 262:3826–3832.
2. Aravind, L., and E. V. Koonin. 2000. Eukaryote-specific domains in transla-
tion initiation factors: implications for translation regulation and evolution
of the translation system. Genome Res. 10:1172–1184.
3. Bauer, C., I. Diesinger, N. Brass, H. Steinhart, H. Iro, and E. U. Meese. 2001.
Translation initiation factor eIF-4G is immunogenic, overexpressed, and
amplified in patients with squamous cell lung carcinoma. Cancer 92:822–829.
4. Brown, E. J., and S. L. Schreiber. 1996. A signaling pathway to translational
control. Cell 86:517–520.
5. Cmarik, J. L., H. Min, G. Hegamyer, S. Zhan, M. Kulesz-Martin, H. Yoshi-
naga, S. Matsuhashi, and N. H. Colburn. 1999. Differentially expressed
protein Pdcd4 inhibits tumor promoter-induced neoplastic transformation.
Proc. Natl. Acad. Sci. USA 96:14037–14042.
6. Daga, R. R., and J. Jimenez. 1999. Translational control of the cdc25 cell
cycle phosphatase: a molecular mechanism coupling mitosis to cell growth.
J. Cell Sci. 112:3137–3146.
7. De Benedetti, A., and A. L. Harris. 1999. eIF4E expression in tumors: its
possible role in progression of malignancies. Int. J. Biochem. Cell. Biol.
8. De Gregorio, E., T. Preiss, and M. W. Hentze. 1998. Translational activation
of uncapped mRNAs by the central part of human eIF4G is 5? end-depen-
dent. RNA 4:828–836.
9. Dong, Z., M. J. Birrer, R. G. Watts, L. M. Matrisian, and N. H. Colburn.
1994. Blocking of tumor promoter-induced AP-1 activity inhibits induced
transformation in JB6 mouse epidermal cells. Proc. Natl. Acad. Sci. USA
10. Eberle, J., K. Krasagakis, and C. E. Orfanos. 1997. Translation initiation
factor eIF-4A1 mRNA is consistently overexpressed in human melanoma
cells in vitro. Int. J. Cancer 71:396–401.
11. Fukuchi-Shimogori, T., I. Ishii, K. Kashiwagi, H. Mashiba, H. Ekimoto, and
K. Igarashi. 1997. Malignant transformation by overproduction of transla-
tion initiation factor eIF4G. Cancer Res. 57:5041–5044.
12. Gingras, A. C., B. Raught, and N. Sonenberg. 1999. eIF4 initiation factors:
effectors of mRNA recruitment to ribosomes and regulators of translation.
Annu. Rev. Biochem. 68:913–963.
13. Haghighat, A., Y. Svitkin, I. Novoa, E. Kuechler, T. Skern, and N. Sonen-
berg. 1996. The eIF4G-eIF4E complex is the target for direct cleavage by the
rhinovirus 2A proteinase. J. Virol. 70:8444–8450.
14. Hershey, J. W. B., and W. C. Merrick. 2000. Pathway and mechanism of
initiation of protein synthesis, p. 33–38. In N. Sonenberg, J. W. B. Hershey,
and M. B. Mathews (ed.), Translational control of gene expression. Cold
Spring Harbor Press, Cold Spring Harbor, N.Y.
15. Hsu, T. C., M. R. Young, J. Cmarik, and N. H. Colburn. 2000. Activator
protein 1 (AP-1)- and nuclear factor ?B (NF-?B)-dependent transcriptional
events in carcinogenesis. Free Radic. Biol. Med. 28:1338–1348.
16. Imataka, H., and N. Sonenberg. 1997. Human eukaryotic translation initia-
tion factor 4G (eIF4G) possesses two separate and independent binding sites
for eIF4A. Mol. Cell. Biol. 17:6940–6947.
17. Iost, I., M. Dreyfus, and P. Linder. 1999. Ded1p, a DEAD-box protein
required for translation initiation in Saccharomyces cerevisiae, is an RNA
helicase. J. Biol. Chem. 274:17677–17683.
18. Kang, M. J., H. S. Ahn, J. Y. Lee, S. Matsuhashi, and W. Y. Park. 2002.
Up-regulation of PDCD4 in senescent human diploid fibroblasts. Biochem.
Biophys. Res. Commun. 293:617–621.
19. Korneeva, N. L., B. J. Lamphear, F. L. Hennigan, W. C. Merrick, and R. E.
Rhoads. 2001. Characterization of the two eIF4A-binding sites on human
eIF4G-1. J. Biol. Chem. 276:2872–2879.
20. Koromilas, A. E., A. Lazaris-Karatzas, and N. Sonenberg. 1992. mRNAs
containing extensive secondary structure in their 5? non-coding region trans-
late efficiently in cells overexpressing initiation factor eIF-4E. EMBO J.
21. Lawson, T. G., B. K. Ray, J. T. Dodds, J. A. Grifo, R. D. Abramson, W. C.
Merrick, D. F. Betsch, H. L. Weith, and R. E. Thach. 1986. Influence of 5?
proximal secondary structure on the translational efficiency of eukaryotic
mRNAs and on their interaction with initiation factors. J. Biol. Chem.
22. Lazaris-Karatzas, A., K. S. Montine, and N. Sonenberg. 1990. Malignant
transformation by a eukaryotic initiation factor subunit that binds to mRNA
5? cap. Nature 345:544–547.
23. Li, Q., H. Imataka, S. Morino, G. W. Rogers, Jr., N. J. Richter-Cook, W. C.
Merrick, and N. Sonenberg. 1999. Eukaryotic translation initiation factor
4AIII (eIF4AIII) is functionally distinct from eIF4AI and eIF4AII. Mol.
Cell. Biol. 19:7336–7346.
24. Li, W., G. J. Belsham, and C. G. Proud. 2001. Eukaryotic initiation factors
4A (eIF4A) and 4G (eIF4G) mutually interact in a 1:1 ratio in vivo. J. Biol.
25. Linder, P., P. F. Lasko, M. Ashburner, P. Leroy, P. J. Nielsen, K. Nishi, J.
Schnier, and P. P. Slonimski. 1989. Birth of the D-E-A-D box. Nature
26. Lomakin, I. B., C. U. Hellen, and T. V. Pestova. 2000. Physical association of
eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances bind-
ing of eIF4G to the internal ribosomal entry site of encephalomyocarditis
36 YANG ET AL.MOL. CELL. BIOL.
virus and is required for internal initiation of translation. Mol. Cell. Biol. Download full-text
27. Mader, S., H. Lee, A. Pause, and N. Sonenberg. 1995. The translation
initiation factor eIF-4E binds to a common motif shared by the translation
factor eIF-4 gamma and the translational repressors 4E-binding proteins.
Mol. Cell. Biol. 15:4990–4997.
28. Maeshima, Y., A. Sudhakar, J. C. Lively, K. Ueki, S. Kharbanda, C. R. Kahn,
N. Sonenberg, R. O. Hynes, and R. Kalluri. 2002. Tumstatin, an endothelial
cell-specific inhibitor of protein synthesis. Science 295:140–143.
29. Maeshima, Y., U. L. Yerramalla, M. Dhanabal, K. A. Holthaus, S. Bar-
bashov, S. Kharbanda, C. Reimer, M. Manfredi, W. M. Dickerson, and R.
Kalluri. 2001. Extracellular matrix-derived peptide binds to ?v?3integrin
and inhibits angiogenesis. J. Biol. Chem. 276:31959–31968.
30. Manders, E. M. M., F. J. Verbeek, and J. A. Aten. 1993. Measurement of
colocalization of objects in dual-color confocal images. J. Microsc. 169:375–
31. Matsuhashi, S., H. Yoshinaga, H. Yatsuki, A. Tsugita, and K. Hori. 1997.
Isolation of a novel gene from a human cell line with Pr-28 MAb which
recognizes a nuclear antigen involved in the cell cycle. Res. Commun. Bio-
chem. Cell. Mol. Biol. 1:109–120.
32. Morino, S., H. Imataka, Y. V. Svitkin, T. V. Pestova, and N. Sonenberg. 2000.
Eukaryotic translation initiation factor 4E (eIF4E) binding site and the
middle one-third of eIF4GI constitute the core domain for cap-dependent
translation, and the C-terminal one-third functions as a modulatory region.
Mol. Cell. Biol. 20:468–477.
33. Nielsen, P. J., and H. Trachsel. 1988. The mouse protein synthesis initiation
factor 4A gene family includes two related functional genes which are dif-
ferentially expressed. EMBO J. 7:2097–2105.
34. Onishi, Y., and H. Kizaki. 1996. Molecular cloning of the genes suppressed
in RVC lymphoma cells by topoisomerase inhibitors. Biochem. Biophys.
Res. Commun. 228:7–13.
35. Pause, A., N. Methot, and N. Sonenberg. 1993. The HRIGRXXR region of
the DEAD box RNA helicase eukaryotic translation initiation factor 4A is
required for RNA binding and ATP hydrolysis. Mol. Cell. Biol. 13:6789–
36. Pause, A., N. Methot, Y. Svitkin, W. C. Merrick, and N. Sonenberg. 1994.
Dominant negative mutants of mammalian translation initiation factor
eIF-4A define a critical role for eIF-4F in cap-dependent and cap-indepen-
dent initiation of translation. EMBO J. 13:1205–1215.
37. Pause, A., and N. Sonenberg. 1992. Mutational analysis of a DEAD box
RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J.
38. Pestova, T. V., C. U. Hellen, and I. N. Shatsky. 1996. Canonical eukaryotic
initiation factors determine initiation of translation by internal ribosomal
entry. Mol. Cell. Biol. 16:6859–6869.
39. Ponting, C. P. 2000. Novel eIF4G domain homologues linking mRNA trans-
lation with nonsense-mediated mRNA decay. Trends Biochem. Sci. 25:423–
40. Proud, C. G. 1994. Translation: turned on by insulin. Nature 371:747–748.
41. Richter-Cook, N. J., T. E. Dever, J. O. Hensold, and W. C. Merrick. 1998.
Purification and characterization of a new eukaryotic protein translation
factor, eukaryotic initiation factor 4H. J. Biol. Chem. 273:7579–7587.
42. Rogers, G. W., Jr., W. F. Lima, and W. C. Merrick. 2001. Further charac-
terization of the helicase activity of eIF4A: substrate specificity. J. Biol.
43. Rogers, G. W., Jr., N. J. Richter, W. F. Lima, and W. C. Merrick. 2001.
Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F.
J. Biol. Chem. 276:30914–30922.
44. Rogers, G. W., Jr., N. J. Richter, and W. C. Merrick. 1999. Biochemical and
kinetic characterization of the RNA helicase activity of eukaryotic initiation
factor 4A. J. Biol. Chem. 274:12236–12244.
45. Rozen, F., I. Edery, K. Meerovitch, T. E. Dever, W. C. Merrick, and N.
Sonenberg. 1990. Bidirectional RNA helicase activity of eukaryotic transla-
tion initiation factors 4A and 4F. Mol. Cell. Biol. 10:1134–1144.
46. Shibahara, K., M. Asano, Y. Ishida, T. Aoki, T. Koike, and T. Honjo. 1995.
Isolation of a novel mouse gene, MA-3, that is induced upon programmed
cell death. Gene 166:297–301.
47. Svitkin, Y. V., A. Pause, A. Haghighat, S. Pyronnet, G. Witherell, G. J.
Belsham, and N. Sonenberg. 2001. The requirement for eukaryotic initiation
factor 4A (elF4A) in translation is in direct proportion to the degree of
mRNA 5? secondary structure. RNA 7:382–394.
48. Watkins, S. J., and C. J. Norbury. 2002. Translation initiation and its dereg-
ulation during tumorigenesis. Br. J. Cancer 86:1023–1027.
49. Yang, H. S., A. P. Jansen, R. Nair, K. Shibahara, A. K. Verma, J. L. Cmarik,
and N. H. Colburn. 2001. A novel transformation suppressor, Pdcd4, inhibits
AP-1 transactivation but not NF-?B or ornithine decarboxylase transactiva-
tion. Oncogene 20:669–676.
50. Yoder-Hill, J., A. Pause, N. Sonenberg, and W. C. Merrick. 1993. The p46
subunit of eukaryotic initiation factor (eIF)-4F exchanges with eIF-4A.
J. Biol. Chem. 268:5566–5573.
51. Yoshinaga, H., S. Matsuhashi, J. Ahaneku, Z. Masaki, and K. Hori. 1997.
Expression and identification of H731 gene product in HeLa cells. Res.
Commun. Biochem. Cell Mol. Biol. 1:121–131.
52. Yoshinaga, H., S. Matsuhashi, C. Fujiyama, and Z. Masaki. 1999. Novel
human PDCD4 (H731) gene expressed in proliferative cells is expressed in
the small duct epithelial cells of the breast as revealed by an anti-H731
antibody. Pathol. Int. 49:1067–1077.
53. Young, M. R., J. J. Li, M. Rincon, R. A. Flavell, B. K. Sathyanarayana, R.
Hunziker, and N. Colburn. 1999. Transgenic mice demonstrate AP-1 (acti-
vator protein-1) transactivation is required for tumor promotion. Proc. Natl.
Acad. Sci. USA 96:9827–9832.
VOL. 23, 2003 NOVEL eIF4A BINDING PROTEIN Pdcd437