Molecular Biology of the Cell
Vol. 18, 4420–4437, November 2007
ARL4D Recruits Cytohesin-2/ARNO to Modulate Actin
Chun-Chun Li,* Tsai-Chen Chiang,* Tsung-Sheng Wu,*
Gustavo Pacheco-Rodriguez,†Joel Moss,†and Fang-Jen S. Lee*
*Institute of Molecular Medicine, College of Medicine, National Taiwan University, and Department of
Medical Research, National Taiwan University Hospital, Taipei 100, Taiwan; and†Pulmonary-Critical Care
Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD
Submitted February 20, 2007; Revised August 21, 2007; Accepted August 27, 2007
Monitoring Editor: Francis Barr
ARL4D is a developmentally regulated member of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of
Ras-related GTPases. Although the primary structure of ARL4D is very similar to that of other ARF/ARL molecules, its
function remains unclear. Cytohesin-2/ARF nucleotide-binding-site opener (ARNO) is a guanine nucleotide-exchange
factor (GEF) for ARF, and, at the plasma membrane, it can activate ARF6 to regulate actin reorganization and membrane
ruffling. We show here that ARL4D interacts with the C-terminal pleckstrin homology (PH) and polybasic c domains of
cytohesin-2/ARNO in a GTP-dependent manner. Localization of ARL4D at the plasma membrane is GTP- and N-terminal
myristoylation-dependent. ARL4D(Q80L), a putative active form of ARL4D, induced accumulation of cytohesin-2/ARNO
at the plasma membrane. Consistent with a known action of cytohesin-2/ARNO, ARL4D(Q80L) increased GTP-bound
ARF6 and induced disassembly of actin stress fibers. Expression of inactive cytohesin-2/ARNO(E156K) or small interfer-
ing RNA knockdown of cytohesin-2/ARNO blocked ARL4D-mediated disassembly of actin stress fibers. Similar to the
results with cytohesin-2/ARNO or ARF6, reduction of ARL4D suppressed cell migration activity. Furthermore, ARL4D-
induced translocation of cytohesin-2/ARNO did not require phosphoinositide 3-kinase activation. Together, these data
demonstrate that ARL4D acts as a novel upstream regulator of cytohesin-2/ARNO to promote ARF6 activation and
modulate actin remodeling.
ADP-ribosylation factors (ARFs) are small GTPases involved
in membrane transport, maintenance of organelle integrity,
and activation of phospholipase D and phosphatidylinositol
4-phosphate 5-kinase (Moss and Vaughan, 1998; Chavrier
and Goud, 1999; Takai et al., 2001; D’Souza-Schorey and
Chavrier, 2006). ARF1 is mainly associated with the Golgi
apparatus, and it regulates vesicle budding of transport events
(Stearns et al., 1990; Balch et al., 1992). ARF6 can regulate
peripheral membrane dynamics and actin rearrangements at
the plasma membrane (Donaldson, 2003; Sabe, 2003) such as
stress fibers disassembly (D’Souza-Schorey et al., 1997; Boshans
et al., 2000), formation of plasma membrane protrusions and
ruffles (Radhakrishna et al., 1996; D’Souza-Schorey et al., 1997;
Franco et al., 1999), cell migration (Palacios et al., 2001; Santy
and Casanova, 2001), cell adhesion (Palacios et al., 2001), and
regulation of endosomal membrane traffic (D’Souza-Schorey et
al., 1995; Radhakrishna and Donaldson, 1997). Similar to other
guanosine triphosphate (GTP)-binding proteins, ARF function
depends on the highly controlled binding and hydrolysis of
GTP. The conformational changes that accompany the binding
GTPase for proteins, lipids, and membranes. Interconversion
guanine nucleotide-exchange factors (GEFs) and GTPase-acti-
vating proteins (GAPs) (Moss and Vaughan, 1998; Donaldson
and Jackson, 2000; Jackson and Casanova, 2000).
ARF-GEFs are linked to vesicular trafficking and cytoskeletal
events underlying cell movement, secretion, and endocytosis
(Jackson et al., 2000; Shin and Nakayama, 2004). ARF-GEFs that
have homology with the Sec7 domain of yeast Sec7p have been
identified by their ability to catalyze the exchange of GDP for
GTP on ARF proteins in vitro (Chardin et al., 1996; Jackson and
Casanova, 2000); the Sec7 domain is the only region of signif-
icant sequence similarity among ARF-GEFs, and the Sec7 do-
main alone is sufficient for guanine nucleotide-exchange activ-
ity (Chardin et al., 1996; Beraud-Dufour et al., 1998; Cherfils et
al., 1998; Mossessova et al., 1998; Kremer et al., 2004). To date,
four members of cytohesin ARF-GEFs that are ?77% identical
have been identified, including cytohesin-1, cytohesin-2/ARF
nucleotide-binding-site opener (ARNO), cytohesin-3/general
This article was published online ahead of print in MBC in Press
on September 5, 2007.
DThe online version of this article contains supplemental material
at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Fang-Jen S. Lee (email@example.com.
Abbreviations used: AD, activation domain; ARF, ADP-ribosylation
factor; ARL, ARF-like protein; ARNO, ARF nucleotide-binding-site
opener; BD, binding domain; GAP, GTPase-activating protein; GEF,
guanine nucleotide-exchange factor; GST, glutathione S-transferase;
NLS, nuclear localization signal; PAGE, polyacrylamide gel electro-
phoresis; PBS, phosphate-buffered saline; PCR, polymerase chain
reaction; PH, pleckstrin homology; PI3K, phosphoinositide 3-ki-
nase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; TBS, Tris-buff-
ered saline; GRP1, general receptor for phosphoinositides 1.
4420 © 2007 by The American Society for Cell Biology
receptor for phosphoinositides 1, and cytohesin-4. This family
is characterized by a molecular structure that consists of an
by a pleckstrin homology (PH) domain and an adjacent car-
boxy-terminal polybasic c domain (Nagel et al., 1998a; Santy et
al., 1999; Jackson and Casanova, 2000; Ogasawara et al., 2000;
Dierks et al., 2001). The coiled-coil domain of the cytohesin pro-
protein interaction. PH domains of cytohesins bind the lipid sec-
phosphatidylinositol bisphosphate in vitro. The cytohesins seem
to be involved in ARF6-mediated membrane trafficking and cy-
toskeletal reorganization (Jackson et al., 2000; Dierks et al., 2001;
Santy and Casanova, 2001; Moss and Vaughan, 2002). In cells
inositide 3-kinase (PI3K)-dependent manner that requires both
the PH and Sec7 domains of the cytohesin (Hemmings, 1997;
1999; Cullen and Chardin, 2000; Venkateswarlu, 2003).
ARLs share 40–60% amino acid sequence identity with
ARFs and are highly conserved throughout eukaryotic evo-
lution (Burd et al., 2004; Kahn et al., 2006). The best-charac-
terized ARL protein, ARL1 is localized to the trans-Golgi
network (TGN) and regulates trafficking in the TGN-endo-
somal pathways (Lu et al., 2004). ARL2 regulates the folding
of ?-tubulin (Bhamidipati et al., 2000). ARL4A, ARL4C, and
ARL4D, whose expression is differentiation dependent, de-
velopmentally regulated, and tissue specific, may function
in the nucleus (Schurmann et al., 1994; Jacobs et al., 1999; Lin
et al., 2000, 2002). Disruption of the ARL4A gene in mice can
reduce sperm count and a role(s) for ARL4A in the early
stages of spermatogenesis in adults is inferred (Schurmann
et al., 2002). ARL4D was identified as an open-reading frame
in a genomic region containing the BRCA1 gene (Harshman
et al., 1995; Smith et al., 1995) and was reported to interact
with HP1 (Lin et al., 2002). Nevertheless, until now, rela-
tively little is known about the cellular functions of ARL4D.
To obtain additional clues to its physiological role(s), we
attempted to identify interacting proteins, which may be
Here, we present evidence that the ARF6-GEF, ARNO, is
an effector of ARL4D. ARL4D interacted with ARNO in a
GTP-dependent manner and the interaction required the
C-terminal PH and polybasic c domains of ARNO. Localiza-
tion of ARL4D to the plasma membrane was GTP dependent
but it was not altered in ARNO knockdown cells or those
overexpressing ARNO(E156K). In addition, ARL4D induced
translocation of ARNO to the plasma membrane, with acti-
vation of ARF6 and loss of actin stress fibers. Knockdown of
ARNO suppressed the effect of activated ARL4D on actin
remodeling. ARL4D-induced redistribution of ARNO to the
plasma membrane was not dependent on PI3K activation.
These findings demonstrate a key role for ARL4D in the
recruitment of cytohesin-2/ARNO to the plasma membrane
along with ARF6 activation and actin remodeling.
MATERIALS AND METHODS
ARL4D antibodies were raised by immunizing rabbits with peptide N or peptide
B corresponding to amino acids (a.a.) 2-18 (GNHLTEMAPTASSFLPC) or a.a.
139-155 (QPGALSAAEVEKRLAVR) of ARL4D, respectively. Rabbit anti-myc
and FLAG antibodies were generated against the epitope tags, and a calnexin
antibody was prepared against a peptide DTSAPTSPKVTYKAPVPSGC corre-
sponding to a.a. 50-68 of calnexin. Other monoclonal antibodies used were myc
9E10 (BAbCO, Richmond, CA), FLAG M2, ?-tubulin, ARNO (Sigma-Aldrich, St.
Louis, MO), ARF6, Na?/K?ATPase (Santa Cruz Biotechnology, Santa Cruz,
CA), Akt, phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA), and
plasma membrane calcium pump pan ATPase (PMCA) (Abcam, Cambridge,
MA). Horseradish peroxidase-conjugated sheep anti-rabbit or anti-mouse immu-
noglobulin (IgG) antibodies were from GE Healthcare (Little Chalfont, Bucking-
hamshire, United Kingdom). Alexa Fluor 594, 488, or 350-conjugated anti-rabbit
and anti-mouse antibodies were purchased from Invitrogen (Carlsbad, CA).
ARL4D and mutants (Lin et al., 2002) were subcloned into the mammalian
expression vector pSG5 (Stratagene, La Jolla, CA). For expression as fusion with
the LexA DNA-binding domain (BD) in yeast, the cDNA fragments of ARL4D
and other ARF/ARLs cloned into the pBTM116 plasmid were used as described
previously (Lin et al., 2002). ARNO and its deletion mutant cDNA sequences
were amplified by polymerase chain reaction (PCR) by using ARNO/pACT2
plasmid as a template (obtained from yeast two-hybrid screen) and oligonucle-
otide primers incorporating restriction sites. To introduce site-specific mutations
in ARNO, a two-step recombinant PCR procedure was used. To generate
with alanine (386RKKRISVKKKQEQP3993386AAAAISVAAAQEQP399), by us-
ing a 3? primer in which the basic residues were replaced by alanine codons (5?
CTC GAG TCA GGG CTG CTC CTG TGC TGC TGC GAC TGA AAT TGC TGC
TGC TGC CGC TGC CAG CAT CTC ATA 3?). These constructs were subcloned
in pACT2 for yeast two-hybrid assay. For expression as GST-fusion protein in
Escherichia coli and N-terminal FLAG-fusion protein in mammalian cells, ARNO
was cloned into pGEX-4T (GE Healthcare) and pCMV-Tag2 (Stratagene) vectors,
respectively. For production of recombinant His-tagged proteins, ARNO and
ARNO(E156K) were subcloned into the bacterial expression vector pET15b (No-
vagen, Madison, WI). ARF6 and ARL4D were cloned into the expression vector
pET43a (Novagen). pACYC177/ET3d/yNMT, which encodes yeast (Saccharomy-
ces cerevisiae) N-myristoyltransferase (Lin et al., 2002) was used to myristoylate
ARF6 and ARL4D in Escherichia coli. Cytohesin-3 and cytohesin-4 cDNA were
synthesized by PCR from a prostatic adenocarcinoma cDNA pool (Clontech,
Mountain View, CA), respectively. The GGA3 cDNA was amplified from a HeLa
cell cDNA pool. Cytohesin-1 cDNA was kindly provided by Dr. M. Vaughan
(National Institutes of Health, Bethesda, MD). Plasmid expressing Akt-PH-green
fluorescent protein (GFP), which contains AKT1 (a.a. 1-164) with the PH domain
(a.a. 6-107), was obtained from Dr. Z.-F. Chang (National Taiwan University,
Taipei, Taiwan). Sequences of all constructs were confirmed by double-stranded
Yeast Two-Hybrid Screen and Interaction Assay
The yeast strain L40 was constructed with two readouts for an interaction of
histidine auxotrophy and ?-galactosidase expression with the use of the LexA
DNA-binding domain and GAL4-activation domain system. Using a lithium
acetate transformation protocol (Clontech), mouse embryonic day 7 pACT2
cDNA library (Clontech) was screened using ARL4D(Q80L) as bait. The yeast
two-hybrid screen and interaction assay was performed essentially as de-
scribed previously (Lin et al., 2000, 2002). For histidine auxotrophy and
?-galactosidase expression, we screened 6 ? 106clones, and we obtained 73
clones that specifically interacted with ARL4D(Q80L).
Guanosine 5?-O-(3-thio)triphosphate (GTP?S) Binding
Preparation and purification of recombinant proteins were carried out as
described previously (Pacheco-Rodriguez et al., 1998; Lin et al., 2000) The
GTP?S-binding assay was carried out in a rapid filtration system as described
previously (Pacheco-Rodriguez et al., 1998; Lin et al., 2000; Ogasawara et al.,
2000) with minor modification. Briefly, 50 ?l of the reaction buffer (20 mM
Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM NaN3, 250 mM sucrose,
2 mM MgCl2, and 2 mM dithiothreitol [DTT]) containing 1 ?g (?1 ?M) of
ARF6 or ARL4D; 40 ?g of bovine serum albumin; 10 ?g of l-?-phosphatidyl-
l-serine; 0.1 ?g each of aprotinin, leupeptin, and soybean, and lima bean
trypsin inhibitors; 0.5 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydro-
chloride; ARNO or ARNO(E156K) (1 ?g unless otherwise specified); and 4
?M [35S]GTP?S (?2 ? 106cpm) were incubated for the indicated time at 30°C.
The exchange reaction was terminated by cooling the sample on ice and
transferring to nitrocellulose filters followed by washing six times with 2 ml
of ice-cold wash buffer (25 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 100 mM NaCl,
1 mM DTT, and 1 mM EDTA). Scintillation fluid was added to the filters, and
they were counted in an LS 5000 beta-counter (Beckman Coulter, Fullerton,
CA) to quantify the amount of [35S]GTP?S-bound protein. Data are reported
as means ? SD of values from triplicate assays in a representative experiment.
All observations have been replicated at least twice with different prepara-
tions of recombinant proteins.
SDS-Polyacrylamide Gel Electrophoresis (PAGE) and
SDS-PAGE was performed according to the method of Laemmli (1970). Pro-
tein was visualized by Coomassie Brilliant Blue stain. For immunoblotting
analysis, proteins were separated on SDS gel and electrotransferred to poly-
vinylidene difluoride membranes (Millipore, Billerica, MA). Blocking and
ARL4D Protein Interacts with Cytohesin-2/ARNO
Vol. 18, November 2007 4421
antibody dilutions were made in 5% low-fat dry milk in Tris-buffered saline
(TBS) containing 0.1% Tween 20. Washing steps were in TBS containing 0.1%
Tween 20. Membranes were blocked overnight at 4°C; primary and secondary
antibodies were each incubated for 1 h at room temperature. Blots were
developed using secondary antibodies coupled to horseradish peroxidase (GE
Healthcare), and the immunoreactive bands were detected using the ECL
system (GE Healthcare) after exposure to x-ray film. In peptide competition
experiments, ?30 ?g of cell was loaded for each lane. The diluted primary
antibody solution was preincubated overnight with equal amounts of im-
munogen (immunized peptide dissolved in dimethyl sulfoxide [DMSO]) or a
Cell Culture and Transfection
COS-7 and human embryonic kidney (HEK) 293T cells were maintained in
DMEM (Hyclone Laboratories, Logan, UT), supplemented with 10% fetal
bovine serum (FBS) (Hyclone Laboratories), 100 U/ml penicillin, and 100
?g/ml streptomycin (Invitrogen) in a humidified incubator with 5% CO2at
37°C. Transient transfections were performed using the Lipofectin reagent
(Invitrogen) according to the manufacturer’s protocol or calcium phosphate
method. Cells were harvested 24–48 h later for analysis.
Glutathione S-Transferase (GST)-Fusion Protein
GST pull-down analysis was performed essentially as described previously
(Lin et al., 2002). In brief, 293T cells transfected with ARL4D or mutants were
lysed in radioimmunoprecipitation assay (RIPA) buffer containing 50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1%
SDS, 50 ?g/ml N?-?-tosyl-l-lysine chlorometyl ketone, 1 mg/ml benzami-
dine, and 1 ?g/ml each pepstatin A, aprotinin, antipain, and leupeptin. The
lysates were incubated with 10 ?g of GST or GST-ARNO coupled to gluta-
thione beads for 3 h at 4°C. The beads were then washed three times with the
RIPA buffer, and the bound protein was analyzed by Western blot.
293T or COS-7 cells were cotransfected with ARL4D and FLAG-ARNO con-
structs by the calcium-phosphate method. After 48 h, cells were treated with
2 mM dithiobis succinimidylpropionate (DSP; Pierce Chemical, Rockford, IL),
a thiol-cleavable cross-linker, to stabilize the protein complex, and lysed in
NP-40 lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1% NP-40).
Lysates were cleared by centrifugation at 13,000 ? g for 10 min at 4°C,
incubated with M2 anti-FLAG affinity gel (Sigma-Aldrich) for 2 h at 4°C, and
then washed three times in NP-40 lysis buffer and once in RIPA buffer. The
coimmunoprecipitated proteins were analyzed by Western blot.
Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS)
for 15 min and permeabilized with 0.01% Triton X-100 and 0.05% SDS in PBS
for 5 min, and blocked with blocking buffer (0.1% saponin, 0.2% bovine serum
albumin [BSA] in PBS) for an additional 30 min. Cells were incubated with
primary antibodies in blocking solution for 1 h. After extensive washing with
PBS, cells were incubated with the Alexa Fluor-conjugated secondary antibodies
in blocking solution for 1 h and mounted in 90% glycerol in PBS containing 1
mg/ml ?-phenylenediamine. For competition experiments, diluted primary an-
tibody was preincubated overnight with equal amounts of immunogen (immu-
nized peptide dissolved in DMSO) or a control peptide.
The plasma membrane localization of ARNO was determined as described
previously (Ueda et al., 2007). The ARNO membrane localization was evalu-
ated by the fluorescence intensity profile of a typical line scan. The ratios of
the average of two fluorescence signals of the plasma membrane to the
average signal of the cytosol were measured using Axiovision 4.6 software
(Carl Zeiss, Thornwood, NY). In the present study, we defined plasma mem-
brane translocation as a ratio of ?1.0. At least 50 different cells were analyzed for
each condition. Results are the means ? SD of three independent experiments.
The quantification of F-actin fluorescence was performed as described
previously (Poupel et al., 2000; Yamashiro et al., 2001). F-actin was stained
with Alexa Fluor 594-conjugated phalloidin (Invitrogen). Mock-transfected,
ARL4D, ARNO, or ARF6 mutant-expression cells were double labeled with
the antibody and fluorescent phalloidin. Phalloidin-stained images were
taken under the same conditions, and any images showing intensity satura-
tion were excluded from analysis. The area around each cell was delineated,
and the mean fluorescence intensity was measured in pixels. Background
fluorescence was obtained from a cell-free field of each image and subtracted.
Cells expressing exogenous proteins were randomly selected, and mean
fluorescent intensities (to compensate for differences in cell size) of phalloidin
staining were quantified using the Axiovision 4.6 software (Carl Zeiss). More
than 50 cells were examined in each group, and two independent experiments
were performed. The results were presented as the mean of the fluorescence
intensity for each group of cells ? SD.
For stimulation with EGF, cells were serum starved for 14 h, and 100 ng/ml
EGF (Upstate Biotechnology, Lake Placid, NY) was added for 10 min at 37°C.
To inhibit PI3K-Akt activation, serum-starved cells were pretreated with 100
nM wortmannin or 1 ?M LY294002 (Calbiochem, San Diego, CA) for 1 h, and
then they were incubated with EGF in the continuous presence of wortman-
nin and LY294002. The phosphorylation level of Akt (Ser473) was used to
confirm the inhibition of PI3K-Akt activation in each experiment. The staining
was examined with a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss)
with a 40?/1.3 numerical aperture (NA) oil objective lens. The images were
acquired with a charge-coupled device (CCD) camera (AxioCam HRm; Carl
Zeiss) operated by the Axiovision imaging software (Carl Zeiss). Some slides
were optically sectioned, and the out-of-focus signals were removed using the
ApoTome system (Carl Zeiss). The Apotome system provides an optical
section slice view reconstructed from fluorescent samples, by using a series of
“grid projection“ acquisitions to reject signals belonging to regions of the
sample that were outside the best focus position of the microscope. For
confocal microscopy, cells were viewed on a Nikon C1 confocal microscope or
LSM 510 META laser confocal microscope (Carl Zeiss) with 60?/1.4 NA oil
objective lens and 488- or 543-nm lasers. Pinholes were set to scan layers ?1
?m, at a resolution of 1024 ? 1024 pixels. Both projection view and optical
sections were processed digitally using CLSM5 Zeiss Browse Image software.
Figures were assembled and labeled using Adobe Photoshop 7.0 (Adobe
Systems, Mountain View, CA).
Small Interference RNA (siRNA)
Twenty one-nucleotide (nt) RNA duplexes were purchased from Dharmacon
RNA Technologies (Lafayette, CO) for targeting ARL4D or from Ambion
(Austin, TX) for targeting ARNO. The sequences of designed siRNAs are as
follows (only sense sequences are shown): ARL4D siRNA, 5?-GGGAACCA-
CUUGACUGAGAUU-3?; and ARNO siRNA, 5?-GAUGGCAAUGGGCAG-
GAAGtt-3?. Control siRNA (nontargeting siRNA pool) were purchased from
Dharmacon RNA Technologies. Sixty percent confluent COS-7 or HeLa cells
were transfected with siRNA at 100 nM concentration by using Lipofectamine
2000 reagent (Invitrogen) according to the manufacturer’s protocol. Cells were
harvested after 48 h for immunoblotting and immunofluorescence assays.
Subcellular fractionation was performed according to a method described
previously (Fournier et al., 2005), with a slight modification. HeLa cells were
washed with PBS, resuspended in homogenization buffer (0.25 M sucrose, 1
mM EDTA, and 20 mM HEPES-KOH, pH 7.4) and incubated on ice for 15 min
before disruption of cells by passing through 26-gauge needle 35 times until
?70% of the cells were broken. All processing steps were performed at 4°C.
After low-speed centrifugation (800 ? g; 6 min) to isolate nuclei and unbroken
cells, the postnuclear supernatant (PNS) was then centrifuged at 100,000 ? g
for 1 h to separate into crude cytosolic and membrane fractions. The mem-
brane pellet and 10% PNS were resuspended in the cytosol volume of ho-
mogenization buffer, precipitated with trichloroacetic acid. The sedimented
materials were resuspended in SDS sample buffer to the same volume, and an
equal volume of different fraction was subjected to SDS-PAGE and Western
blot analysis using various antibodies.
For ARNO translocation assay, transfected COS-7 cells (?1 ? 106cells)
were harvested, washed with PBS, and treated with 1 mM DSP, and then the
cytosol and membrane fractions of COS-7 cells were prepared using a CNM
compartment protein extraction kit (BioChain Institute, Hayward, CA) ac-
cording to the manufacturer’s instructions.
Pull-Down Assay for ARF6-GTP
The activation levels of expressed ARF6 were assayed as described previously
(Santy and Casanova, 2001). COS-7 cells grown in 10-cm dishes were cotrans-
fected with the indicated constructs and lysed in 1 ml of ice-cold lysis buffer
(50 mM Tris-HCl, pH 7.5; 100 mM NaCl; 2 mM MgCl2; 0.1% SDS; 0.5%
sodium deoxycholate; 1% Triton X-100; 10% glycerol, 50 ?g/ml N?-?-tosyl-
l-lysine chlorometyl ketone, 1 mg/ml benzamidine; and 1 ?g/ml each pep-
statin A, aprotinin, antipain, and leupeptin). Cell extracts were incubated with
20 ?g of glutathione Sepharose-bound GST-GGA3 (a.a. 1-316) for 1 h at 4°C.
The beads were then washed three times with 50 mM Tris-HCl, pH 7.5, 100
mM NaCl, 2 mM MgCl2, 1% NP-40, and 10% glycerol. Bound proteins were
analyzed by immunoblotting using ARF6 or myc antibodies. Immunoblots
were scanned, and the GTP-bound ARF6 precipitated with GST-GGA3 was
normalized to total ARF levels in the lysates to compare ARF6-GTP levels in
cells transfected with the indicated constructs.
Haptotactic migration assay were performed using a modified Boyden cham-
ber assay, essentially as described previously (Palacios et al., 2001). The lower
surface of Transwell polycarbonate membranes (6.5 mm diameter, 8-?m pore
size; Corning Life Sciences, Acton, MA) were coated with fibronectin (10
?g/ml in DMEM) overnight at 4°C. The membrane was washed in PBS to
remove excess ligand, and the lower chamber was filled with 600 ?l of
migration medium (DMEM with 1% BSA or 10% FBS). Cells were serum
starved for 16 h, and then 105cells were suspended in 0.1 ml of DMEM
containing 1% BSA were added to the upper chamber and allowed to migrate
to the lower side for 6 h. Nonmigratory cells were removed using a cotton
C.-C. Li et al.
Molecular Biology of the Cell 4422
swab, whereas migratory cells were fixed with 4% formaldehyde, stained with
1% crystal violet and count by phase-contrast microscopy by using a microscope
(Eclipse TS-100; Nikon, Tokyo, Japan) equipped with a digital camera (DS-5M;
Nikon). Migratory cells in five fields per well (Nikon Plan Fluor 10 ? 0.30
objective) were counted for two individual wells per condition.
For wound-healing migration assays, cells were seeded on six-well plates at
a density of 7 ? 105cells/well in culture medium. Thirty hours after seeding,
confluent cells were scratched with a fine pipette tip, washed with PBS, and
time-lapse microscopy was performed using a microscope (Axiovert 200M;
Carl Zeiss) equipped with a temperature and CO2controller. Cell movement
was recorded at 20-min intervals over 18 h with a CCD video camera (CoolSNAP
HO; Roper Scientific, Trenton, NJ) operated by MetaMorph 7.0 image analysis
software (Molecular Devices, Sunnyvale, CA). Quantitation of cellular migration
was performed as described previously (Santy and Casanova, 2001). The area
covered by the monolayer (decreased wound area) was traced and measured
using MetaMorph 7.0 software.
Identification of Cytohesin-2/ARNO as an ARL4D Interactor
To identify potential effectors of ARL4D, we used a yeast
two-hybrid system and used the ARL4D(Q80L), a puta-
tive GTP-bound form of ARL4D, as bait to screen a mouse
embryonic cDNA library. We identified several candidate
genes, including eight independent clones for cytohesin-
2/ARNO. We next assessed specificity among ARF/ARL
family members by yeast two-hybrid assays (Figure 1, A
and B). Our data showed that ARNO interacted specifi-
cally with ARL4D and ARL4A, but not with ARF1 or other
ARLs. Furthermore, ARNO interacted with ARL4D(WT),
not with ARL4D?C, the GTP-binding-defective mutant
ARL4D(T35N) or ARL4A(T34N). These results indicated
that the interactions were specific, nucleotide dependent,
and involved the C terminus of ARL4D.
To confirm the ARL4D and ARNO interaction, we per-
formed in vitro GST pull-down assays. We tested whether
purified, bacterially expressed GST-ARNO could bind to
ARL4D or its mutants (Figure 1C). GST-ARNO pulled
down ARL4D, ARL4D(Q80L), and ARL4D(G2A), but not
ARL4D(T35N) or ARL4D?C. GST alone failed to pull
down ARL4D constructs. Next, we used coimmunoprecipi-
tation experiments to demonstrate that ARL4D interacts
with ARNO. Proteins immunoprecipitated from lysates of
293T cells coexpressing ARL4D or mutants and FLAG-
ARNO with anti-FLAG M2 affinity gel were immunoblotted
with antibodies against ARL4D or FLAG. As shown in Fig-
important for binding to phosphate/Mg2?and the guanine base. ARL4D(Q80L) is a putative GTPase-defective mutant, and it is predicted
to exist in the GTP-bound active form. ARL4D(T35N) is a putative GTP-binding-defective mutant, and it is predicted to be in a GDP-bound
form. ARL4D?C lacks 16 a.a. (a.a. 186-201) at the C terminus, which contain the bipartite NLS. ARL4D(G2A) is a N-myristoylation–deficient
mutant. (B) ARL4D and ARL4A interacted with ARNO in a yeast two-hybrid system. Yeast reporter strain L40 cotransformed with the Gal4
AD fusion constructs of ARNO and the LexA BD fusion of the indicated constructs were grown on synthetic histidine-containing medium
lacking leucine and tryptophan (His? plate) and assayed for ?-galactosidase activity by a filter assay. Colonies from His? plates were also
grown on His-minus selective medium lacking histidine, leucine, and tryptophan (His? plate) for a growth assay. (C) ARL4D coprecipitated
with ARNO in vitro. Lysates of 293T cells expressing ARL4D or its mutants were incubated with either GST or GST-ARNO immobilized on
glutathione beads. Bead-bound ARL4D was probed using anti-ARL4D antibody. Ten percent of the cell lysate (Input) is shown for each of
the experiments. The equal input of GST fusion proteins used in the assay and visualized by Coomassie staining is shown on the bottom. (D)
Interaction between ARL4D and ARNO. 293T cells cotransfected with ARL4D constructs and FLAG-ARNO were lysed and immunopre-
cipitated with anti-FLAG M2 affinity agarose gel. Bound proteins were separated by SDS-PAGE and subjected to immunoblot with anti-FLAG
and anti-ARL4D antibodies. Ten percent of the cell lysate (Input) was loaded to show the expression level.
ARNO interacts with ARL4D. (A) Schematic representation of ARL4D molecule and mutants. G1–G3 are conserved regions
ARL4D Protein Interacts with Cytohesin-2/ARNO
Vol. 18, November 2007 4423
ure 1D, ARL4DWT, ARL4D(Q80L), or ARL4D(G2A), but not
ARL4D(T35N) or ARL4D?C, was coimmunoprecipitated
with FLAG-ARNO. Thus, we demonstrated interactions of
recombinant ARNO and ARL4D that were nucleotide de-
pendent and involved in the C-terminal NLS domain by
using proteins synthesized in bacteria or in 293T cells.
The PH Domain and the Polybasic C Domain of ARNO Are
Both Necessary and Sufficient for Interaction with ARL4D
All ARNO clones isolated from the yeast two-hybrid screen-
ing contained an intact PH domain and partial Sec7 domain,
suggesting that the C-terminal PH domain of ARNO may be
involved in the interaction with ARL4D. To identify the
specific domains of ARNO responsible for this interaction, a
series of ARNO-deletion mutants was generated (Figure 2A)
and tested for the ability to interact with ARL4D in yeast
two-hybrid assays (Figure 2B). We found that the C-terminal
140 amino acids (ARNOCT) alone were sufficient for the
interaction; the N-terminal coiled-coil and central Sec7 do-
mains were not required. ARL4D interaction with the
ARNO PH domain (ARNOPH) was much weaker than that
of the C-terminal PH domain and polybasic c domain
a coiled-coil domain (a.a. 10-63), Sec7 domain (a.a. 72-201), PH domain, and polybasic c domain (a.a.386-399). (B) ARL4D and its activated
mutants fused to the DNA BD of LexA were cotransformed with deletion mutants of ARNO fused to the Gal4 AD into yeast strain L40, and
the transformants were tested for their ability to grow in the absence of histidine (left) and to express ?-galactosidase (right). Mammalian
STE20-like protein kinase 3 (MST3) was a negative control. (C) Interaction of ARL4D and ARNO is mediated by the C terminus of ARNO.
COS-7 cells transfected with the indicated expression vectors were treated with the reducible cross-linker DSP before cells were lysed.
Proteins immunoprecipitated from cell lysates with an anti-FLAG M2 affinity agarose gel were separated by SDS-PAGE and immunoblotted
with anti-ARL4D or anti-FLAG antibodies. ARL4D(Q80L) was selectively coimmunoprecipitated with FLAG-ARNO constructs containing
the C-terminal PH domain and polybasic c domain. The arrows indicate the immunoglobulin light chain (LC) of antibody.
The PH domain of ARNO interacts with ARL4D. (A) Schematic representation of ARNO and its deletion mutants. ARNO contains
C.-C. Li et al.
Molecular Biology of the Cell4424
(ARNOCT) (Figure 2B), indicating that, besides the PH do-
main, the C-terminal polybasic c domain is also important.
The C-terminal polybasic stretch of cytohesin-1 and ARNO
was reported to be important for its collaboration with the
PH domain in membrane recruitment and phospholipids
PIP3binding (Nagel et al., 1998a; Macia et al., 2000; Dierks et
al., 2001). To determine whether the basic charge amino
acids in the C-terminal polybasic stretch is important for
the interaction between ARL4D and ARNO, we used site-
directed mutagenesis to generate an ARNO mutant,
ARNOC7A, in which the seven basic residues were replaced
with alanine. Interestingly, ARNOC7A, like ARNO, showed
similar interaction ability for ARL4D (Figure 2B). This result
indicates that the C-terminal basic amino acids are not in-
volved in the interaction between ARL4D and ARNO. These
constructs were expressed in relatively equal amounts in the
transformed yeast (unpublished data).
By coimmunoprecipitation, we further showed that
ARL4D(Q80L) was coimmunoprecipitated with FLAG-ARNO
and FLAG-ARNOCT, but not FLAG-ARNO?CT (Figure 2C).
Thus, our results demonstrate that the PH domain and poly-
basic c domain of ARNO are necessary and sufficient for inter-
action with ARL4D.
ARNO Does Not Catalyze Nucleotide Exchange on ARL4D
ARNO has previously been shown to catalyze guanine nu-
cleotide exchange on ARF6 in vitro (Frank et al., 1998a). To
determine whether ARL4D is a substrate of ARNO, we
measured the ability of ARNO to catalyze the binding of
GTP?S on ARF6 and ARL4D. Consistent with the previous
observation (Frank et al., 1998a), ARF6 undergoes spontane-
ous nucleotide exchange even in the absence of ARNO and
addition of ARNO results in a stimulation of GTP?S binding
(Supplemental Figure S1A and S1B). However, no stimula-
tion of GTP?S binding was observed when using ARL4D as
substrate. Our data indicated that ARNO could not catalyze
nucleotide exchange on ARL4D in the GTP?S binding assay.
Localization of ARL4D at the Plasma Membrane Is GTP
Our previous studies showed that N-terminally enhanced
green fluorescent protein (EGFP)-tagged ARL4D is located
in nuclei and partially in nucleoli and can interact with
importin-? through its C-terminal bipartite NLS (Lin et al.,
2002). However, ARL4D has a myristoylation site at its N
terminus and a NLS in its C terminus; thus, epitope tags at
either end might sterically hinder and change its conforma-
tion, localization, and function. To examine the physiologi-
cal phenotype of ARL4D, we first used two unique peptides
(peptide N corresponding to a.a. 2-18 and peptide B a.a.
139-155 of ARL4D) to generate peptide-specific antibodies
(Supplemental Figure S2A). A Blast search using the peptide
sequences of ARL4D-N and ARL4D-B did not reveal any
homologous peptides. The antibody against peptide B was
more sensitive in detecting purified recombinant human
ARL4D in low nanogram amounts, whereas no reaction was
detected with 100 ng of recombinant human ARL4A, ARF1,
or ARF6 proteins (Supplemental Figure S2B). Using this
antibody, endogenous ARL4D was detected in three cell
lines (Supplemental Figure S2C). The detected ?25-kDa band
was similar in size to recombinant ARL4D protein expressed in
To determine the subcellular localization of ARL4D, we
first analyzed overexpression of ARL4D and its mutants in
transiently transfected COS-7 cells by indirect immunofluo-
rescence microscopy (Figure 3A). The stacked images were
obtained by using a confocal microscope. The single plane
images of transfected cells are shown in Supplemental Fig-
ure S3. Interestingly, ARL4D was detected at the plasma
membrane, in addition to the nucleus and cytoplasm. Like
ARL4D(WT), ARL4D(Q80L) was detected in the nucleus
and diffusely throughout the cytoplasm but concentrated
most intensely at the plasma membrane, where it associated
with areas of membrane folding or ruffles along the periph-
ery of the cell. Notably, cells transfected with ARL4D(Q80L)
also demonstrated membranous protrusion structures from
their dorsal surface, and ARL4D(Q80L) was also concen-
trated within these structures. Nuclear, perinuclear punc-
tate, and diffuse cytoplasmic labeling, but much less plasma
membrane-associated signals, were detected in cells express-
ing ARL4D(T35N). Our results indicated that subcellular lo-
calization of ARL4D was guanine nucleotide dependent. We
further showed that ARL4D(G2A), containing Ala substituted
for Gly at position 2, was diffusely distributed in the cytoplasm
(Figure 3A), indicating that N-terminal myristoylation is im-
portant for association with the plasma membrane.
We next used the ARL4D antibody to detect endogenous
ARL4D. Figure 3B shows the result of an immunoblot anti-
body competition analysis of total HeLa cell lysate. The
detection of a 25-kDa protein was abolished by preincuba-
tion of the antibody with the ARL4D-B peptide immunogen
but not with ARL4D-N peptide. Moreover, endogenous
ARL4D distributed mainly in the membrane fraction after
cytosol-membrane fractionation of HeLa cells (Figure 3C).
We also observed that an ?42-kDa band on immunoblots,
which was only seen with nuclear fractions, was abolished
by the addition of a specific ARL4D-B antigen (Figure 3, B
and C). Whether the ?42-kDa band is the source of the
nuclear staining due to endogenous proteins needs to be
further investigated. We also examined the subcellular lo-
calization of endogenous ARL4D in interphase HeLa cells.
Endogenous ARL4D, like overexpression of ARL4D, was
detected at the plasma membrane, in addition to the nucleus
and cytoplasm (Figure 3D). PMCA was used as a control.
The signals of ARL4D were abolished when the antibody
was preincubated with the ARL4D-B peptide used for im-
munization, but not with DMSO or ARL4D-N peptide.
Both the PH Domain and Polybasic c Domain, but Not
GEF Activity of ARNO, Are Required for ARL4D-Induced
Recruitment of ARNO to the Plasma Membrane
The PH domain of ARNO is required for its membrane
targeting or translocation (Venkateswarlu et al., 1998). We
examined whether ARL4D could affect the subcellular local-
ization of ARNO in transiently transfected COS-7 cells. Fig-
ure 4A showed that FLAG-ARNO was detected as a diffuse
signal throughout the cytoplasm and was not clearly ob-
served at the periphery plasma membrane in COS-7 cells.
Coexpression of FLAG-ARNO and ARL4D(WT) or its mu-
tants reveals that part of FLAG-ARNO was colocalized with
ARL4D(WT) or ARL4D(Q80L) at plasma membrane ruffles
and dorsal membranous protrusions. This translocation of
ARNO was not observed in cells coexpressing ARL4D(G2A)
or ARL4D(T35N) (Figure 4B). We confirmed the effect of
ARL4D(Q80L) on ARNO translocation to the membrane
fraction by subcellular fractionation (Supplemental Figure S4).
We also observed similar effects of ARL4D on ARNO in HeLa
and Madin Darby canine kidney (MDCK) cells (unpublished
data). The localization of ARL4D or its mutants was not altered
when coexpressed with FLAG-ARNO (compare with Figures 3
and 4B). Quantitation of the ARNO fluorescence signal con-
firmed that ARL4D(WT) or ARL4D(Q80L) induced ARNO
redistribution to the plasma membrane (Figure 4, C and D).
Moreover, overexpression of ARF1(Q71L) or ARL1(Q71L) did
ARL4D Protein Interacts with Cytohesin-2/ARNO
Vol. 18, November 2007 4425
coverslips were transiently transfected with plasmids encoding ARL4D or its mutants. Forty-eight hours after transfection, cells were fixed,
permeabilized, and processed for immunofluorescence with anti-ARL4D antibody. The stacked images were obtained by using a confocal
microscope. (B) Detection of endogenous ARL4D by immunoblotting. Total HeLa cell lysate was separated by SDS-PAGE and probed with
anti-ARL4D-B antibody alone, or anti-ARL4D-B competed by 1 ?g of immunized ARL4D-B peptide, or nonimmunized ARL4D-N peptide
dissolved in DMSO. DMSO was used as a mock control. (C) Subcellular distribution of endogenous ARL4D by fractionation. HeLa cells were
homogenized and nuclear (N), PNS, cytosolic (C), and membrane (M) fractions prepared as described in Materials and Methods. Equivalent
amounts of proteins were separated by SDS-PAGE and analyzed by Western blot by using specific antibodies against ARL4D-B, HP1?
(nuclear marker), ?-tubulin (cytosol marker), and Na?/K?ATPase (membrane marker). Asterisk (*) indicates a nonspecific band detected in
cytosolic fraction. This band might be a degradation product from an unknown protein (?40-kDa), which was cross-reacted with the ARL4D
antibody in the total cell lysate. (D) Subcellular localization of endogenous ARL4D in HeLa cells. Endogenous ARL4D or plasma membrane
calcium pump pan PMCA ATPase was detected by immunofluorescence staining with anti-ARL4D-B or anti-PMCA antibody, respectively.
The plasma membrane labeling (arrow) was abolished when the antibody was preincubated with ARL4D-B peptide but not by DMSO (mock
control) or ARL4D-N peptide. Bar, 10 ?m. Peptide competition assays were performed as described in Materials and Methods.
Subcellular localization of ARL4D and its mutants. (A) Localization of overexpressed untagged ARL4D. COS-7 cells grown on
C.-C. Li et al.
Molecular Biology of the Cell4426
ARL4D Protein Interacts with Cytohesin-2/ARNO
Vol. 18, November 20074427
not affect the distribution of FLAG-ARNO (unpublished data).
redistribution to the plasma membrane and this effect is GTP
We next examined whether the PH domain or polybasic c
domain is required for inducing redistribution of ARNO by
ARL4D. COS-7 cells were transfected with ARNO?CT or
ARNOCT and/or ARL4D(Q80L). Similar to that of wild-
type ARNO, the localization of ARNO?CT or ARNOCT is
detected in the cytoplasm, and it did not seem to concentrate
at the lateral margins (Supplemental Figure S5A and S5B).
Although ARL4D(Q80L) could not induce redistribution of
ARNO?CT to the plasma membrane, ARNOCT and ARNOC7A
were translocated to ARL4D(Q80L)-enriched plasma membrane
in a manner similar to full-length FLAG-ARNO (Supplemental
less amounts of ARNOPH than full-length ARNO to the
plasma membrane (Supplemental Figure S5D). Our data dem-
onstrate that ARL4D mediates redistribution of ARNO
through its C-terminal PH domain and polybasic c domain.
ARNO is a GEF for ARF1 and ARF6 (Chardin et al., 1996;
Frank et al., 1998a); thus, we were interested to learn
whether ARL4D-regulated translocalization of ARNO is de-
pendent on ARNO GEF activity. To test this, we constructed
a catalytically inactive GEF form of ARNO, ARNO(E156K).
When expressed in COS-7 cells, ARNO(E156K) exhibited a
distribution similar to that of wild-type ARNO (Supplemen-
tal Figure S6A). ARNO(E156K) was coprecipitated by
ARL4D(Q80L) (Figure 2C), and, like the wild-type ARNO,
ARNO(E156K) was also recruited to the ARL4D(Q80L)-en-
riched plasma membrane region (Supplemental Figure S6B).
Moreover, coexpression of ARNO(E156K) or down-regula-
tion of endogenous ARNO by siRNA in COS-7 cells did not
affect the distribution of ARL4D(WT) (unpublished data).
These data suggest that induced redistribution of ARNO by
ARL4D does not require ARNO GEF activity.
ARL4D Promotes Activation of ARF6
Translocation of ARNO to the plasma membrane is a critical
event for ARNO GEF activity and ARF6 activation. We exam-
ined whether ARL4D-induced ARNO membrane-targeting
can promote the activation of ARF6. We showed in Figure 5A
that cells cotransfected with ARF6 and ARL4D(Q80L) exhib-
ited colocalization along plasma membrane ruffles and protru-
sions. Conversely, ARF6 showed little or no colocalization with
ARL4D(T35N). We carried out a pull-down assay to detect
ARF6 states by using a GST fusion construct containing the
VPS27, Hrs, and STAM- and ARF-binding domains of GGA3
(Santy and Casanova, 2001). ARF6(Q67L) bound to GST-GGA3
was used as a control (Figure 5B). In COS-7 cells, wild-type
ARF6-myc was coexpressed with either hemagglutinin (HA)-
ARNO, ARL4D(Q80L), or ARL4D(T35N). ARL4D(Q80L), but
not ARL4D(T35N), stimulated ARF6 activation. Consistent
with a previous report (Santy and Casanova, 2001), ARF6
activation was stimulated by ARNO (Figure 5B). Moreover,
cells coexpressed with HA-ARNO and ARL4D(Q80L) in-
Together, these results indicate that ARL4D recruits ARNO to
the plasma membrane and thus promotes activation of ARF6.
ARL4D(Q80L) Induces Disassembly of Actin Stress Fibers
Both ARNO and ARF6 have been demonstrated to modulate
the assembly and organization of the actin cytoskeleton
(Radhakrishna et al., 1996; D’Souza-Schorey et al., 1997;
Frank et al., 1998b; Boshans et al., 2000). Next, we investi-
gated whether the downstream effect of ARL4D-induced
ARNO translocation and ARF6 activation might be involved
in the organization of the actin cytoskeleton (Figure 6). Con-
sistent with previous reports (D’Souza-Schorey et al., 1997;
Frank et al., 1998b; Boshans et al., 2000), expression of ARNO,
but not the catalytically inactive ARNO(E156K) mutant, re-
sulted in the reduction of stress fibers, and a decrease in
actin stress fibers was elicited by overexpression of
ARF6(Q67L), but not ARF6(T27N) (Figure 6, A and C). As
expected, examination of actin organization in cells express-
ing ARL4D(Q80L) revealed a loss of stress fibers, and this
phenotype was not observed in mock-transfected cells, in-
dicating that the loss of stress fibers is a consequence of
ARL4D(Q80L) expression (Figure 6, A and C). A similar
phenotype was seen with the expression of relatively higher
levels of wild-type ARL4D (Figure 6C; data not shown).
Expression of ARL4D(T35N) or ARL4D(G2A) did not result
in the reduction of stress fibers (Figure 6, A and C). Quan-
titative fluorescence analyses for F-actin intensity are shown
in Figure 6C. Together, our data indicate that ARL4D(Q80L),
similar to ARNO and ARF6(Q67L), induces disassembly of
actin stress fibers.
ARL4D Modulates Actin Remodeling through ARNO
Having demonstrated that overproduction of ARL4D manip-
ulates the downstream effect in a manner similar to that of
ARNO and ARF6, we next examined whether ARL4D may act
coordinately with ARNO and ARF6 to modulate actin organi-
zation. Two experiments for abolishing GEF activity and re-
ducing the expression level of ARNO helped dissect this pos-
sibility. First, cells transfected with ARNO(E156K) showed no
effect on stress fiber organization and inhibited the decrease in
ARL4D(Q80L)-induced actin remodeling (Figure 6, B and C).
Second, we introduced ARNO or ARL4D siRNAs into COS-7
cells, and we showed that expression of endogenous ARNO
and ARL4D were markedly reduced when cells were treated
with ARNO-specific and ARL4D-specific siRNAs (Figure 6D).
These siRNAs were specific, and they did not interfere with
expression of other proteins, such as calnexin, ?-tubulin, or
ARF6. We further used siRNA knockdown to ask whether
reduction of ARNO could block ARL4D(Q80L)-mediated actin
remodeling. The localization of ARL4D(WT) or ARL4D(Q80L)
in ARNO knockdown cells remained unchanged; however,
ARL4D(Q80L)-induced stress fiber reduction was significantly
decreased (Figure 6E; data not shown). In contrast, reduction of
ARL4D had no affect on ARNO-mediated decrease in actin stress
downstream effector of ARL4D on the actin remodeling. Consis-
tent with the result from ARNO GEF inactivation, an inactive
Figure 4 (cont).
membrane protrusions and ruffles. COS-7 cells were transfected
with FLAG-ARNO alone (A) or cotransfected with FLAG-ARNO
and ARL4D mutants (B). Cells were fixed, permeabilized, labeled
with anti-FLAG M2 and anti-ARL4D antibody and examined by
confocal microscopy. Staining intensities measured according to
pixel brightness were evaluated by the fluorescence intensity (F.I.)
profile of a typical line scan for ARNO in a representative positively
stained cell. (C) Quantification of ARNO localization in ARL4D-
coexpressing cells. The ratios of the average of the two fluorescence
signals of the plasma membrane (PM) to the average signal of the
cytosol (CS) were evaluated. (D) Quantification of translocation of
ARNO to plasma membrane protrusions and ruffles in ARL4D-
expressing cells. The method for quantifying the ARNO plasma
membrane localization is described in Materials and Methods. At least
50 cells were analyzed for each condition. Results are the means ?
SD of three independent experiments. *p ? 0.001 compared with
ARNO alone. Bars, 10 ?m.
ARL4D induces ARNO redistribution to plasma
C.-C. Li et al.
Molecular Biology of the Cell 4428
form of ARF6 (T27N), blocked ARL4D(Q80L)-induced actin
remodeling. ARL4D(T35N) did not block ARF6(Q67L)-medi-
ated cytoskeletal rearrangements (Figure 6, B and C) or inter-
fere with the ARNO-mediated reduction of actin stress fibers
(Figure 6, B and C). These data demonstrate that ARL4D effects
on actin remodeling lie upstream of ARNO and ARF6.
Requirement for ARL4D in Cell Migration
Expression of ARNO or activation of ARF6 stimulates
MDCK cell migration (Palacios et al., 2001; Santy and
Casanova, 2001), and suppression of ARF6 blocks invasive
and migration activities of breast cancer cells (Hashimoto et
al., 2004). To investigate the potential role of ARL4D in cell
migration, we assessed whether knockdown of the endoge-
nous ARL4D would impair cell motility. We used a Trans-
well migration assay with control siRNA, ARL4D siRNA,
and ARNO siRNA in HeLa cells. The cells were plated in the
upper chamber containing filters that had been coated with
fibronectin on the underside, and they were allowed to
migrate for 6 h. As shown in Figure 7, B and C, HeLa cells
transfected with either ARL4D siRNA or ARNO siRNA
showed a significantly reduced motility compared with con-
trol siRNA. We also used a wound-healing assay to monitor
cell migration and obtained similar results. Namely, knock-
down of ARL4D or ARNO expression caused a delay in
wound closure (Figure 7D). Quantification of the wound
area covered by the migrating monolayer cell is shown in
Figure 7E. Together, these results provide evidence that
ARL4D play a physiological role in cell motility.
ARL4D-induced Translocation of ARNO to the Plasma
Membrane Is Independent of PI3-Kinase Signaling
It has been demonstrated that cytohesin-2/ARNO responds
to the PI3-kinase signaling cascade through the selectivity of
PH domains for binding PIP3, and cells stimulated with
agonists such as epidermal growth factor (EGF), nerve
growth factor (NGF), or insulin showed translocation of
cytohesin-2/ARNO from the cytosol to the plasma mem-
brane; this redistribution was inhibited by PI3K inhibitors
(wortmannin and LY294002) (Venkateswarlu et al., 1998,
2003; Mansour et al., 2002). To investigate whether the effects
of ARL4D on inducing redistribution of ARNO to the
plasma membrane were dependent on PI3K, we first exam-
ined the subcellular localization of ARL4D and ARNO in
transfected cells when PI3K activity was stimulated by EGF
or was blocked by PI3K inhibitors (Figure 8, A and B). To
confirm the inhibition of PI3K–Akt activation, the phosphor-
ylation level of Akt (Ser473) was examined in each experi-
ment (data not shown). As shown in a previous report
(Mansour et al., 2002), EGF stimulated plasma membrane
ruffling and translocation of ARNO to membrane ruffles
(Figure 8A). This translocation of ARNO was abrogated by
the addition of wortmannin or LY294002 (Figure 8, A and
C). However, the localization of ARL4D at the plasma mem-
brane was not affected by wortmannin or LY294002, indicat-
ing that localization of ARL4D is not dependent on PI3K
signaling (Figure 8A). Consistent with this finding, ARL4D-
mediated redistribution of ARNO to the plasma membrane
was not blocked by wortmannin when cells were incubated
ARF6. (A) ARL4D(Q80L) and ARF6 colocalize
along plasma membrane ruffles and protru-
sions. COS-7 cells cotransfected with vectors
encoding ARL4D(Q80L) or ARL4D(T35N) to-
gether with ARF6-myc were processed for im-
munofluorescence with ARL4D and myc anti-
bodies and examined by confocal microscopy.
Bar, 10 ?m. (B) Expression of ARL4D(Q80L)
results in increased levels of ARF6-GTP.
COS-7 cells transfected with plasmids encod-
ing ARF6-myc and/or the indicated proteins
were lysed. ARF6-GTP was precipitated using
GST-GGA3 coupled to glutathione beads, and
the precipitates were immunoblotted with an-
ti-myc antibody. Samples of cell lysates (2%
input) were also immunoblotted with myc,
HA, and ARL4D antibodies. (C) Overexpres-
sion of HA-ARNO increased endogenous
ARF6-GTP in HeLa cells when ARL4D(Q80L)
was coexpressed. ARF6-GTP levels were cal-
culated as the ratio of the ARF6-GTP to total
ARF6 (5% input), and they are reported rela-
tive to that in the absence a HA-ARNO ? 1.0.
ARL4D promotes activation of
ARL4D Protein Interacts with Cytohesin-2/ARNO
Vol. 18, November 20074429
C.-C. Li et al.
Molecular Biology of the Cell4430
with or without EGF (Figure 8, B and C). Quantitative flu-
orescence analyses confirm that ARNO redistribution to the
plasma membrane by ARL4D was independent on PI3K
activation (Figure 8C).
A previous report showed that a mutation (R279C) in the
PH domain of ARNO abolishes PIP3binding and does not
show EGF-stimulated translocation of the R279C mutant to
the plasma membrane (Venkateswarlu and Cullen, 2000).
Thus, we examined whether ARL4D could induce translo-
cation of ARNO(R279C) to the plasma membrane (Figure 8,
E–G). ARNO(R279C) was able to interact with ARL4D by
yeast two-hybrid analysis (Figure 8D). Consistent with a
previous report, ARNO(R279C) resides primarily in the cy-
tosol (Figure 8E, left; Venkateswarlu and Cullen, 2000).
When coexpressed with ARL4D(Q80L), we observed that
ARNO(R279C) translocated to the plasma membrane (Fig-
ure 8E, right). Moreover, ARL4D-mediated redistribution of
ARNO(R279C) to the plasma membrane was not blocked by
wortmannin when cells were incubated with or without EGF
(Figure 8, F and G). Collectively, these data suggested that
ARL4D-mediated association of ARNO to the plasma mem-
brane is dependent on interaction with plasma membrane-
associated ARL4D, but not in a PIP3-dependent manner.
ARL4D Recruits Other Members of Cytohesin Family, but
Not Akt PH Domain, to the Plasma Membrane
Because of highly structural conservation of the PH domain in
the cytohesin family (Ogasawara et al., 2000), we next exam-
ined whether ARL4D can induce redistribution of other cyto-
Figure 6 (facing page).
disassembly. (A) COS-7 cells were transfected with an expression
vector encoding FLAG-ARNO, FLAG-ARNO(E156K), ARL4D(Q80L),
ARL4D(T35N), ARL4D(G2A), ARF6(Q67L)-myc, and ARF6(T27N)-
myc, respectively. Forth-eight hours after transfection, cells were fixed
and stained with phalloidin to visualize F-actin and with anti-FLAG,
ARL4D, or myc antibody. (B) ARNO(E156K) suppressed decrease of
stress fibers induced by ARL4D(Q80L) expression. COS-7 cells cotrans-
fected with a combination of either ARL4D(Q80L) and FLAG-
ARNO(E156K), ARL4D(T35N) and FLAG-ARNO, ARL4D(Q80L) and
ARF6(T27N)-myc, or ARL4D(T35N) and ARF6(Q67L)-myc. Cells were
fixed and labeled with anti-ARL4D and FLAG M2 or myc 9E10 anti-
body and with phalloidin to visualize F-actin. (C) Quantification of the
average fluorescence intensity of F-actin in cells expressing the indi-
cated proteins is described in Materials and Methods. Data are given as
the mean ? SD and expressed as arbitrary units (AU). For each pop-
ulation, at least 50 cells were scored. *p ? 0.05, calculated by t test and
compared with control cells. (D) Depletion of ARNO or ARL4D in
COS-7 cells by siRNA. Extracts were prepared 48 h posttransfection,
and immunoblots were performed with antibodies against ARNO,
ARL4D, ARF6, ?-tubulin, or calnexin. Relative ARL4D or ARNO ex-
pression was compared after setting the ratio of ARL4D or ARNO
signal to ?-tubulin signal in the mock-transfected cells as 1.0. (E and F)
COS-7 cells were transfected with ARNO siRNAs together with
ARL4D(Q80L) or with ARL4D siRNA and FLAG-ARNO. Forty-eight
hours after transfection, cells were fixed and stained with phalloidin to
visualize F-actin and with anti-ARL4D or FLAG antibody to detect
ARL4D(Q80L) and FLAG-ARNO. Cells transfected with each indi-
cated construct to reduced stress fibers were assayed as described
above. Error bars represent the SD of three independent experiments.
*p ? 0.05, calculated by t test and compared with ARL4D(Q80L) alone
transfected cells. Bars, 10 ?m.
ARL4D(Q80L) induces actin stress fiber
gration. (A) HeLa cells transfected with
ARL4D siRNA, ARNO siRNA, or a control
siRNA were lysed 48 h after transfection, and
then they were assayed for expression by im-
munoblotting. (B and C) HeLa cells trans-
fected with the indicated siRNAs were sub-
jected to a Transwell migration assay. Cells
were plated in the upper chamber of the filters
that had been coated on the underside with
fibronectin, and their migrations were as-
sessed in the presence or absence of FBS as
indicated. Six hours after plating, cells that
had migrated to the underside of the filters
were fixed, stained with crystal violet, and
counted as described in Materials and Methods.
Error bars represent the SD of three indepen-
dent experiments. Bar, 100 ?m. (D) Time-lapse
microscopy of wound healing migration of
HeLa cells transfected with indicated siRNA.
Cells were cultured to confluence and then
scratched. Cell migration into wounds was
monitored and images were captured at the
indicated time after wounding. Bar, 200 ?m.
(E) Quantification of wound-healing migra-
tion assays. Migration was measured by cal-
culating the change in the area between mi-
grating cell sheets using MetaMorph software
and five repeats per data point. The increase in
cellular monolayer area over time is shown.
Results are the means ? SD.
Requirement for ARL4D in cell mi-
ARL4D Protein Interacts with Cytohesin-2/ARNO
Vol. 18, November 20074431
membrane localization of ARL4D. COS-7 cells transfected with ARL4D or FLAG-ARNO were serum starved, treated with EGF or
wortmannin alone, or pretreated with wortmannin followed by EGF. The cells were then fixed immediately and stained with ARL4D or
FLAG antibodies. (B) ARL4D recruitment of ARNO to the plasma membrane is PI3K independent. COS-7 cells cotransfected with ARL4D
or FLAG-ARNO were examined as described above. (C) Quantification of ARNO localization in ARL4D-coexpressing cells as described in
Materials and Methods. The fluorescence signals of ARNO were examine after EGF or wortmannin treatment from transfected FLAG-ARNO
alone (black bar) or cotransfected with ARL4D (white bar) cells. At least 50 cells were analyzed for each condition from two separate
experiments and error bars represent SD (D) ARL4D(Q80L) binds to ARNO(R279C) in yeast two-hybrid system. (E) ARL4D(Q80L) recruits
ARNO(R279C) to the plasma membrane. COS-7 cells transfected with FLAG-ARNO(R279C) alone or cotransfected with ARL4(Q80L) were
probed with antibodies against with ARL4D and FLAG. (F) ARNO(R279C), a PIP3-binding abolished mutant, can be recruited to the plasma
membrane by overexpressed ARL4D. COS-7 cells cotransfected with ARL4D or FLAG-ARNO(R279C) were examined as described above. (H)
ARL4D did not recruit Akt-PH-GFP to the plasma membrane. COS-7 cells cotransfected with ARL4D and Akt-PH-GFP were examined as
above. The membrane distribution of FLAG-ARNO(R279C) (G) and Akt-PH-GFP (I) were defined and quantified as per the methods used
in Figure 4. More than 50 transfected cells were counted in each group. Data represent means ? SD of two independent experiments. Bar,
ARL4D-induced translocation of ARNO is not dependent on PI3K signaling. (A) Inhibitors of PI3K do not inhibit the plasma
C.-C. Li et al.
Molecular Biology of the Cell4432
hesins, including cytohesin-1, cytohesin-3, and cytohesin-4, to
the plasma membrane. All FLAG-tagged cytohesins were
diffusely localized throughout the cytoplasm (Supplemental
Figure S7). When coexpressed with ARL4D(Q80L), the redis-
tribution of FLAG-cytohesin-1, FLAG-cytohesin-3, and FLAG-
cytohesin-4 to plasma membrane ruffles and protrusions was
detected (Supplemental Figure S7). Together, these results il-
lustrate that the effect of ARL4D on the subcellular localization
of all members of the cytohesin family is similar.
Despite high primary sequence variability, PH domains
retain a conserved three-dimensional organization consist-
ing of seven-stranded ?-sandwich structure, with one corner
capped off by a C-terminal ?-helix and another by three
interstrand loops. However, different PH domains have dif-
ferent affinities to several kinds of phospholipids (Lemmon,
2004; Balla, 2005). There has been speculation about the key
regulator(s) of their specificity (Lemmon, 2004; Balla, 2005;
Varnai et al., 2005), especially concerning the protein–protein
interaction. Similar to the PH domain of ARNO, the PH
domain of Akt showed growth-factor-stimulated and wort-
mannin-sensitive translocation from the cytosol to the
plasma membrane (Gray et al., 1999). Thus, we next exam-
ined whether ARL4D can induce translocation of Akt-PH-
GFP to the plasma membrane. However, a yeast two-hybrid
assay showed that ARL4D could not interact with Akt-PH
(unpublished data). In control, Akt-PH-GFP is localized to
the cytoplasm and the nucleus (Figure 8H, top). In EGF-
stimulated cells with or without coexpressing ARL4D, we
found that Akt-PH-GFP localized to the plasma membrane
(Figure 8, H and I). However, unlike the effect on ARNO,
ARL4D Protein Interacts with Cytohesin-2/ARNO
Vol. 18, November 20074433
ARL4D did not induce redistribution of Akt-PH-GFP to the
plasma membrane in the presence of wortmannin (Figure 8,
H and I). This suggests that the interaction between ARL4D
and cytohesin family proteins does not extend to another
PIP3 membrane-associated PH domain-containing protein,
and it reflects a novel and specific relationship between
ARL4D and cytohesin family proteins.
In this study, we demonstrated that ARL4D could act as a
novel upstream regulator of cytohesin-2/ARNO to modu-
late actin remodeling. Based on several lines of evidence, we
suggest that ARL4D directly interacts with cytohesin-2/
ARNO and recruits it to the plasma membrane in a PIP3-
independent manner to modulate actin remodeling. First,
ARL4D bound to cytohesin-2/ARNO, and this interaction
was dependent on the guanine-nucleotide bound by ARL4D
and the C-terminal PH and polybasic c domains of ARNO.
Second, localization of ARL4D at the plasma membrane was
GTP- and N-terminal myristoylation dependent. Third,
ARL4D induced translocation of ARNO to the plasma mem-
brane and promoted activation of ARF6, resulting in the
decrease of actin stress fibers. Fourth, siRNA-mediated
down-regulation of ARL4D lead to a suppression in cell
migration. Finally, ARL4D-induced translocation of cytohe-
sin-2/ARNO to the plasma membrane did not require the
activation of PI3K. We infer that ARL4D is activated by a yet
to be identified signal to recruit cytohesin-2/ARNO to the
plasma membrane, where ARNO activates ARF6 to induce
actin reorganization (Figure 9).
Localization of ARL4D at the Plasma Membrane
Our previous studies showed that N-terminal EGFP- or
C-terminal myc-tagged ARL4D was present in nuclei and
partially in nucleoli, possibly through the interaction of its
C-terminal bipartite NLS with importin-? (Lin et al., 2002;
our unpublished data). In this study, we detected endoge-
nous and nontagged recombinant ARL4D at the plasma
membrane in addition to the nucleus and cytoplasm. This
plasma membrane localization was GTP- and N-terminal
myristoylation dependent. The differences in the intracellu-
lar distribution may result from the effects of epitope tags at
either the N-terminal myristoylation site or near the C-
terminal bipartite NLS, both of which are important for
ARL4D localization. The staining of cells for endogenous
ARL4D (Figure 3D) and overexpressed ARL4D (Figure 3A)
is somewhat different. The staining pattern of overexpressed
ARL4D seems to show less nuclear staining and more punc-
tate cytosolic signals. Because the level of overexpressed
ARL4D compared with the endogenous protein could be
greater than 10-fold, increased overexpressed ARL4D pro-
tein might saturate nuclear sites or a nuclear import system,
thereby increasing cytosolic signals. Recently, Barrios-
Rodiles et al. (2005) showed that transforming growth fac-
tor-? signaling could stimulate interaction of ARL4D with
Smad2 and subsequently recruit ARL4D to a Smad2/Smad4
complex. This observation indicates that ARL4D may play
another role in the nucleus and/or nucleoli under specific
Cytohesin-2/ARNO Is a Downstream Effector of ARL4D
It is generally accepted that GEFs bind preferentially to
GDP-bound or nucleotide-free GTPase, and GAPs to GTP-
bound GTPase. The binary protein complex of nucleotide-
free GTPase and GEF is thought to be an enzymatic reaction
intermediate (Cherfils et al., 1998; Day et al., 1998). It is well
established that ARNO stimulates nucleotide exchange on
both ARF1 and ARF6 (Chardin et al., 1996; Frank et al.,
1998a) through the Sec7 domain, with its hydrophobic sur-
face groove for interaction with the switch 1 and switch
2 regions of ARFs (Betz et al., 1998; Goldberg, 1998;
Mossessova et al., 1998; Pacheco-Rodriguez et al., 1999).
ARNO-Sec7 formed a stable complex with the nucleotide-
free form of N?17ARF1 (Paris et al., 1997; Beraud-Dufour et
al., 1998). Unlike the typical association of a GEF with its
substrate, an association between ARL4D and ARNO was
found with ARL4D is in a GTP-bound state. This observa-
tion contrasts sharply with findings for the association be-
tween GEF and its bona fide substrates. The binding site for
ARL4D(Q80L) in ARNO lies in the C-terminal PH domain
and polybasic c domain, but not the Sec7 domain, indi-
cating that ARNO may serve as an ARL effector rather
than an activator. Moreover, our data showed that ARNO
is not a GEF for ARL4D. Consistent with this notion, we
showed that ARNO and ARNO(E156K) were translocated to
the plasma membrane when they were coexpressed with
ARL4D(Q80L). In addition, the amount of ARNO localized at
the plasma membrane was not altered in cells coexpressing the
membrane localization-defective mutant, ARL4D(G2A), al-
though ARL4D(G2A) interacted with ARNO. We suggest that
ARL4D might directly serve as a determinant for ARNO tar-
geting to the plasma membrane.
ARL4D Modulates Actin Remodeling via Regulating
ARNO and ARF6 Activity
In cells overexpressing ARL4D or ARL4D(Q80L), we ob-
served a paucity of actin stress fibers. The effects of ARL4D
on the actin cytoskeleton depend on its localization and the
guanine nucleotide-bound state. Mutants with decreased
affinity for GTP (T35N) or unable to localize at the plasma
membrane (lacking the myristorylation site [G2A]) are un-
able to modulate actin remodeling.
ARF6-GTP initiates cortical actin rearrangement at the cell
periphery, accompanied by a depletion of stress fibers
(D’Souza-Schorey et al., 1997; Boshans et al., 2000). Previous
and activation of ARF6. We speculate that ARL4D can be activated
by an unidentified GEF. Through protein–protein interaction, the
active form of ARL4D can recruit ARNO to the plasma membrane
where ARNO efficiently activates ARF6. ARF6-GTP induces actin
reorganization and membrane ruffling formation. ARL4D recruits
ARNO to the plasma membrane through a PH domain and poly-
basic c domain-mediated interaction. The PI3K pathway is not
involved in the regulation of ARL4D-mediated translocation of
Model for the role of ARL4D in recruitment of ARNO
C.-C. Li et al.
Molecular Biology of the Cell 4434
studies had indicated that ARNO might be the GEF for
ARF6. Overexpression of ARNO lead to disassembly of actin
stress fibers, remodeling of the cortical actin cytoskeleton in
HeLa cells (Frank et al., 1998b), and development of broad
lamellipodia in MDCK cells with a dramatic increase in
migratory behavior (Santy and Casanova, 2001). Coexpres-
sion of ARF6(T27N) or ARNO(E156K), or depletion of
ARNO content with siRNA, prevented ARL4D(Q80L)-in-
duced actin disassembly, suggesting that the activation of
ARF6 and ARNO are downstream effects of ARL4D. ARNO-
induced actin reorganization did not differ significantly in
cells with reduced ARL4D expression, further supporting
the idea. However, the amounts of actin stress fibers in
ARNO-depleted cells overexpressing ARL4D(Q80L) were
not as high as those cells overexpressing ARL4D(T35N) or
inactive ARNO. Perhaps the action of ARNO remaining in
siRNA-treated cells or of the other similar cytohesin/ARNO
proteins may account for only partial decrease of actin stress
The overexpression of ARNO or activation of ARF6 in-
duced a migratory phenotype (Palacios et al., 2001; Santy
and Casanova, 2001). It was also reported that expression of
ARNO(E156K), which cannot activate ARF6, caused a de-
crease in cell mobility (Santy and Casanova, 2001), and
suppression of ARF6 blocked invasive and migration activ-
ities of breast cancer cells (Hashimoto et al., 2004). This is
consistent with our finding of reduced cell mobility in
ARL4D siRNA knockdown cells. Our results are consistent
with the notion that ARL4D increases activation of ARF6 by
recruiting ARNO to the plasma membrane, followed by
reorganization of the actin cytoskeleton and enhanced cell
Some Ras superfamily GTPases have been shown to reg-
ulate each another via modulated GEF activities. Ras acti-
vated Ral through the direct association with the Ral-GEF
RalGDS (Feig, 2003) and activated Rac by stimulating the
activity of the Rac1-specific GEF Tiam1 (Lambert et al., 2002).
In Schizosaccharomyces pombe, GTP-Ras interacted with Scd1,
a RhoGEF, to promote Cdc42 activation (Chang et al., 1994).
To our knowledge, this is the first report describing the
coordination of GTPase cascade through an ARF-GEF.
ARL4D-induced Translocation of ARNO via the PH
Domain Is Independent of PI3-Kinase Signaling
PH domains are well-known for their ability specifically to
teins. Membrane targeting of PH-containing protein is often
important for functional activation in precise spatial and tem-
poral regulation. The PH domain of the cytohesin family pro-
teins is necessary for membrane association, PIP3binding, and
recruitment to the plasma membrane of cells stimulated with
growth factor in a PI3K-dependent manner (Klarlund et al.,
1997; Nagel et al., 1998b; Venkateswarlu et al., 1998; Klarlund et
al., 2000). Our data provide a new mechanism for regulation of
ARNO by protein–protein interaction. ARL4D overexpression
facilitated ARNO translocation to the plasma membrane even
in the presence of PI3K inhibitor, and PIP3-binding defective
mutant ARNO(R279C) could still be recruited to the plasma
membrane upon ARL4D overexpression. ARL4D did not in-
teract with PH domain from Akt, proving that its interaction
with cytohesin family PH domain is specific. Several PH do-
main-containing proteins are reported to interact via their PH
domains with small GTPases (Lemmon, 2004). The PH domain
of FAPP1 bound directly to ARF1 and targets it to Golgi (Godi
et al., 2004) and that of PLC-?2 acted as an effector site for Rac
(Snyder et al., 2003), whereas RhoA bound to the N-terminal
PH domain of the Btk family tyrosine kinase Etk (Kim et al.,
cytohesin family proteins are in agreement with these results.
We also showed that ARL4A and ARL4C recruited ARNO/
cytohesin2 to the plasma membrane (Supplemental Figure S8).
While this manuscript was under revision, Munro and col-
leagues (Hofmann et al., 2007; published online ahead of print
on March 28, 2007, in the Current Biology) reported that the
ARL4 family of small G proteins could recruit the cytohesin
ARF6 exchange factors to the plasma membrane, consistent
with our results. Although the ARL4 family can interact with
all cytohesin members, the differential expression of ARL4s
and cytohesins in different tissues and during different stages
of development (Schurmann et al., 1994; Jacobs et al., 1999; Lin
et al., 2000, 2002; Ogasawara et al., 2000) provides a means to
regulate the elaborate signaling pathway in eukaryotes. Inter-
estingly, Donaldson and colleagues (Cohen et al., 2007; pub-
lished online ahead of print on April 4, 2007, in the Mol. Biol.
Cell) showed that ARF6 could also bind to the PH domain of
ARNO and recruit it to the plasma membrane. Therefore, it is
plausible that recruitment of ARNO family GEFs to the plasma
membrane for further activation of other ARF isoforms might
be dependent on differential expression of ARL4s and ARF6 in
different cell types.
The precise subcellular localization of GTPase activation is
required for the proper initiation of downstream signaling
events. The biochemical mechanism upstream of ARL4D-
mediated regulation of ARNO signaling is not known yet.
Knowing that the expression of ARL4A and ARL4D are
developmentally regulated and tissue specific (Lin et al.,
2000, 2002), and that ARNO can regulate dendritic develop-
ment (Hernandez-Deviez et al., 2002), it will be important to
define precisely the signaling events upstream of ARL4 ac-
tivation and their functional consequences during develop-
We thank Drs. Randy Haun (University of Arkansas for Medical Sciences,
Little Rock, AR), Martha Vaughan (National Institutes of Health), and Chun-
Fang Huang for critical review of this manuscript. Experiments and image
analysis were performed in part through the use of the Cell Imaging Center
(Core lab 2) at National Taiwan University Hospital and with the assistance
of Shu-Chen Shen. We also thank laboratory members at Core lab 6 (espe-
cially to Shu-Tieng You, Yueh-Tso Tsai, and Yi-Jiun Chen) for help purifying
recombinant proteins. This work was supported by grants from the National
Science Council, R.O.C. (NSC 91GMP032-2, NSC 95GMP032-2, NSC 96-2752-
B-002-007-PAE), National Taiwan University Hospital (94A12-1, 95A12-1,
96A12-1), and the Yung-Shin Biomedical Research Funds (YSP-86-019) to
F.-J.S.L. This research was supported in part by the Intramural Research
Program, National Heart, Lung, and Blood Institute, National Institutes of
Balch, W. E., Kahn, R. A., and Schwaninger, R. (1992). ADP-ribosylation factor
is required for vesicular trafficking between the endoplasmic reticulum and
the cis-Golgi compartment. J. Biol. Chem. 267, 13053–13061.
Balla, T. (2005). Inositol-lipid binding motifs: signal integrators through pro-
tein-lipid and protein-protein interactions. J. Cell Sci. 118, 2093–2104.
Barrios-Rodiles, M. et al. (2005). High-throughput mapping of a dynamic
signaling network in mammalian cells. Science 307, 1621–1625.
Beraud-Dufour, S., Robineau, S., Chardin, P., Paris, S., Chabre, M., Cherfils, J.,
and Antonny, B. (1998). A glutamic finger in the guanine nucleotide exchange
factor ARNO displaces Mg2? and the beta-phosphate to destabilize GDP on
ARF1. EMBO J. 17, 3651–3659.
Betz, S. F. et al. (1998). Solution structure of the cytohesin-1 (B2–1) Sec7
domain and its interaction with the GTPase ADP ribosylation factor 1. Proc.
Natl. Acad. Sci. USA 95, 7909–7914.
Bhamidipati, A., Lewis, S. A., and Cowan, N. J. (2000). ADP ribosylation
factor-like protein 2 (Arl2) regulates the interaction of tubulin-folding cofactor
D with native tubulin. J. Cell Biol. 149, 1087–1096.
ARL4D Protein Interacts with Cytohesin-2/ARNO
Vol. 18, November 20074435
Boshans, R. L., Szanto, S., van Aelst, L., and D’Souza-Schorey, C. (2000).
ADP-ribosylation factor 6 regulates actin cytoskeleton remodeling in coordi-
nation with Rac1 and RhoA. Mol. Cell Biol. 20, 3685–3694.
Burd, C. G., Strochlic, T. I., and Gangi Setty, S. R. (2004). Arf-like GTPases: not
so Arf-like after all. Trends Cell Biol. 14, 687–694.
Chang, E. C., Barr, M., Wang, Y., Jung, V., Xu, H. P., and Wigler, M. H. (1994).
Cooperative interaction of S. pombe proteins required for mating and mor-
phogenesis. Cell 79, 131–141.
Chardin, P., Paris, S., Antonny, B., Robineau, S., Beraud-Dufour, S., Jackson,
C. L., and Chabre, M. (1996). A human exchange factor for ARF contains Sec7-
and pleckstrin-homology domains. Nature 384, 481–484.
Chavrier, P., and Goud, B. (1999). The role of ARF and Rab GTPases in
membrane transport. Curr. Opin. Cell Biol. 11, 466–475.
Cherfils, J., Menetrey, J., Mathieu, M., Le Bras, G., Robineau, S., Beraud-
Dufour, S., Antonny, B., and Chardin, P. (1998). Structure of the Sec7 domain
of the Arf exchange factor ARNO. Nature 392, 101–105.
Cohen, L. A., Honda, A., Varnai, P., Brown, F. D., Balla, T., and Donaldson.
J. G. (2007). Active Arf6 recruits ARNO/cytohesin GEFs to the PM by binding
their PH domains. Mol. Biol. Cell 18, 2244–2253.
Cullen, P. J., and Chardin, P. (2000). Membrane targeting: what a difference a
G makes. Curr. Biol. 10, R876–R878.
D’Souza-Schorey, C., Boshans, R. L., McDonough, M., Stahl, P. D., and Van
Aelst, L. (1997). A role for POR1, a Rac1-interacting protein, in ARF6-medi-
ated cytoskeletal rearrangements. EMBO J. 16, 5445–5454.
D’Souza-Schorey, C., and Chavrier, P. (2006). ARF proteins: roles in mem-
brane traffic and beyond. Nat. Rev. Mol. Cell Biol. 7, 347–358.
D’Souza-Schorey, C., Li, G., Colombo, M. I., and Stahl, P. D. (1995). A
regulatory role for ARF6 in receptor-mediated endocytosis. Science 267, 1175–
Day, G. J., Mosteller, R. D., and Broek, D. (1998). Distinct subclasses of small
GTPases interact with guanine nucleotide exchange factors in a similar man-
ner. Mol. Cell Biol. 18, 7444–7454.
Dierks, H., Kolanus, J., and Kolanus, W. (2001). Actin cytoskeletal association
of cytohesin-1 is regulated by specific phosphorylation of its carboxyl-termi-
nal polybasic domain. J. Biol. Chem. 276, 37472–37481.
Donaldson, J. G. (2003). Multiple roles for Arf 6, sorting, structuring, and
signaling at the plasma membrane. J. Biol. Chem. 278, 41573–41576.
Donaldson, J. G., and Jackson, C. L. (2000). Regulators and effectors of the ARF
GTPases. Curr. Opin. Cell Biol. 12, 475–482.
Feig, L. A. (2003). Ral-GTPases: approaching their 15 minutes of fame. Trends
Cell Biol. 13, 419–425.
Fournier, H. N., Dupe-Manet, S., Bouvard, D., Luton, F., Degani, S., Block,
M. R., Retta, S. F., and Albiges-Rizo, C. (2005). Nuclear translocation of
integrin cytoplasmic domain-associated protein 1 stimulates cellular prolifer-
ation. Mol. Biol. Cell 16, 1859–1871.
Franco, M., Peters, P. J., Boretto, J., van Donselaar, E., Neri, A., D’Souza-
Schorey, C., and Chavrier, P. (1999). EFA6, a sec7 domain-containing ex-
change factor for ARF6, coordinates membrane recycling and actin cytoskel-
eton organization. EMBO J. 18, 1480–1491.
Frank, S., Upender, S., Hansen, S. H., and Casanova, J. E. (1998a). ARNO is a
guanine nucleotide exchange factor for ADP-ribosylation factor 6. J. Biol.
Chem. 273, 23–27.
Frank, S. R., Hatfield, J. C., and Casanova, J. E. (1998b). Remodeling of the
actin cytoskeleton is coordinately regulated by protein kinase C and the
ADP-ribosylation factor nucleotide exchange factor ARNO. Mol. Biol. Cell 9,
Godi, A., Di Campli, A., Konstantakopoulos, A., Di Tullio, G., Alessi, D. R.,
Kular, G. S., Daniele, T., Marra, P., Lucocq, J. M., and De Matteis, M. A. (2004).
FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and
PtdIns(4)P. Nat. Cell Biol. 6, 393–404.
Goldberg, J. (1998). Structural basis for activation of ARF GTPase: mecha-
nisms of guanine nucleotide exchange and GTP-myristoyl switching. Cell 95,
Gray, A., Van Der Kaay, J., and Downes, C. P. (1999). The pleckstrin homology
domains of protein kinase B and GRP1 (general receptor for phosphoinositi-
des-1) are sensitive and selective probes for the cellular detection of phos-
phatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphos-
phate in vivo. Biochem. J. 344, 929–936.
Harshman, K. et al. (1995). Comparison of the positional cloning methods
used to isolate the BRCA1 gene. Hum. Mol. Genet. 4, 1259–1266.
Hashimoto, S., Onodera, Y., Hashimoto, A., Tanaka, M., Hamaguchi, M.,
Yamada, A., and Sabe, H. (2004). Requirement for Arf6 in breast cancer
invasive activities. Proc. Natl. Acad. Sci. USA 101, 6647–6652.
Hemmings, B. A. (1997). PH domains–a universal membrane adapter. Science
Hernandez-Deviez, D. J., Casanova, J. E., and Wilson, J. M. (2002). Regulation
of dendritic development by the ARF exchange factor ARNO. Nat. Neurosci.
Hofmann, I., Thompson, A., Sanderson, C. M., and Munro, S. (2007). The Arl4
family of small G proteins can recruit the cytohesin Arf6 exchange factors to
the plasma membrane. Curr. Biol. 17, 711–716.
Jackson, C. L., and Casanova, J. E. (2000). Turning on ARF: the Sec7 family of
guanine-nucleotide-exchange factors. Trends Cell Biol. 10, 60–67.
Jackson, T. R., Kearns, B. G., and Theibert, A. B. (2000). Cytohesins and
centaurins: mediators of PI 3-kinase-regulated Arf signaling. Trends Biochem.
Sci. 25, 489–495.
Jacobs, S., Schilf, C., Fliegert, F., Koling, S., Weber, Y., Schurmann, A., and
Joost, H. G. (1999). ADP-ribosylation factor (ARF)-like 4, 6, and 7 represent a
subgroup of the ARF family characterization by rapid nucleotide exchange
and a nuclear localization signal. FEBS Lett. 456, 384–388.
Kahn, R. A., Cherfils, J., Elias, M., Lovering, R. C., Munro, S., and Schurmann,
A. (2006). Nomenclature for the human Arf family of GTP-binding proteins:
ARF, ARL, and SAR proteins. J. Cell Biol. 172, 645–650.
Kim, O., Yang, J., and Qiu, Y. (2002). Selective activation of small GTPase
RhoA by tyrosine kinase Etk through its pleckstrin homology domain. J. Biol.
Chem. 277, 30066–30071.
Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and
Czech, M. P. (1997). Signaling by phosphoinositide-3,4,5-trisphosphate
through proteins containing pleckstrin and Sec7 homology domains. Science
Klarlund, J. K., Tsiaras, W., Holik, J. J., Chawla, A., and Czech, M. P. (2000).
Distinct polyphosphoinositide binding selectivities for pleckstrin homology
domains of GRP1-like proteins based on diglycine versus triglycine motifs.
J. Biol. Chem. 275, 32816–32821.
Kremer, W., Steiner, G., Beraud-Dufour, S., and Kalbitzer, H. R. (2004).
Conformational states of the small G protein Arf-1 in complex with the
guanine nucleotide exchange factor ARNO-Sec7. J. Biol. Chem. 279, 17004–
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227, 680–685.
Lambert, J. M., Lambert, Q. T., Reuther, G. W., Malliri, A., Siderovski, D. P.,
Sondek, J., Collard, J. G., and Der, C. J. (2002). Tiam1 mediates Ras activation
of Rac by a PI(3)K-independent mechanism. Nat. Cell Biol. 4, 621–625.
Lemmon, M. A. (2004). Pleckstrin homology domains: not just for phosphoi-
nositides. Biochem. Soc. Trans. 32, 707–711.
Lin, C. Y., Huang, P. H., Liao, W. L., Cheng, H. J., Huang, C. F., Kuo, J. C.,
Patton, W. A., Massenburg, D., Moss, J., and Lee, F. J. (2000). ARL4, an
ARF-like protein that is developmentally regulated and localized to nuclei
and nucleoli. J. Biol. Chem. 275, 37815–37823.
Lin, C. Y., Li, C. C., Huang, P. H., and Lee, F. J. (2002). A developmentally
regulated ARF-like 5 protein (ARL5), localized to nuclei and nucleoli, inter-
acts with heterochromatin protein 1. J. Cell Sci. 115, 4433–4445.
Lu, L., Tai, G., and Hong, W. (2004). Autoantigen Golgin-97, an effector of
Arl1 GTPase, participates in traffic from the endosome to the trans-Golgi
network. Mol. Biol. Cell 15, 4426–4443.
Macia, E., Paris, S., and Chabre, M. (2000). Binding of the PH and polybasic
C-terminal domains of ARNO to phosphoinositides and to acidic lipids.
Biochemistry 39, 5893–5901.
Mansour, M., Lee, S. Y., and Pohajdak, B. (2002). The N-terminal coiled coil
domain of the cytohesin/ARNO family of guanine nucleotide exchange fac-
tors interacts with the scaffolding protein CASP. J. Biol. Chem. 277, 32302–
Moss, J., and Vaughan, M. (1998). Molecules in the ARF orbit. J. Biol. Chem.
Moss, J., and Vaughan, M. (2002). Cytohesin-1 in 2001. Arch. Biochem. Bio-
phys. 397, 156–161.
Mossessova, E., Gulbis, J. M., and Goldberg, J. (1998). Structure of the guanine
nucleotide exchange factor Sec7 domain of human arno and analysis of the
interaction with ARF GTPase. Cell 92, 415–423.
Nagel, W., Schilcher, P., Zeitlmann, L., and Kolanus, W. (1998a). The PH
domain and the polybasic c domain of cytohesin-1 cooperate specifically in
C.-C. Li et al.
Molecular Biology of the Cell4436
plasma membrane association and cellular function. Mol. Biol. Cell 9, 1981– Download full-text
Nagel, W., Zeitlmann, L., Schilcher, P., Geiger, C., Kolanus, J., and Kolanus,
W. (1998b). Phosphoinositide 3-OH kinase activates the beta2 integrin adhe-
sion pathway and induces membrane recruitment of cytohesin-1. J. Biol.
Chem. 273, 14853–14861.
Ogasawara, M., Kim, S. C., Adamik, R., Togawa, A., Ferrans, V. J., Takeda, K.,
Kirby, M., Moss, J., and Vaughan, M. (2000). Similarities in function and gene
structure of cytohesin-4 and cytohesin-1, guanine nucleotide-exchange pro-
teins for ADP-ribosylation factors. J. Biol. Chem. 275, 3221–3230.
Pacheco-Rodriguez, G., Meacci, E., Vitale, N., Moss, J., and Vaughan, M.
(1998). Guanine nucleotide exchange on ADP-ribosylation factors catalyzed
by cytohesin-1 and its Sec7 domain. J. Biol. Chem. 273, 26543–26548.
Pacheco-Rodriguez, G., Patton, W. A., Adamik, R., Yoo, H. S., Lee, F. J.,
Zhang, G. F., Moss, J., and Vaughan, M. (1999). Structural elements of ADP-
ribosylation factor 1 required for functional interaction with cytohesin-1.
J. Biol. Chem. 274, 12438–12444.
Palacios, F., Price, L., Schweitzer, J., Collard, J. G., and D’Souza-Schorey, C.
(2001). An essential role for ARF6-regulated membrane traffic in adherens
junction turnover and epithelial cell migration. EMBO J. 20, 4973–4986.
Paris, S., Beraud-Dufour, S., Robineau, S., Bigay, J., Antonny, B., Chabre, M.,
and Chardin, P. (1997). Role of protein-phospholipid interactions in the
activation of ARF1 by the guanine nucleotide exchange factor Arno. J. Biol.
Chem. 272, 22221–22226.
Poupel, O., Boleti, H., Axisa, S., Couture-Tosi, E., and Tardieux, I. (2000).
Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters
actin monomers and caps actin filaments. Mol. Biol. Cell 11, 355–368.
Radhakrishna, H., and Donaldson, J. G. (1997). ADP-ribosylation factor 6
regulates a novel plasma membrane recycling pathway. J. Cell Biol. 139,
Radhakrishna, H., Klausner, R. D., and Donaldson, J. G. (1996). Aluminum
fluoride stimulates surface protrusions in cells overexpressing the ARF6
GTPase. J. Cell Biol. 134, 935–947.
Sabe, H. (2003). Requirement for Arf6 in cell adhesion, migration, and cancer
cell invasion. J. Biochem. 134, 485–489.
Santy, L. C., and Casanova, J. E. (2001). Activation of ARF6 by ARNO
stimulates epithelial cell migration through downstream activation of both
Rac1 and phospholipase D. J. Cell Biol. 154, 599–610.
Santy, L. C., Frank, S. R., Hatfield, J. C., and Casanova, J. E. (1999). Regulation
of ARNO nucleotide exchange by a PH domain electrostatic switch. Curr.
Biol. 9, 1173–1176.
Schurmann, A., Breiner, M., Becker, W., Huppertz, C., Kainulainen, H., Kentrup,
H., and Joost, H. G. (1994). Cloning of two novel ADP-ribosylation factor-like
proteins and characterization of their differential expression in 3T3–L1 cells.
J. Biol. Chem. 269, 15683–15688.
Schurmann, A., Koling, S., Jacobs, S., Saftig, P., Krauss, S., Wennemuth, G.,
Kluge, R., and Joost, H. G. (2002). Reduced sperm count and normal fertility
in male mice with targeted disruption of the ADP-ribosylation factor-like 4
(Arl4) gene. Mol. Cell Biol. 22, 2761–2768.
Shin, H. W., and Nakayama, K. (2004). Guanine nucleotide-exchange factors
for arf GTPases: their diverse functions in membrane traffic. J. Biochem. 136,
Smith, S. A., Holik, P. R., Stevens, J., Melis, R., White, R., and Albertsen, H.
(1995). Isolation and mapping of a gene encoding a novel human ADP-
ribosylation factor on chromosome 17q12–q21. Genomics 28, 113–115.
Snyder, J. T., Singer, A. U., Wing, M. R., Harden, T. K., and Sondek, J. (2003).
The pleckstrin homology domain of phospholipase C-beta2 as an effector site
for Rac. J. Biol. Chem. 278, 21099–21104.
Stearns, T., Willingham, M. C., Botstein, D., and Kahn, R. A. (1990). ADP-
ribosylation factor is functionally and physically associated with the Golgi
complex. Proc. Natl. Acad. Sci. USA 87, 1238–1242.
Takai, Y., Sasaki, T., and Matozaki, T. (2001). Small GTP-binding proteins.
Physiol. Rev. 81, 153–208.
Ueda, H., Fujita, R., Yoshida, A., Matsunaga, H., and Ueda, M. (2007). Iden-
tification of prothymosin-alpha1, the necrosis-apoptosis switch molecule in
cortical neuronal cultures. J. Cell Biol. 176, 853–862.
Varnai, P., Bondeva, T., Tamas, P., Toth, B., Buday, L., Hunyady, L., and Balla,
T. (2005). Selective cellular effects of overexpressed pleckstrin-homology do-
mains that recognize PtdIns(3,4,5)P3 suggest their interaction with protein
binding partners. J. Cell Sci. 118, 4879–4888.
Venkateswarlu, K. (2003). Interaction protein for cytohesin exchange factors 1
(IPCEF1) binds cytohesin 2 and modifies its activity. J. Biol. Chem. 278,
Venkateswarlu, K., and Cullen, P. J. (2000). Signalling via ADP-ribosylation
factor 6 lies downstream of phosphatidylinositide 3-kinase. Biochem. J. 345,
Venkateswarlu, K., Gunn-Moore, F., Tavare, J. M., and Cullen, P. J. (1999).
EGF-and NGF-stimulated translocation of cytohesin-1 to the plasma mem-
brane of PC12 cells requires PI 3-kinase activation and a functional cytohe-
sin-1 PH domain. J. Cell Sci. 112, 1957–1965.
Venkateswarlu, K., Oatey, P. B., Tavare, J. M., and Cullen, P. J. (1998).
Insulin-dependent translocation of ARNO to the plasma membrane of adipo-
cytes requires phosphatidylinositol 3-kinase. Curr. Biol. 8, 463–466.
Yamashiro, S., Chern, H., Yamakita, Y., and Matsumura, F. (2001). Mutant
Caldesmon lacking cdc2 phosphorylation sites delays M-phase entry and
inhibits cytokinesis. Mol. Biol. Cell 12, 239–250.
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