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INTRODUCTION |
Overexpression of EGFR1
and EGF, as well as constitutive activation of mutant EGFR, give rise
to the deregulation of this EGFR-dependent signaling
network, which is involved in the development and malignancy of
numerous types of human cancers, including cancer of the head and neck,
lung, bladder, breast, colon, prostate, kidney, ovary, brain, and
pancreas (1). The EGF receptor is the prototypical receptor with
intrinsic protein tyrosine kinase activity (2-4). Upon ligand binding
by its extracellular domain, EGFR forms homodimers or heterodimers with
the closely related ErbB receptors (such as ErbB2/Her2) and begins
tyrosine autophosphorylation of its cytoplasmic domain (2-4). This
autophosphorylation of the intracellular domain of EGFR is critical for
signal transduction, since it creates docking sites for various
signaling molecules, such as molecules containing Src homology 2 or
phosphotyrosine binding domains. These interactions with downstream
molecules create signaling complexes that lead to oncogenic responses
by linking EGFR activation to numerous cytoplasmic signaling pathways
(2-4). In cancer cells, the signaling domain of EGFR mediates and
regulates the proliferative signals generated from this receptor.
Therefore, identifying molecules that interact with the signaling
domain of EGFR will contribute not only to the elucidation of the
regulatory mechanism of EGFR in cancer progression but also to the
development of new treatments for uncontrolled growth of human cancers.
To further understand the molecular mechanisms by which the
intracellular region of EGFR transmits signals to various intracellular signaling pathways, we used the cytoplasmic domain of EGFR as bait in
yeast two-hybrid screening of a human carcinoma cDNA library. Here
we identify an intracellular protein ARF4 that interacts with the
cytoplasmic domain of EGFR. ARFs, members of the Ras superfamily, are
small 20-kDa guanine nucleotide-binding proteins. The inactive
GDP-bound form of ARF is soluble, although it can weakly associate with
the cell membrane. On the other hand, the active GTP-bound form binds
tightly to the membrane (5). On the basis of deduced amino acid
sequences and phylogenetic analysis, ARFs have been divided into three
classes: class I (ARF1, -2, and -3), class II (ARF4 and -5), and class
III (ARF6) (6). Although all three classes of ARF proteins are capable
of activating cellular phospholipase D, the specific roles of each ARF
seem to be different. Class I is involved in the trafficking pathway that links the endoplasmic reticulum, Golgi, and endosomal systems, and
class III, instead, takes part in the trafficking pathway that links
the endosome and plasma membrane system (7). Many researchers believe
that class II ARFs are only supplementary to class I ARFs and, as such,
have focused their attention on class I ARFs while nearly ignoring the
cellular functions of the class II ARF4 and ARF5 proteins. Here, we
show that EGF does not activate PLD1 but does activate cellular PLD2
through the activation of ARF4. Moreover, this
ARF4-dependent activation of PLD2 is required for inducing
the EGF-induced transcriptional activity of activator protein 1 (AP-1).
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EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Screening--
The EcoRI fragment
encoding the cytoplasmic domain of EGFR was fused in frame with the
GAL4 DNA binding domain (BD) of the pGBKT7 fusion vector
(Clontech) to create the BD/EGFR construct. A human
epithelial carcinoma cDNA library was separately fused with the
GAL4 activation domain (AD) of the pGAD-GH vector
(Clontech). The yeast strain pJ69-2A was
co-transformed, by a lithium acetate-based method, with this library
and the BD/EGFR construct. The transformed yeast cells were screened,
according to the manufacturer's protocols, for their ability to grow
on plates lacking histidine and adenine.
DNA Constructs--
PCR was used to obtain full-length cDNAs
encoding ARF4, PLD1, PLD2, and EGFR by use of the
Rapid-ScreenTM arrayed human placenta cDNA library,
which is constructed in the pCMV6-XL4 (ORIGENE, Rockville, MD) vector.
The ARF4 wild-type and mutant cDNA fragments were cloned either
into the mammalian expression vector pEBG, which also encodes the
glutathione S-transferase tag (8), or into the multiple
cloning site of the vector pRK5, which encodes three copies of the
hemagglutinin (HA) epitope at its carboxyl terminus (9). All mutations
in the ARF4 gene were generated by PCR-based mutagenesis as previously
described (9).
Cell Lines and Culture Conditions--
293T cells overexpressing
EGFR (293T/EGFR) were obtained by transfecting, via the calcium
phosphate method, the hygromycin selection vector pCMV6-XL4 into 293T
cells, which express undetectable levels of endogenous EGFR.
Transfected cultures were selected with hygromycin (300 µg/ml) for
10-14 days at 37 °C. All cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum
(HyClone) as described previously (10). Cells were grown to
subconfluence and then starved overnight by replacing medium with
serum-free Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine and nonessential amino acids before initiating
the 30-min stimulation with EGF (50 ng/ml).
Immunoprecipitation--
Cells were solubilized in lysis buffer
containing 2 mM HEPES (pH 7.6), 1% Triton X-100, 137 mM NaCl, 0.1 mM Na3VO4,
25 mM
-glycerophosphate, 3 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride as described (10, 11).
Cell lysates were then incubated with protein G-coupled Sepharose beads
(Amersham Biosciences) for 1 h, after which the beads were
discarded, and the supernatants were incubated with specific antibodies
(2 µg/ml anti-EGFR antibody or IgG from preimmune serum) for 1 h
followed by overnight incubation with protein G-agarose at 4 °C.
Samples were collected and washed four times with lysis buffer. The
precipitated samples were analyzed by Amersham Bioscience's ECL
blotting system with specific antibodies (200 ng/ml unless mentioned).
The R891 anti-ARF4 antiserum and monoclonal 1D9 antibody against the
other ARFs (ARF1, -3, -5, and -6) were kindly provided by Dr. R. A. Kahn (7) and used for probing immunoblots for endogenous ARFs.
Immunofluorescence--
Cells were plated on 25-mm coverslips
(Fisher). After transient transfection experiments, cells were fixed
with 4% formaldehyde for 15 min, washed three times with PBS, and
permeabilized in PBS containing 0.5% Triton X-100 for 5 min. Cells
were washed three times in PBS containing 3% bovine serum albumin for
1 h. The anti-EGFR antibody and anti-HA antibody were added to
cells in PBS containing 3% bovine serum albumin for 1 h, washed
four times with PBS, then incubated 1 h with Alexa Fluor 568 goat-anti-rabbit (red-orange) or Alexa Fluor 488 anti-mouse antibody
(green) (Molecular Probes, Inc., Eugene, OR). Cells were washed and
mounted on coverslips with Prolong (Molecular Probes) mounting medium
for immunofluorescence.
Digitonin Treatment--
Briefly, 293T/EGFR cells were
transfected with the plasmid expressing wild-type ARF4 with three HA
tags at its C terminus. After serum starvation, cells were scraped
gently and resuspended in PBS. The samples were treated with 8 µM digitonin (WAKO Chemicals, Richmond, VA), in the
presence of GTP
S (100 µM) with or without EGF (100 ng/ml) at 37 °C for 20 min. ATP (1 mM) and magnesium (2 mM) were included in all experiments. Intracellular
proteins were released from digitonin-treated cells by
microcentrifugation for 20 min. Supernatants and pellets were collected
separately and resolved by SDS-PAGE. The ARF4-HA3 protein was detected
by probing immunoblots with the anti-HA antibody.
Membrane Preparations--
Cell membranes were prepared from ten
150-mm dishes of confluent cells. Cells were washed in PBS, scraped,
and centrifuged. Pellets were resuspended in 50 mM HEPES
(pH 7.6), 8% sucrose, 10 mM EDTA, 10 mM EGTA,
and 1 mM phenylmethylsulfonyl fluoride and then disrupted
with a glass homogenizer. Cell extracts were layered onto a 41%
sucrose cushion and centrifuged at 95,000 × g for 90 min. Membrane material at the interface was collected, diluted to 10 ml, and centrifuged at 60,000 × g for 60 min. Membrane pellets were resuspended in 50 mM HEPES for the following
ARF activation assays.
ARF Activation Assays--
For the nucleotide binding assay,
recombinant human ARF4 (2.5 µg) was incubated at 37 °C with
[35S]GTP
S, with or without EGF, in the presence of
membranes as previously described (5). Protein-bound nucleotides were
separated by filtration through nitrocellulose filters. Bound
nucleotide was determined by scintillation counting. Nonspecific
binding was determined by using excess, unlabeled GTP
S.
PLD Assays--
Cells were starved and labeled overnight with
[3H]palmitate (5 µCi/ml) in 0.1% fetal bovine serum.
Cells were stimulated with EGF (50 ng/ml) in the presence of 0.3%
butanol for 30 min. PLD-catalyzed transphosphatidylation was performed
as described previously (5, 13). The lipid phase was extracted and
developed by TLC on silica gel 60 plates by using the upper phase of a
mixture of ethyl acetate/2,2,4-trimethylpentane/acetic acid (9:5:2) as
the solvent. PhosphorImager technology (Molecular Dynamics) was used to
determine the amount of labeled phosphatidylbutanol and total lipids.
Reporter Gene Assays--
Reporter plasmids (Stratagene) encoded
a luciferase gene whose expression was driven by one of the following
cis-acting elements: seven tandem repeats of the AP-1
binding site; four tandem repeats of interferon
-activated sequence
(GAS), nuclear factor of activated T cells (NFAT), or serum response
element (SRE). Cells were grown on 35-mm multiwell plates (Nunc,
Naperville, IL) and transiently transfected with 8 µg of total
plasmid DNA as described previously (10, 11). Briefly, in each
transfection, 0.05 µg of the
-galactosidase expression plasmid
(pCMV-
Gal; Clontech, Palo Alto, CA) was included to determine the transfection efficiency. At 24 h
posttransfection, cells were starved overnight and treated with or
without stimuli as described in the figure legends. Cell extracts were
prepared, and the activities of
-galactosidase and luciferase were
measured as described previously (10, 11). The luciferase activity in
each experiment was divided by the activity of
-galactosidase to
correct for transfection efficiency.
Electrophoretic Mobility Shift Assay--
Nuclear extracts were
prepared as described (14). The AP-1 oligonucleotide was labeled with
[
-32P]ATP and incubated with nuclear extracts for 30 min by using the gel shift assay system kit (Promega, Madison, WI). The
specificity of binding was determined by adding homologous or mutated
unlabeled synthetic oligonucleotides (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA). For gel supershift assays, rabbit affinity-purified antibodies (2 µg) to Jun and Fos families of nuclear factors (Santa Cruz Biotechnology) were incubated for 30 min with nuclear extracts and
with synthetic oligonucleotides at room temperature. DNA-protein complexes were resolved on nondenaturing, nonreducing 4% acrylamide gels, and the complexes were visualized by PhosphorImager technology.
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RESULTS |
Identification of ARF4 as an EGFR-interacting Protein by Yeast
Two-hybrid Screening--
To identify molecules that act as mediators
of EGFR-dependent signaling, we used the cytoplasmic domain
of EGFR as bait in a yeast two-hybrid screen. From a human carcinoma
cDNA library, 2 × 107 transformants were
screened, and 102 positive clones were selected for sequence analysis
based on their potential interaction with EGFR. In this GAL4 system,
growth of transformed yeast in medium lacking histidine and adenine is
completely dependent on the presence of sequences from both ARF4 and
EGFR (Fig. 1A). Using the
BLAST algorithm and the nucleotide data base at the National Library of
Medicine, we found one clone that encoded the small GTPase ARF4.

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Fig. 1.
EGF receptor physically interacts with ARF4
GTPase. A, the yeast strain PJ69-2A was cotransformed
with vectors encoding the indicated GAL4 DNA BD and GAL4 AD fusion
proteins. Transformed yeast cells were cultured on plates containing
selective medium. B, 293T/EGFR cells were transfected with
expression vectors encoding wild-type ARF4-HA3 or the ARF4(Q71L)-HA3 or
ARF4(T31N)-HA3 mutants. After transfection, cells were starved
overnight and then treated with or without EGF for 30 min. Cells were
lysed, precipitated with the anti-HA antibody, and electroblotted onto
nitrocellulose filters. The top gel shows co-immunoprecipitation of
EGFR and ARF4, as detected by Western blot analysis with the anti-EGFR
antibody, EGFR (1005) (Santa Cruz Biotechnology). The
bottom, control gel shows corresponding samples of each
immunocomplex separated by SDS-PAGE, electrotransferred, and analyzed
by Western blot with the anti-HA antibody. C, lysates from
293T/EGFR cells were immunoprecipitated with either anti-EGFR or
control antibodies before or after EGF stimulation as indicated.
Immunocomplexes were electroblotted onto nitrocellulose filters, and
precipitated EGFR was detected with the anti-EGFR antibody
(top panel). Endogenous ARF4 (second
panel) and endogenous ARF1, -3, -5, and -6 (third
panel) proteins were detected in these immunocomplexes with
antibodies R891 and 1D9, respectively. In the total cell lysates, the
presence of endogenous ARF4 (fourth panel) and
endogenous ARF1, -3, -5, and -6 (fifth panel)
proteins were detected with R891 and 1D9, respectively. D,
293T/EGFR cells were transfected with expression vectors encoding
ARF4-HA3 or ARF4(Q71L)-HA3. After transfection, cells were starved
overnight followed by stimulation with or without EGF for 10 min. For
detecting the tyrosine autophosphorylation of EGFR, cell lysates were
immunoprecipitated with anti-EGFR antibody and immunoblotted with
anti-PY20 (top panel). EGFR in the immunocomplex
(middle panel) and the expression of ARF4-HA3 or
ARF4(Q71L)-HA3 in whole cell lysates were detected with anti-EGFR or
anti-HA antibodies, respectively. Data are representative of three
independent experiments.
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To study the interaction between EGFR and ARF4 in mammalian cells, we
transfected expression vectors encoding either wild-type or mutant ARF4
with three C-terminal HA epitope tags (ARF4-HA3) into 293T/EGFR cells
(Fig. 1B). These transfected cells were treated with or
without EGF and later lysed to prepare total cell lysates. From these
cell lysates, anti-HA antibody was used to immunoprecipitate HA-tagged
ARF4s and other cellular molecules complexed with these ARF4s. The
ARF4-HA3 complex was recovered by centrifugation, and the pellets were
separated by SDS-PAGE and analyzed by Western blot with an anti-EGFR
antibody. As shown in Fig. 1B, EGFR co-purified with
wild-type ARF4 only in lysates from EGF-treated cells but not from
cells without EGF stimulation. In contrast, EGF treatment was not
required for the co-purification of EGFR with the active form of the
ARF4 mutant, ARF4(Q71L), which theoretically constitutively binds to
GTP (Fig. 1B). The inactive form of ARF4, ARF4(T31N), which
constitutively binds to GDP, did not co-immunoprecipitate with EGFR
regardless of EGF stimulation of the cells (Fig. 1B). In
order to test whether EGFR interacts with endogenous ARF4 or other
members of the ARF family, extracts from cells stimulated with or
without EGF were immunoprecipitated with anti-EGFR or control
antibodies. The resulting immunocomplexes were analyzed by
immunoblotting with R891 anti-ARF4 antiserum to detect endogenous ARF4
or with the 1D9 monoclonal antibody to detect the other ARF family
members (ARF1, -3, -5, and -6) (7). Fig. 1C shows that ARF4
was present in the immunocomplex derived from EGF-stimulated cells,
whereas other ARFs (ARF1, -3, -5, and -6) were not detected in the
immunocomplex from EGF-stimulated cells (Fig. 1C). In cells without EGF stimulation, we were unable to detect any endogenous ARFs
(ARF1, -3, -4, -5, and -6) that co-immunoprecipitated with EGFR (Fig.
1C). These results suggest that in mammalian cells, EGFR or
the EGFR complex preferentially interacts with GTP-bound ARF4 but does
not interact with GDP-bound ARF4.
We next tried to determine whether the interaction between EGFR and
activated ARF4 as well as the activation of ARF4 could potentially
affect the EGFR activation by EGF. We examined tyrosine autophosphorylation of the EGFR signaling domain before or after stimulation of EGF in 293T/EGFR cells expressing wild-type ARF4 or
ARF4(Q71L) (Fig. 1D). No difference in the tyrosine
autophosphorylation of EGFR was detected in the presence of the
wild-type or constitutively active form of ARF, indicating that the
association of ARF4 with EGFR and ARF4 activation do not have a
detectable effect on EGFR activity before or after EGF induction.
EGF Activates the Binding of GTP
S to ARF4 and Induces the
Translocation of ARF4 Protein to Cell Membranes--
After we
demonstrated that the affinity between ARF4 and EGFR is induced by EGF
treatment and that association of ARF4 with GTP is critical for this
EGF-induced interaction of EGFR and ARF4, we next examined whether ARF4
translocates to cell membranes and colocalizes with EGFR after EGF
stimulation. Before and after EGF induction, the subcellular
localization of ARF4-HA3 and EGFR in 293T/EGFR cells was visualized by
immunofluorescence microscopy (Fig. 2).
Without the addition of EGF, cellular AFR4 was mainly localized in the
cytosol, whereas, after EGF induction, cells expressing ARF4 formed
membrane ruffles and ARF4 together with EGFR became concentrated in the
ruffles. This result suggests that, after EGF induction, ARF4
translocates to membrane ruffles and colocalizes with EGFR.

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Fig. 2.
Colocalization of ARF4 and EGFR after EGF
induction. 293T/EGFR cells were transiently transfected with the
ARF4-HA3 expression vector. After transfection, cells were starved
overnight and then treated with or without EGF (100 ng/ml) at 37 °C
for 10 min as indicated. The cells were processed and immunostained
with the anti-EGFR antibody to detect the localization of cellular EGFR
(left panels, red
fluorescence) or anti-HA antibodies to detect the
localization of ARF4-HA3 (middle panels,
green fluorescence). The merge of red
and green fluorescence is shown in the right
panels (yellow color).
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Next, we examined whether EGF-dependent cell activation
would enhance the GTP binding of ARF4. The purified recombinant ARF4 protein was separately incubated with membranes prepared from 293T/EGFR
or 293T cells together with GTP
S and treated with or without EGF.
The amount of GTP
S binding to the ARF4 protein was analyzed by a
reconstitution assay as previously described (5). Briefly, cell
membranes were prepared from 293/EGFR or 293 cells followed by
incubation with EGF and/or recombinant ARF4 in the presence of GTP
S.
Protein-bound GTP
S was separated by filtration through
nitrocellulose filters and was quantitated by scintillation counting.
In comparison with the GTP
S binding to the 293T/EGFR cell membranes
alone, no significant enhancement of GTP
S binding to cell membranes
occurred by incorporating either ARF4 or EGF proteins in these assays
(Fig. 3A). Conversely,
treating this membrane preparation in the presence of both ARF4 and EGF
notably increased the level of GTP
S association with ARF4 (Fig.
3A), indicating that EGF treatment of membranes boosted the
binding of GTP
to ARF4. In similar experiments, we used membranes
obtained from 293T cells, in which EGFR expression is undetectable.
Fig. 3B shows that with these EGFR-deficient membranes,
there was no increase of GTP
S binding to ARF4, neither by adding
ARF4 nor by adding ARF4 and EGF. These results demonstrate that EGF
enhances the binding of GTP
S to ARF4.

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Fig. 3.
EGF enhances the
GTP S binding of ARF and the association
between ARF4- and EGFR-enriched cellular membranes. Membranes
(Mem) of 293/EGFR (A) or 293T (B)
cells treated with or without EGF were incubated with ARF4 protein for
30 min in the presence of [35S]GTP . As negative
controls, membranes were boiled at 100 °C for 5 min to denature
membrane proteins. Protein-bound nucleotides were separated by
filtration through nitrocellulose filters. The amount of bound
nucleotide was determined by liquid scintillation counting of the
material retained on nitrocellulose filters. Experiments were repeated
at least three times. Samples were analyzed statistically by analysis
of variance. C, 293T/EGFR cells were transfected with the
expression plasmid encoding ARF4-HA3. After serum starvation, 293T/EGFR
cells were gently scraped, resuspended in PBS, and permeabilized with
digitonin (Dig; 8 µM). GTP S (100 µM) and EGF (100 ng/ml) were included in the
permeabilization buffer for the indicated samples. Treated cells were
centrifuged, and the resulting pellets and supernatants were separated
by SDS-PAGE and analyzed by immunoblot with the anti-HA antibody. The
control sample shows results from cells treated with culture medium
only, without digitonin. On Western blots, the relative densities of
each ARF4 band in C were measured by using the Personal
Densitometer SI (Molecular Dynamics). The density of the ARF4 band in
digitonin-treated cell pellets was set arbitrarily as 1.
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We then examined whether the active GTP-bound form of ARF4 proteins
attaches to EGFR-enriched membranes and whether the inactive GDP-bound
form of ARF4 remains cytosolic. ARF4 with three C-terminal HA tags
(ARF4-HA3) was expressed in 293T/EGFR cells. After serum starvation,
cells were collected and incubated with GTP
S in the presence or
absence of EGF. These cells were then treated with digitonin, a
detergent that permeabilizes cell membranes and allows cytosolic
ARF4-HA3 to leak out of the cells. The cytosolic and membrane-containing cell pellet fractions of these cells were separated
by SDS-PAGE and analyzed by Western blot with an anti-HA antibody to
examine the distribution of ARF4-HA3 between these two fractions. The
digitonin-treated cells released most of their ARF4-HA3 into the
supernatant, indicating that most ARF4-HA3 proteins were cytosolic,
whereas without digitonin treatment, ARF4-HA3 remained inside the cells
(Fig. 3C). In the presence of either GTP
S or EGF,
digitonin-treated cells only partially released ARF4-HA3 into
supernatant (Fig. 3C). However, when these cells were
treated with EGF in combination with GTP
S, almost all ARF4-HA3 was
retained in the cell membrane-containing cell pellet fraction. These
data, together with the EGF-induced ARF4 translocation data shown in
Fig. 2, indicate that the activation of ARF4 either by EGF or by
association with GTP
S induces the translocation of ARF4 from the
cytosol to EGFR-enriched cell membranes.
ARF4 Mediates EGF-induced Activation of PLD2--
Previous studies
have shown that agonist-induced ARF activation and its consequent
translocation to cell membranes stimulate PLD activity (13, 15, 16). To
investigate whether EGF-induced ARF4 activation is able to stimulate
PLD activity in cells, we transiently co-expressed PLD1 or PLD2
together with ARF4 or its mutants in 293T/EGFR cells, followed by EGF
stimulation of particular samples. Activation of cellular PLD was
determined by the unique ability of PLD to produce, in the presence of
butanol, phosphatidylbutanol, which cannot be further metabolized. In
cells overexpressing PLD2 and ARF4, PLD activity was dramatically
stimulated by EGF treatment (Fig.
4A). However, in cells
overexpressing PLD1 and ARF4, this EGF-induced up-regulation of PLD
activity was not observed (Fig. 4B). In addition,
overexpressing ARF4(Q71L), the dominant active form of ARF4,
constitutively stimulated the activity of PLD2 but did not have the
same effect on PLD1 (Fig. 4, A and B). These data
suggest that ARF4 mediates EGF-induced cellular activation of PLD2 but
does not mediate activation of PLD1. As a positive control in these
experiments, we used the small GTPase RalA, which mediates EGF-induced
PLD1 activity and, to a lesser extent, PLD2 activity (17) (Fig. 4,
A and B).

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Fig. 4.
ARF4 mediates EGF-induced activation of PLD2
but does not mediate EGF-induced activation of PLD1. 293T/EGFR
cells (2 × 105) were cotransfected transiently with
expression plasmids encoding PLD2 (A) or PLD1 (B)
along with expression plasmids encoding ARF4, ARF4(Q71L), ARF4(T31N),
or RalA as indicated. After transfection, cells were starved overnight
and then treated with or without EGF for 15 min. The efficiency of
transfection was monitored by cotransfecting cells with pEGFP-N1
and ARF4, ARF4(Q71L), ARF4(T31N), or RalA expression plasmids. The
percentage of transfected cells was estimated 48 h after
transfection by comparing the number of fluorescent cells to the total
number of cells in the same field. The transfection efficiency for
293T/EGFR was maintained at 70 80%, and only plates with this range
of efficiency were used. PLD activity was measured by the accumulation
of phosphatidylbutanol (PtdBtd) relative to total
lipids. Error bars represent the S.D. of three
independent experiments.
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ARF4 GTPase Regulates Transcriptional Activity of AP-1 through
Activation of PLD2--
PLD activity, in cooperation with EGFR, has
been reported to be involved in the transformation of cells (17). One
of the major causes of EGFR-dependent cell transformation
is the altered expression of specific target genes that results from
changing the activities of certain transcriptional factors by
unrestrained signaling effectors. The role of PLD2 in the EGF-mediated
modulation of gene expression through transcriptional activation has
not been thoroughly examined. We tested whether ARF4-mediated PLD2 activation regulates gene transcription through specific enhancer elements, such as AP-1, interferon GAS, NFAT, or SRE. For these experiments, we used expression plasmids encoding the luciferase gene,
driven by a basic promoter element (TATA box) joined to tandem repeats
of AP-1, GAS, NFAT, or SRE binding elements. These plasmids were
individually transfected into 293T/EGFR cells. Expression of activated
PLD2 significantly induced luciferase expression driven by the promoter
containing AP-1; in contrast, luciferase expression was not induced by
promoters containing GAS, NFAT, and SRE binding sites (Fig.
5A). These data suggest that
PLD2 activation stimulated by the active form of ARF can enhance AP-1 activity.

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Fig. 5.
ARF4 GTPase enhances transcriptional activity
of AP-1 through activation of PLD2. A, 293T/EGFR cells
were transfected with the indicated PLD2 and ARF4 expression vectors
together with the luciferase reporter gene driven by a minimal promoter
containing the cis-acting element of AP-1, GAS, NFAT, or
SRE. The luciferase activities were normalized against 293T/EGFR cells
transfected with each cis-reporter plasmid alone, whose
value was taken as 1. Bars, S.E. of average values from
three independent experiments performed in duplicate. 293T/EGFR cells
were transfected with the AP-1 (B) or
SRE-dependent (C) luciferase reporter plasmid,
with or without ARF4(T31N), as indicated. After serum starvation, cells
were treated with or without EGF for 10 h, and the luciferase
activities were normalized against cells transfected with the AP-1-Luc
(B) or SRE-Luc (C) plasmid alone without EGF
stimulation, whose value was taken as 1. Bars represent the
S.E. of average values from three independent experiments performed in
duplicate. In each transfection, 0.05 µg of the -galactosidase
expression plasmid (pCMV- Gal) was included to determine the
transfection efficiency. The luciferase activity in each experiment was
divided by the activity of -galactosidase to correct for
transfection efficiency.
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We further tested whether this ARF4-mediated PLD2 activation is
involved in EGF-induced cellular AP-1 activation. 293T/EGFR cells were
transiently transfected with the AP-1- or SRE-driven luciferase
reporter constructs and, in some cases, cotransfected with the
expression vector encoding ARF4(T31N), followed by EGF treatment (Fig.
5B). EGF induced AP-1
dependent reporter gene expression
more than 3-fold, and this induction of luciferase expression was
inhibited considerably by dominant negative ARF4(T31N). In contrast,
SRE-dependent reporter gene expression stimulated by EGF
was not blocked by ARF4(T31N) (Fig. 5C).
To further confirm and characterize the AP-1 activation by
ARF4-mediated PLD2 activation, we used nuclear extracts generated from
cells transfected with the combination of expression plasmids as
indicated in Fig. 5 for electrophoretic mobility shift assay experiments, and the resultant nuclear extracts were incubated with
radiolabeled oligonucleotides containing AP-1 sequences. Binding
studies revealed that the AP-1/nuclear protein complex increased
dramatically in cells containing activated PLD2 due to the
co-expression of the ARF4 active form, ARF4(Q71L) (Fig. 6A). Unlabeled homologous AP-1
oligonucleotides prevented the binding of the radiolabeled AP-1
sequences to nuclear proteins, whereas mutated AP-1 oligonucleotides
did not prevent this binding (Fig. 6A). In addition, to
examine the individual components within the AP-1-nuclear protein
binding complex, we included antibodies against c-Jun or c-Fos in some
of these electrophoretic mobility shift assay experiments (Fig.
6B). We found that these antibodies significantly up-shifted
the binding of nuclear proteins from cells containing activated PLD2 to
the AP-1 consensus sequence, suggesting the presence of c-Jun and c-Fos
in these DNA-protein complexes (Fig. 6B). In 293T/EGFR
cells, EGF induces the binding of nuclear proteins to AP-1 (Fig.
6C). We tested whether ARF4 activity is required for
EGF-dependent AP-1 activation. We found that the cellular
expression of dominant negative forms of ARF4, ARF4(T31N), dramatically
reduced this EGF-mediated binding of nuclear proteins to AP-1
oligonucleotides (Fig. 6C), suggesting a critical role of
ARF4 activity in EGF-induced AP-1 activation. Taken together, these
results suggest an important function of both ARF4 and PLD2 in
regulating the transcriptional activity of AP-1 in the EGF-induced cell
activation.

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Fig. 6.
EGF-induced ARF4/PLD2 activation enhances the
binding of nuclear proteins to AP-1 sequences. 293T/EGFR cells
were transfected with different combinations of expression vectors as
indicated: none, PLD2, and PLD2 plus ARF4(Q71L). Nuclear extracts
prepared from these transfected cells were incubated with
[ -32P]oligonucleotides. A, incubations were
performed in the absence or presence of a 100-fold excess of unlabeled
wild-type or mutated oligonucleotides as noted. The arrow
indicates the specific DNA-protein complex. B, incubations
were performed in the absence (None) or presence of control
nonspecific antibodies (NS) or with antibodies specific to
the c-Jun (c-Jun (D)) or to c-Fos (c-Fos (K-25)) families of nuclear
proteins (Santa Cruz Biotechnology). C, 293T/EGFR cells were
transiently transfected either with an empty expression vector or
vector encoding ARF4(T31N). After transfection, cells were starved
overnight followed by stimulation with or without EGF (100 ng/ml)
for 10 min. Nuclear extracts were prepared and incubated with
labeled oligonucleotides alone or together with a 100-fold excess
of unlabeled wild-type or mutated oligonucleotides.
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Mitogen-activated protein (MAP) kinases have been implicated in the
regulation of AP-1 activity (18, 19). To explore the possible
involvement of two MAP kinase pathways, BMK1/ERK5 and ERK1/2, in
mediating PLD2-induced AP-1 activation, we transfected expression
vectors encoding ARF(Q71L) and PLD2 into 293T/EGFR cells and examined
the activity of endogenous BMK1/ERK5 and ERK1/2. We found that the
activation of PLD2 by ARF4(Q71L) had no detectable effect on endogenous
BMK1/ERK5 and ERK1/2 activities (Fig. 7). MEK5(D) and MEK1(E), dominant active forms of MEK5 and MEK1, are known
to specifically activate BMK1/ERK5 and ERK1/2, respectively. When
MEK5(D) or MEK1(E) were expressed in 293T/EGFR cells, the endogenous
BMK1/ERK5 or ERK1/2 was activated correspondingly (Fig. 7). These
results suggest that these two MAP kinase cascades are not critically
involved in AP-1 activation by the ARF4/PLD2 pathway.

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Fig. 7.
ARF4-dependent PLD2 activation
does not up-regulate activities of BMK1/ERK5 and ERK1/2 MAP
kinases. 293T/EGFR cells were transiently transfected with
expression vectors encoding ARF4(Q71L), PLD2, MEK5(D), or MEK1(E), and
the activity of endogenous BMK1/ERK5 and ERK1/2 was determined after
48 h. The phosphorylation and consequent activation of endogenous
BMK1 resulted in a shift in its electrophoretic mobility, detected by
Western blot analysis with the anti-BMK1 antibody (top
panel) (12). Activation of endogenous ERK1/2 was
measured in cell lysates by a phospho-specific anti-ERK1/2 antibody
(middle panel) and endogenous ERK1/2 was detected
by an anti-ERK1/2 antibody (bottom panel). The
transfection efficiency for 293T/EGFR was maintained at 70 80% with
the pEGFP-N1 plasmid as described in the legend to Fig. 4, and only
plates with this range of efficiency were used.
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DISCUSSION |
In this study, we have identified ARF4 as an intermediate molecule
that interacts with EGFR and have shown that EGFR-mediated ARF4
activation specifically up-regulates cellular PLD2 but does not
up-regulate PLD1, activity. Moreover, we have demonstrated that this
ARF4-dependent PLD2 activation significantly enhances the
transcriptional activity of AP-1. EGFR is known to cooperate with PLD
in cellular transformation, yet the molecular mechanism of this
oncogenic process is unknown. One possible route, as indicated by our
findings, is via the dramatic enhancement of the activity of the AP-1
oncoprotein, resulting from EGFR-dependent PLD activation.
ARFs are usually modified by myristoylation at Gly2 through
the action of N-myristoyltransferase (20). Nevertheless,
Kanoh et al. (21) have shown that a mutant form of ARF3,
which is mutated at Gly2 to eliminate myristoylation, does
not abolish its interaction with arfaptin 1. In our yeast two-hybrid
analysis (Fig. 1A), affinity between the EGFR cytoplasmic
domain and ARF4 does not seem to require N-terminal myristoylation. In
yeast two-hybrid-based assay, the ARF4 fusion protein that we used
contained an N-terminal GAL4-activating domain, which should have
prohibited the ARF4 part of this GAL4-AD/ARF4 fusion protein from being
modified by N-myristoyltransferase, an enzyme that can act
only at the N-terminal end of a protein (22). Without its N-terminal
myristoylation, ARF4 appears to have some affinity with the cytoplasmic
domain of EGFR; however, drawing any conclusion is difficult without
obtaining additional information about the N-terminal modification of
GAL4/ARF4 fusion proteins in yeast. More detailed studies are needed to
decipher the contribution of N-terminal myristoylation of ARF4 in its
binding to the cytoplasmic domain of activated EGFR.
In general, ARF proteins are involved with vesicle transport within the
Golgi complex, between the endoplasmic reticulum and Golgi complex
(23), with endosome fusion (24), and with several other processes
related to intracellular membrane transport. ARF proteins, with the
exception of ARF6, are found either dispersed in the cytosol or
associated with Golgi membranes (25); ARF6 is permanently bound to
plasma membranes. Our discovery indicates that the ARF4 protein is
activated by EGFR-mediated cellular activation and is simultaneously
translocated from the cytosol to the cellular membrane. Similarly, ARF1
is known to be activated and translocated to the cell membrane by
activation of another receptor tyrosine kinase, the insulin receptor
(5). Since ARF1 and ARF4 belong to class I and II ARFs, classes that
appear to be similar in both their cellular localization and functions,
it is not surprising that both ARF1 and ARF4 are activated by receptor
tyrosine kinase-mediated cellular activation and translocated to
cellular membranes. However, it would be interesting to know whether
the other members of class I and II ARFs behave similarly in receptor
tyrosine kinase-dependent stimulation of cells.
PLD, consisting of PLD1 and PLD2 isoforms, hydrolyzes
phosphatidylcholine to choline and to the second messenger,
phosphatidic acid, which can serve as a signal transducer. PLD2 is a
106-kDa protein that shares 50
55% homology with PLD1 but lacks the
116-amino acid loop region following the first HKD motif, a highly
conserved catalytic motif
(HXKX4D) that is essential for
the enzymatic activity of phosphatidyltransferase (26). PLD2 localizes
primarily to plasma membranes, in contrast to PLD1, which localizes
solely to perinuclear regions (the endoplasmic reticulum, Golgi
apparatus, and late endosomes) (27). In comparison with PLD1, the
membrane localization of PLD2 makes it a logical mediator for
delivering signals generated from receptors that reside in the cellular
membrane. Indeed, other than mediating signaling from activated EGFR,
as we have demonstrated, PLD2 can also relay intracellular signals descended from activation of the insulin receptor.
AP-1 activity is modulated by a variety of dimers that are composed of
members of the Fos, Jun, and ATF families (28). Whereas the Fos
proteins (c-Fos, FosB, Fra-1, and Fra-2) can only form heterodimers
with members of the Jun family, the Jun proteins (c-Jun, JunB, and
JunD) can form both homodimers and heterodimers with Fos members to
form transcriptionally active complexes. Moreover, AP-1 activity has
been implicated in many biological processes, including cell
differentiation, proliferation, and oncogenic transformation (29-31).
The MAP kinase pathways regulate both the amounts and transactivating
capacities of the component Fos, Jun, and ATF proteins of AP-1 in a
stimulus-specific manner. For instance, treatment of HeLa cells with
platelet-derived growth factor, serum, or phorbol esters predominantly
activates the extracellular signal-regulated kinase (ERK) members of
the MAP kinase family, leading to strong stimulation of Jun and Fos
activity. On the other hand, treatment with stress-inducing stimuli,
such as ultraviolet light, activates the c-Jun N-terminal
kinase/stress-activated protein kinase members of the MAP kinase
family, which preferentially enhance Jun and ATF activity via
phosphorylation of c-Jun serines 63 and 73 and ATF2 threonines 69 and
71, which are located in their respective transactivation domains (32).
In contrast, activation of protein kinase A by cAMP strongly enhances
only de novo synthesis of JunB and c-Fos (33). Here, we show
that AP-1 activity is up-regulated by EGF-induced PLD2 activation (Fig.
5). Still, the intermediate signaling pathways by which PLD2 induces
the activation of AP-1 are presently unknown. We have explored the
possible involvement of two MAP kinase pathways, ERK1/2 and BMK1/ERK5,
in mediating PLD2-induced AP-1 activation and have found that the
ARF4-dependent activation of PLD2 has no effect on
endogenous BMK1/ERK5 and ERK1/2 activities (Fig. 7), indicating that
these two kinase cascades have no involvement in AP-1 activation by the
ARF4/PLD2 pathway. We are currently evaluating other signaling pathways
to identify the molecular mechanisms responsible for mediating
PLD2-dependent AP-1 activation.