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INTRODUCTION |
Four principal steps in vesicular membrane traffic can be
distinguished. First, the coated membrane is invaginated by the assembly of coat proteins and formation of the coated pits. This is
followed by a pinching off of the coated vesicles, transport of the
vesicles to the appropriate acceptor membrane, and fusion of the
vesicles with this membrane. Numerous proteins and lipids are involved
in and regulated properly in a molecular cascade (1). In mammalian
cells, endocytosis of vesicles can occur via clathrin-coated pits, a
clathrin-independent pathway, or caveolae. It is thought that
endocytosis occurs at rafts, which are plasma membrane domains enriched
in cholesterol and sphingolipids (2, 3). Rafts also contain
glycosylphosphatidylinositol-anchored proteins and various
transmembrane proteins (4-6).
Activation of receptors by ligands results in internalization of
receptor complexes, and these complexes are rapidly recycled from
endosomes back to the cell surface (7, 8). Endocytosis of epidermal
growth factor (EGF)1 receptor
served as a model system for studies of ligand-dependent receptor trafficking for many years (9). EGF-activated receptors are
internalized via the clathrin-coated pit pathway (10) through interactions with the clathrin adaptor complex AP2 that recruits the
clathrin triskelion (11). Various accessory proteins, such as Eps15 and
Epsin, are recruited to the AP2-clathrin complex and
subsequently form an endocytic clathrin coat (12). The clathrin-adaptor coats undergo rearrangement, resulting in invagination of the coated
membrane, i.e. the clathrin-coated pit (13). For
constriction from shallow to deeply invaginated coated pits and
fission, endophilin A (EA) and dynamin are recruited to the rim of the
polymerizing clathrin coat (14). EA has lysophosphatidic acid
acyltransferase (LPA-AT) activity that catalyzes transfer of fatty
acids from co-enzyme A to LPA, thereby generating phosphatidic acid
(PA) (15). This shift in the biophysical properties of phospholipids in
the cytoplasmic leaflet of the membrane bilayer would cause inward
distortion of the luminal leaflets and subsequent membrane fission
(16). This finding was confirmed by a recent study (17) showing that
microinjection of an antibody against EA into lamprey reticulospinal
synapses interferes with synaptic vesicle recycling and clathrin-coated
vesicle formation.
The 100-kDa GTPase dynamin acts as an essential factor in the fission
stage of clathrin-mediated endocytosis (18). Dynamin assembles at the
site of fission and garrotes the membrane in a process driven by GTP
hydrolysis (19). It was reported that purified dynamin causes
vesiculation of liposomes in vitro in a
GTP-dependent fashion (20). The newly formed detached
clathrin-coated vesicles were internalized and moved through the
cytoplasm to early endosomes.
Vesicular trafficking at the plasma membrane would require
rearrangement of the cortical actin filaments to remove the barrier to
vesicular fusion or budding events (21). Actin filaments and
actin-based motor proteins play an essential role in endocytosis in
yeast (22-25). It was also reported that treatment of several actin-disrupting agents inhibit receptor-mediated endocytosis in
mammalian cells (26, 27). These data suggest that the actin cytoskeleton plays an essential role in endocytosis. The actin-related protein 2/3 (Arp2/3) complex enhances the nucleation and polymerization of actin filaments to promote filament assembly in vivo (28, 29). Neural Wiskott-Aldrich syndrome protein (N-WASP) plays an
essential role in Arp2/3-dependent actin dynamics by
enhancing Arp2/3 complex-induced nucleation of actin filaments (30).
N-WASP contains a WASP homology domain, a Cdc42 binding domain, a
proline-rich domain, two G-actin-binding verprolin-homology domains, a
cofilin-homology domain, and a carboxyl-terminal acidic segment (31).
The verprolin-homology-cofilin-homology-acidic (VCA) domain of N-WASP
is an essential minimal region for activation of Arp2/3 complex. At
rest, N-WASP is thought to be an auto-inhibited through an
intramolecular interaction between its Cdc42 interaction and
COOH-terminal domains (32). Binding of GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate (PIP2) to N-WASP
causes a conformational change in N-WASP that allows the VCA domain to interact with the Arp2/3 complex and initiate actin polymerization (33).
The yeast homologue of WASPs, Las17, was implicated in endocytosis
(34), and lymphocytes from WASP knockout mice exhibited reduced actin
polymerization and defective T cell receptor endocytosis (35). These
data suggest that N-WASP plays various roles at many steps of
endocytosis. Here we show that EGF induces recruitment of N-WASP from
the cytoplasm to rafts. We also show that N-WASP interacts with EA
through its proline-rich domain, playing an essential role in the
fission step of clathrin-mediated endocytosis.
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EXPERIMENTAL PROCEDURES |
Constructions--
For expression in mammalian cells, several
N-WASP constructs, inducing full-length N-WASP and proline-rich domain
(amino acids 271-385)-deleted N-WASP (N-WASP
P), were constructed in
pcDL-SR
and pEYFP (Clontech). EA3 cDNA (gift
from Dr. T. Endo, University of Chiba, Japan) was amplified by PCR with
primers that introduced 5' BamHI and 3' HindIII
sites. PCR products were cloned into the BamHI-HindIII sites of pCMV-tag3B
(Clontech) and into the BglII and
HindIII sites of pEGFP (Clontech) to
produce proteins tagged with Myc and green fluorescence protein (GFP).
To obtain the glutathione S-transferase (GST) fusion protein
of EA (GST-EA), full-length EA3 cDNA was subcloned into pCMV-tag3B,
cut with BamHI and XhoI, and inserted into
pGEX-2T (Amersham Biosciences). GST fusion proteins of EA3,
GST-EA
SH3 (amino acids 1-254) and GST-SH3 (amino acids 278-348),
were produced by in-frame insertion of the PCR-amplified fragment
corresponding to each sequence into the
BamHI-EcoRI sites of pGEX-2T.
Antibodies--
Anti-Myc antibody was purchased from Santa Cruz
Biotechnology, and the anti-clathrin heavy chain antibody (Ab-1) was
from Oncogene. The anti-dynamin antibody (mouse clone 41), the
anti-caveolin 1, the anti-Rab5, and the anti-phosphotyrosine antibody
(PY20) were from Transduction Laboratories. The anti-N-WASP antibody was prepared as described (36). The secondary antibodies linked to
peroxidase were from Cappel. The secondary antibodies linked to
fluorescein, Texas Red, and Cy5 were from Molecular Probes.
Fractionation of Cell Lysates by Sucrose Gradient
Centrifugation--
HeLa cells plated on 150-mm dishes were
serum-starved for 24 h and then stimulated with 100 ng/ml EGF
(Invitrogen). After two washes with ice-cold phosphate-buffered saline
(PBS), HeLa cells were scraped into 800 µl of 500 mM
sodium carbonate (pH 11.0). The cell lysates were extruded through a
23-gauge needle 10 times and then sonicated for 5 min in a sonicator
bath. One milliliter of the homogenate was then adjusted to 45%
sucrose by the addition of 1 ml of 90% sucrose prepared in MES
buffered saline (MBS) (25 mM MES, pH 6.5, 90% sucrose, 150 mM NaCl) and placed at the bottom of an ultracentrifuge
tube (14 × 89 mm, Beckman Instruments). A discontinuous sucrose
gradient was formed (2 ml of 5% sucrose/3 ml of 25% sucrose/3 ml of
35% sucrose; all in MBS containing 250 mM sodium
carbonate) and centrifuged at 100,000 × g for 3 h
in an SW41 rotor (Beckman Instruments). Proteins at the 5/25, 25/35,
and 35/45% interfaces were collected and separated by SDS-PAGE (10%
acrylamide) followed by Western blot analysis with the ECL detection
system (Pierce).
Cell Culture and Immunofluorescence Microscopy--
HeLa cells,
A431 cells, and COS7 cells were cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. For
immunofluorescence microscopy, 1 × 105 cells were
plated on 2% gelatin-coated coverslips in 35-mm dishes. Cells were
serum-starved for 24 h and then stimulated with 100 ng/ml EGF and
fixed with 3.7% formaldehyde in PBS. Fixed cells were permeabilized
with 0.2% Triton X-100 in PBS for 5 min and then incubated with
primary antibodies for 60 min. After washing, cells were incubated with
secondary antibodies. To stain EGF receptors, biotin-conjugated
anti-EGF receptor (EGFR1, Biogenesis) was used as primary antibody and
ultra avidin-rhodamine (Leinca Technologies, Inc.) as secondary
antibody. To visualize actin filaments, rhodamine-conjugated phalloidin
(Molecular Probes) was also added during the incubation with secondary
antibodies. After a 30-min incubation, coverslips were washed and
mounted on glass slides. Cells were observed with a confocal laser
scanning microscope (MRC 1024; Bio-Rad).
Methyl-
-cyclodextrin Treatment--
HeLa cells plated on
coverslips were incubated with 10 mM
methyl-
-cyclodextrin (CDX, Sigma) in the serum-free DMEM, 50 mM HEPES (pH 7.6) at 37 °C for 1 h. After
incubation in serum-free DMEM without CDX for 15 min, they cells were
stimulated with 100 ng/ml EGF and fixed.
Transfection--
COS7 cells were transfected in Opti-MEM
(Invitrogen) using 6 µl of LipofectAMINE (Invitrogen) and 3 µg of
plasmid DNA per 35-mm dish according to the manufacturer's
instructions. The DNA/LipofectAMINE was maintained on the cells for
4 h, and the medium was then exchanged with maintenance medium.
Twenty-four hours after transfection, cells were subjected to EGF
internalization assay. For EGF stimulation, transfected cells were
cultured for 2 h in maintenance medium and then for 24 h in
serum-free DMEM. To obtain cell lysates, 20 µg of recombinant plasmid
was mixed with 107 cells, and the mixtures were subjected
to electroporation with a Gene Pulser (Bio-Rad).
Immunoprecipitation--
EGF-treated or transfected cells in
100-mm dishes were washed twice with ice-cold PBS and lysed in 200 µl
of TGH buffer (50 mM HEPES (pH 7.6), 50 mM
NaCl, 5 mM EDTA, 1 mM orthovanadate, 10%
glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride). After addition of 800 µl of IP buffer (50 mM
HEPES (pH 7.6), 50 mM NaCl, 5 mM EDTA, 1 mM orthovanadate, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride), lysates were extruded 10 times through
a 23-gauge needle and centrifuged at 100,000 × g for 5 min. Supernatants were mixed with 10 µg of anti-N-WASP antibody or
anti-Myc antibody (Santa Cruz Biotechnology) for 2 h. As a
negative control, normal mouse IgG (Santa Cruz Biotechnology) or
pre-immune rabbit serum was used. Then protein A- and G-agarose beads
(Pierce) were added, and the mixtures were incubated for 1 h.
Immunoprecipitates were washed three times with IP buffer and then
analyzed by Western blotting.
Pull-down Assay--
GST fusion proteins were expressed in
Escherichia coli JM109 and purified from E. coli
lysates with glutathione-Sepharose beads (Amersham Biosciences)
according to standard methods. GST fusion proteins were eluted with 50 mM glutathione in 10 mM HEPES (pH 7.6).
Glutathione in the samples was removed by dialysis with IP buffer.
Protein concentrations were measured by Bradford assays with bovine
serum albumin as a standard. Twenty micrograms of the GST fusion
proteins were immobilized on glutathione-Sepharose beads and mixed with
HeLa cell lysates or COS7 cell lysates. After the beads were washed
with IP buffer, they were suspended in SDS sample buffer and subjected
to SDS-PAGE and Western blot analysis.
Purification of Actin, Arp2/3 Complex, N-WASP, and GST Fusion
Proteins--
Actin was purified from rabbit skeletal muscle, and
monomeric actin was isolated by gel filtration on Superdex 200 (Amersham Biosciences) in G buffer (2 mM Tris-HCl (pH 8.0),
0.2 mM ATP, 0.2 mM CaCl2, 0.5 mM DTT). Arp2/3 complex was purified from bovine brain
extracts as described previously (37). N-WASP was prepared with a
baculovirus system as described previously (30). A GST fusion protein
containing the verplorin homology, cofilin homology, acidic region (VCA
domains) and the GST fusion protein of the Ash/Grb2
NH2-terminal SH3 domain was used as a positive control (36). All GST fusion proteins used in the actin polymerization assay
were eluted with 50 mM glutathione in 10 mM
HEPES (pH 7.6).
Preparation of Lipid Vesicles Preparation--
For the actin
polymerization assay, phospholipid vesicles were prepared from
phosphatidylcholine, egg (PC) (Avanti), phosphatidylinositol (PI)
(Doosan Sedary Research Laboratories), PIP2 (Cell Signals, Inc), phosphatidic acid, dioleoyl (PA) (Sigma), and lysophosphatidic acid, oleoyl (LPA) (Sigma). Chloroform-dissolved phospholipids were
mixed in the appropriate ratios (PC/PI (50:50), PC/PI/PIP2 (48:48:4), PC/PI/PA (48:48:4), or PC/PI/LPA (48:48:4)) and dried under
nitrogen. The dried lipid mixture was resuspended in lipid buffer (10 mM HEPES (pH 7.7), 100 mM NaCl, 5 mM EGTA, 50 mM sucrose) to a final
concentration of 4 mM and then extruded through a 100-nm pore polycarbonate filter (Avanti) with the Mini-Extruder. The lipid
vesicles were used at a final concentration of 2 or 5 µM in the assay.
Actin Polymerization Assay--
Actin polymerization was
measured as the change in the fluorescence intensity of pyrene-labeled
actin as described previously (36). To follow actin polymerization with
purified components, pyrene-labeled G-actin or unlabeled G-actin was
isolated by incubation with freshly thawed proteins in G buffer (5 mM Tris-HCl (pH 8.0), 0.2 mM CaCl2,
0.2 mM ATP, 0.2 mM DTT) for 12 h at
4 °C followed by removal of residual F-actin by centrifugation at
40,000 × g for 1 h. Polymerization reaction
mixtures contained 60 nM Arp2/3 complex and various
proteins and lipids in 95 µl of the assay buffer (10 mM
HEPES (pH 7.6), 100 mM KCl, 1 mM
MgCl2, 0.1 mM EDTA, 1 mM DTT) and
were preincubated for 5 min. The reaction was initiated by adding a
5-µl mixture of 40 µM unlabeled actin, 4 µM labeled actin, and 4 mM ATP to the
preincubated reaction mixtures. The change in fluorescence was measured
at 407 nm with excitation at 365 nm in a fluorescence spectrometer
(Jasco). All kinetic analyses were performed with the software provided
by the manufacturer.
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RESULTS |
N-WASP Recycles between Rafts and the Cytoplasm in an
EGF-dependent Manner--
To study the function of N-WASP,
we examined localization of N-WASP during EGF-induced endocytosis. It
is believed that endocytosis occurs at particular sites in the plasma
membrane called rafts (2, 3). EGF-induced clathrin-coated vesicles are
sorted primarily to early endosomes. Both rafts and early endosomes are rich in cholesterol and sphingolipid and can be separated from the
cytosol by discontinuous sucrose gradient fractionation (38). The
components of rafts and early endosomes were fractionated from lysates
of EGF-stimulated HeLa cells to determine the EGF-dependent distribution of N-WASP. Raft components are enriched at the 5/25% interface of the gradient, whereas the early endosome marker early endosome antigen-1 (EEA1) is found at the 25/35% interface. The rest
of the cell lysates, mainly cytosolic proteins, are localized at the
35/45% interface (38, 39). In the present study, caveolins, marker
proteins of rafts, were detected in all fractions including the 5/25%
interface (Fig. 1A). This
distribution did not change after EGF stimulation (data not shown).
Rab5, a small GTPase associated with EEA1, was localized at the 25/35%
interface (Fig. 1A). These data indicate that raft
components were localized at the 5/25% interface, and those of early
endosomes were at the 25/35% interface. Hereafter, the collections of
proteins at the 5/25, 25/35, and 35/45% interfaces will be referred to
as the rafts fraction, EEA1 fraction, and cytosol fraction,
respectively.

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Fig. 1.
N-WASP is recruited to lipid rafts in
an EGF-dependent manner. A and
B, HeLa cell lysates treated with 100 ng/ml EGF were
fractionated on a discontinuous sucrose gradient loaded with 5, 25, 35, and 45% sucrose. Fractions were collected and analyzed by SDS-PAGE and
Western blotting with antibodies indicated. Proteins collected at the
5/25, 25/35, and 35/45% interfaces are referred to as the raft
fraction, EEA1 fraction, and cytosol fraction, respectively. The
indicated time is the period of incubation with EGF. C,
immunofluorescence microscopy of N-WASP. Serum-starved HeLa cells were
treated with 10 mM CDX at 37 °C for 1 h. Untreated
cells (left) and CDX-treated cells (right) were
incubated with EGF (100 ng/ml) for the indicated times, fixed, and
stained with anti-N-WASP antibody. White arrows indicate
N-WASP recruited to the plasma membranes. D,
immunofluorescence microscopy of N-WASP. Serum-starved A431 cells were
treated with 100 ng/ml EGF for the indicated times, fixed, and stained
with anti-EGF receptor (EGFR) and anti-N-WASP. The
right column is the merged image of EGFR
(red) and N-WASP (green). White
arrows indicate the site where N-WASP is co-localized with
EGFR.
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Activated EGF-receptors were detected with an anti-phosphotyrosine
antibody and used as a marker of internalized clathrin-coated vesicles.
Phosphorylated receptors were detected in the rafts fraction at 1.5 min
after EGF treatment and were then detected in the EEA1 fraction after
15 and 30 min (Fig. 1B). This change in distribution
supports the idea that EGF receptors are internalized from rafts to
early endosomes via a clathrin-dependent pathway. Clathrin,
localized at early endosomes and cytosol in unstimulated cells, was
recruited to rafts and then subsequently moved to early endosomes
similar to EGF receptors. In contrast to clathrin, dynamin was not
detected in early endosomes prior to EGF stimulation. Dynamin was also
recruited to rafts after EGF stimulation, but this recruitment was
later than that of clathrin. This time difference supports the
hypothesis that dynamin is recruited to the edge of the neck of the
clathrin-coated pit (14).
Like dynamin and many other proteins, N-WASP was detected in the
cytosol fraction. After EGF treatment, N-WASP was recruited to rafts
and then internalized into early endosomes (Fig. 1B). The
results of sucrose fractionation indicate that after EGF treatment, N-WASP was recruited approximately at the same time as clathrin.
To visualize the recruitment of N-WASP induced by EGF, HeLa cells were
stained with anti-N-WASP antibody (Fig. 1C) before and after
EGF stimulation. In serum-starved cells, N-WASP was localized in a
dot-like pattern around the perinuclear region. At 1.5-5 min after EGF
stimulation, N-WASP was located throughout the cytoplasm, and a portion
was present at the plasma membrane (Fig. 1C,
left). The translocation of N-WASP was well observed at the cell
periphery (Fig. 1C), where membrane ruffles were induced upon EGF stimulation. After 15 and 30 min, N-WASP had returned to its
resting localization pattern.
CDX is a useful tool to extract cholesterol from biological membranes
with high preference over other lipid species (40, 41). The major
component of rafts is cholesterol, and therefore, CDX treatment should
disrupt rafts in living cells. In the present study, CDX treatment
inhibited uptake of the Texas Red-labeled EGF into HeLa cells (data not
shown), suggesting that clathrin-mediated endocytosis is dependent upon
rafts. In CDX-treated cells, N-WASP remained at the perinuclear region
after EGF stimulation (Fig. 1C, right), and no
translocation to the plasma membrane was observed. These data suggest
that EGF induces the recruitment of N-WASP to rafts in the plasma
membrane and confirm the sucrose fractionation data.
To examine whether N-WASP co-localizes with EGF receptor (EGFR) in
rafts, EGF-treated A431 cells were double-stained with anti-EGFR and
anti-N-WASP antibodies. At 1.5-5 min after EGF stimulation, EGFRs were
accumulated to the plasma membranes together with N-WASP (Fig.
1D) and subsequently internalized with the clathrin-coated vesicles. The internalized EGFRs were co-localized clathrin and dynamin
(data not shown). In CDX-treated cells, neither EGFR nor N-WASP showed
recruitment to the plasma membranes (data not shown). These data
suggest that both EGFR and N-WASP were recruited to raft fractions upon
EGF stimulation.
N-WASP Is Associated with EA--
Some components of the endocytic
machinery, such as Pacsin and Intersectin (42, 43), have been reported
to bind to both dynamin and N-WASP. These proteins have SH3 domains and
associate with dynamin and N-WASP through the proline-rich domains. EA
also has an SH3 domain in its COOH terminus and binds to dynamin (44). EA has LPA-AT activity and plays an essential role in the fission step
of clathrin-mediated endocytosis (45). We hypothesized that EA
associates with N-WASP through its SH3 domain. To evaluate this
hypothesis, we performed co-immunoprecipitation experiments.
Myc-tagged full-length EA expression plasmids (Myc-EA) and full-length
N-WASP expression plasmids were co-transfected transiently into COS7
cells, and the EA/N-WASP interaction was detected by Western blot
analysis after precipitation of cell lysates with anti-N-WASP antibody
or anti-Myc antibody (Fig.
2A). Both immunoprecipitates contained Myc-EA and N-WASP. Positive signals were specific, as preimmune serum and control IgG immunoprecipitates did not yield positive signals. These findings indicate that EA associates with N-WASP in vivo.

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Fig. 2.
N-WASP is associated with the SH3 domain of
EA through the proline-rich domain. A, plasmids
expressing Myc-tagged full-length EA (Myc-EA) and full-length N-WASP
were co-transfected transiently into COS7 cells. Anti-N-WASP
immunoprecipitates (I.P.) (left) and anti-Myc
immunoprecipitates (right) were immunoblotted with
anti-N-WASP antibody (top) and anti-Myc antibody
(bottom). B, structures of GST fusion
proteins of EA. C and D, GST fusion proteins
of EA (C) were immobilized on glutathione-agarose beads and
incubated with COS7 cell lysates. Bound proteins were subjected to
SDS-PAGE and analyzed by Western blotting with anti-N-WASP antibody and
anti-dynamin antibody (D). E, the plasmid
expressing mutant N-WASP with a deletion of the proline-rich domain
(amino acids 271-385) (N-WASP P) were transfected into
COS7 cells, and the cells were then lysed. Immobilized GST-EA and
GST-SH3 were incubated with the resulting lysates. Bound proteins were
detected by Western blotting with anti-N-WASP antibody.
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We then examined whether the SH3 domain of EA can associate with the
proline-rich domain of N-WASP. As shown in Fig. 2B, we constructed a variety of GST fusion proteins of EA, including full-length (GST-EA), SH3 domain-deleted (GST-EA
SH3), and domain only (GST-SH3). These fusion proteins were mixed with COS7 cell lysates
for pull-down assays. Western blotting revealed that the precipitates
collected with GST-EA and GST-SH3 contained dynamin and N-WASP (Fig.
2C). When the same assay was performed with lysates of cells
in which proline-rich domain-deleted N-WASP (N-WASP
P) was
overexpressed, endogenous N-WASP but not N-WASP
P was detected in
precipitates (Fig. 2D). These results show that the SH3
domain of EA interacts with the proline-rich domain.
EA Is Associated with N-WASP in an EGF-dependent
Manner--
As shown in Fig. 2, when both Myc-EA and N-WASP were
expressed transiently, association of EA and N-WASP was revealed by
co-immunoprecipitation. But surprisingly, when only Myc-EA was
transfected into COS7 cells and anti-N-WASP antibody immunoprecipitates
were blotted with anti-Myc antibody, however, no signal was detected
(Fig. 3A). Because EA is a
cytosolic enzyme, it may be recruited to rafts in an
EGF-dependent manner together with N-WASP. Therefore, we tested the possibility that EGF might induce the interaction between N-WASP and EA. COS7 cells transfected with Myc-EA were stimulated with
EGF, and immunoprecipitation with anti-N-WASP antibody or with anti-Myc
antibody was performed. The time course of EA co-immunoprecipitation with N-WASP revealed that the association of EA with N-WASP occurred between 1.5 and 5 min after EGF treatment and disappeared by 15 min
(Fig. 3A). The time course of this association corresponds to that of detection of N-WASP in the rafts fraction (Fig.
1B). Therefore, EGF might induce association of EA with
N-WASP.

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Fig. 3.
Endophilin A is associated with N-WASP in an
EGF-dependent manner. COS7 cells were transfected with
Myc-EA and incubated with 100 ng/ml EGF for the indicated times. Cell
lysates were immunoprecipitated with anti-N-WASP antibody
(A) or anti-Myc antibody (B). Immunoprecipitates
were immunoblotted with anti-Myc, anti-N-WASP, and anti-dynamin
antibodies.
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It has been reported that dynamin interacts with EA and acts as
an essential factor in the fission stage of clathrin-mediated endocytosis. Despite these reports, dynamin was not found in the anti-Myc immunoprecipitates in Myc-EA-expressing cells under
resting conditions (Fig. 3B). After EGF stimulation, dynamin
co-precipitated with Myc-EA (Fig. 3B), suggesting this
interaction is also EGF-dependent. Both immunoprecipitation
studies with anti-Myc and anti-N-WASP indicated that Myc-EA, N-WASP,
and dynamin formed a same complex upon EGF stimulation.
EA Stimulates N-WASP-induced Arp2/3 Complex
Activation--
Because EA associated specifically with N-WASP, we
examined whether EA could further stimulate N-WASP-induced Arp2/3
complex activation in vitro. In the resting form, N-WASP
exists in an auto-inhibited conformation that involves an
intramolecular interaction between the Cdc42-interaction and the
COOH-terminal domains (32). Binding of GTP-bound Cdc42 and
PIP2 to N-WASP causes a conformational change in N-WASP
that exposes the VCA domain, which is the minimal essential region for
activation of the Arp2/3 complex, and initiates actin polymerization.
Actin polymerization can be monitored with pyrene-labeled actin, a
fluorescent derivative of actin that yields higher fluorescence
intensity when assembled into filaments. We used a cell-free system to
measure the effect of N-WASP and EA on Arp2/3 complex-induced actin
polymerization. The GST-VCA fusion protein of N-WASP maximally
activated Arp2/3 complex-induced actin polymerization as described
previously (37). In contrast, full-length N-WASP caused little
acceleration of actin assembly by the Arp2/3 complex. The various GST
fusion proteins of EA used in the present in vitro binding
assays (Fig. 2) had no effect on actin polymerization (Fig.
4A). However, GST-EA and
GST-SH3, which can bind N-WASP, enhanced N-WASP activation of Arp2/3
complex-induced actin polymerization, whereas GST-EA
SH3 did not
(Fig. 4B). A similar phenomenon was observed when various
histidine-tagged forms of EA were used in the place of the GST fusion
proteins (data not shown). The effect of GST-SH3 on N-WASP activation
to Arp2/3 complex was greater than that of GST-EA. The SH3 domain as
well as full-length EA were far more effective than the SH3 domain of
Grb2/Ash, one of known activators of N-WASP. GST-SH3 enhanced the
ability of N-WASP in a dose-dependent manner to the level
evoked by GST-VCA (Fig. 4D). Although the effect of GST-EA
was also dose-dependent, it did not reach the maximal level
like GST-SH3 (Fig. 4C).

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Fig. 4.
Endophilin A stimulates N-WASP-induced Arp2/3
complex activation. Plots of fluorescence intensity
versus time from initiation of the polymerization reaction.
Amounts of G-actin (2 µM unlabeled actin and 0.2 µM labeled actin) and Arp2/3 complex (60 nM)
were maintained throughout the experiments. All reactions were
initiated by addition of actin. Results shown are the means of
triplicate measurements. A, EA has little effect on
Arp2/3 complex-mediated actin polymerization. Curve 1,
actin polymerization in the presence of Arp2/3 complex alone.
Curve 2, addition of 75 nM GST-VCA
(VCA) to the reaction mixture used for curve 1. Curve 3, addition of 75 nM N-WASP
(NW) to the reaction mixture used in curve 1. Curves 4-6, addition of GST fusion proteins to the
reaction mixture used for curve 1. The GST fusion protein of
full-length EA (EA), the SH3 domain of EA (SH3),
and the SH3 domain-deleted EA (EA SH), respectively.
B, effect of various EA GST fusion proteins on
activation of N-WASP. Curve 1, actin polymerization in
the presence of Arp2/3 complex alone. Curve 2, addition
of 75 nM GST-VCA (VCA) to the reaction mixture
used for curve 1. Curve 3, actin
polymerization in the presence of Arp2/3 complex and 75 nM
N-WASP (NW). Curves 4-7, addition of 75 nM of the indicated protein to the reaction mixture used
for curve 3. Ash-SH3 presents the GST fusion
protein of the Ash/Grb2 NH2-terminal SH3 domain.
C and D, EA activates N-WASP in a
dose-dependent manner. Curve 1, actin
polymerization in the presence of Arp2/3 complex alone. Curve
2, addition of 75 nM GST-VCA (VCA) to
the reaction mixture used for curve 1. Curve 3, actin
polymerization in the presence of Arp2/3 complex and 75 nM
N-WASP (NW). Curves 4-8, addition of
full-length EA (C) or the SH3 domain of EA (D) at
the indicated concentrations to the reaction mixture used for
curve 3. E, effect of acidic lipids on
activation of N-WASP. Curve 1, actin polymerization in
the presence of Arp2/3 complex alone. Curve 2, actin
polymerization in the presence of Arp2/3 complex and 75 nM
N-WASP (NW). Curves 3-5, addition of 5 µM PIP2, PA, and LPA to the reaction mixture
used in curve 2, respectively. F, effect of
acidic lipids on activation of N-WASP by EA. Curve 1,
actin polymerization in the presence of Arp2/3 complex alone.
Curve 2, actin polymerization in the presence of Arp2/3
complex and 75 nM N-WASP (NW). Curve
3, addition of 75 nM full-length EA
(EA) to the reaction mixture used for curve 2. Curves
4-6, addition of 5 µM PIP2, PA,
and LPA to the reaction mixture used in curve 3,
respectively.
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|
PA Potentiates the Effect of EA on N-WASP--
PIP2
binds specifically to the basic domain of N-WASP and partially
activates the Arp2/3 complex to induce actin polymerization (33). LPA
and PA are the substrate and product of the LPA-AT activity of EA,
respectively (15). Both are acidic lipids that bind electrostatically
to the basic domain of N-WASP. Therefore, we speculated that LPA and PA
should influence N-WASP activation of the Arp2/3 complex in the
presence of EA. To examine this possibility, we added PA and LPA to
N-WASP in the presence or absence of GST-EA, and we performed Arp2/3
complex-induced actin polymerization assays. In the absence of GST-EA,
PIP2 induced N-WASP activation of actin polymerization more
effectively than LPA or PA (Fig. 4E). In the presence of
GST-EA, PA-induced enhancement of N-WASP-induced actin polymerization
was greater than that with PIP2 (Fig. 4F).
However, increasing concentrations of PA and GST-EA could not increase N-WASP activation of actin polymerization to the level evoked by
GST-VCA (data not shown). In the presence of GST-SH3, PIP2 enhanced N-WASP-induced actin polymerization more significantly than PA
or LPA (data not shown). These data suggest that EA together with PA
could produce greater activation of N-WASP to induce actin polymerization than PIP2, the major activator of N-WASP,
and that the local productions of PA by the LPA-AT activity of EA
cooperate in vivo inducing N-WASP-dependent
actin polymerization.
To examine localization of EA, COS7 cells transfected with GFP-tagged
EA (GFP-EA) were stimulated with EGF. In serum-starved cells, GFP-EA
and N-WASP were localized in the cytoplasm. After EGF stimulation,
GFP-EA was recruited to the plasma membrane and was co-localized with
endogenous N-WASP (Fig. 5). Actin also
accumulated at the plasma membrane where both GFP-EA and N-WASP were
co-localized in an EGF-dependent manner. Fifteen minutes
after EGF stimulation, GFP-EA and N-WASP returned to the cytoplasm.
These findings suggest that EA may regulate the actin cytoskeleton by
activating N-WASP with PA during EGF-induced endocytosis.

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Fig. 5.
EGF induces co-localization of endophilin A,
N-WASP, and actin. COS7 cells were transfected with plasmids
encoding GFP-tagged EA and starved in serum-free medium for 24 h.
Cells were then incubated with 100 ng/ml EGF and stained with
anti-N-WASP antibody and rhodamine phalloidin. The time
(left) indicates the period of incubation with EGF.
The right column is the merged image of
actin (red), EA (green), and N-WASP
(blue). White arrows indicate the site where
N-WASP is co-localized with EA. Note that both N-WASP and EA are
recruited to the site of the plasma membrane where actin has also
accumulated.
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EA May Regulate EGF-induced Recruitment of N-WASP--
As shown in
Fig. 1, EGF induces a recruitment of N-WASP to rafts. To investigate
the influence of clathrin-mediated endocytosis on the recruitment of
N-WASP, COS7 cells were transfected with Myc-tagged ENTH domain-deleted
Epsin (Myc-Epsin
ENTH) and GFP-tagged SH3 domain of EA (GFP-SH3),
stimulated with EGF, and immunostained with anti-N-WASP antibody.
Epsin is a component of clathrin-coated pits that bind to both the
clathrin heavy chain and AP2 (11). In cells overexpressing Epsin
ENTH, AP2 and clathrin were recruited to activated EGF
receptors, but formation of clathrin-coated pits was inhibited (46). It was also reported that injection of the SH3 domain of EA inhibits association of endogenous EA with dynamin in an
EGF-dependent manner, which then blocks fission of
clathrin-coated pits (17, 47). In the present study, uptake of Texas
Red-labeled EGF was inhibited in cells overexpressing Myc-Epsin
ENTH
or GFP-SH3 (data not shown).
We then examined whether N-WASP is localized normally when
clathrin-mediated endocytosis is inhibited. Five minutes after EGF
treatment, N-WASP was recruited to the plasma membranes of non-transfected cells but did not migrate in either Myc-Epsin
ENTH (Fig. 6A) or GFP-SH3
overexpressing cells (Fig. 6B). Quantification of both
results confirmed that the effect was quite noticeable (Fig.
6C). In almost 50% of control cells (untransfected and
GFP-transfected cells), N-WASP was recruited to the plasma membranes by
EGF stimulation. In contrast, only 10% of cells transfected with
GFP-SH3 or Myc-Epsin
ENTH showed the recruitment of N-WASP to the
plasma membranes. These results indicate that both formation of
clathrin-coated pits and fission are necessary for EGF-induced
recycling of N-WASP.

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Fig. 6.
Endophilin A may regulate EGF-induced
recruitment of N-WASP. A and B, COS7
cells were transfected with plasmid encoding the Myc-tagged ENTH
domain-deleted Epsin (Epsin ENTH, A) or
GFP-tagged SH3 domain of EA (GFP-SH3, B) and
incubated with 100 ng/ml EGF for the indicated times. Cells were fixed
and stained with anti-N-WASP antibody and anti-Myc antibody
(A) or with anti-N-WASP antibody (B). Upper
images illustrate the localization of N-WASP (A and
B). Lower images show the localization of
Epsin ENTH (A) or GFP-SH3 (B). White
arrows indicate plasma membranes. N-WASP was recruited to the
plasma membranes in non-transfected cells, but the localization did not
change in transfected cells. C, quantification of the
results by assessing the percentages of cells in which N-WASP was
recruited to the plasma membranes by EGF stimulation. Untransfected
cells: 58.0 ± 13.2%, n = 300; GFP: 47.9 ± 9.5%, n = 228; GFP-SH3: 9.3 ± 0.5%,
n = 122; Epsin ENTH: 11.8 ± 1.7%,
n = 116. D, COS7 cells were transfected
with plasmids encoding mutant N-WASP that is deleted for the
proline-rich domain (N-WASP P) and stimulated with 100 ng/ml EGF for 5 min. Lysates were separated into raft fraction, EEA1
fraction, and cytosol fraction as described in Fig. 1A.
These fractions were analyzed by Western blotting with anti-N-WASP
antibody. Note that endogenous N-WASP but not N-WASP P was recruited
to rafts in an EGF-dependent manner. E,
HeLa cells were transfected with YFP-tagged N-WASP or YFP-tagged
N-WASP P. These cells were starved (left) and stimulated
with EGF for 5 min (right) and then fixed. White
arrows indicate YFP-N-WASP recruited to rafts in the plasma
membrane.
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Sucrose fractionation studies of lysates from N-WASP
P-transfected
COS7 cells revealed that endogenous N-WASP but not N-WASP
P was
recruited to rafts in an EGF-dependent manner (Fig.
6D). To visualize the localization of N-WASP
P, HeLa cells
were transfected with YFP-N-WASP
P and treated with 100 ng/ml EGF for
5 min. As is the case with endogenous protein (Fig. 1), YFP-N-WASP was
recruited to the plasma membranes (Fig. 6E, upper panels),
whereas few cells showed the translocation of YFP-N-WASP
P (Fig.
6E, lower panels). This also confirmed that recruitment of
N-WASP to rafts is regulated through the proline-rich domain of
N-WASP.
 |
DISCUSSION |
N-WASP Is Recruited to Rafts after EGF
Stimulation--
Many recent studies have focused on the connection
between endocytosis and N-WASP; however, the role of N-WASP in
endocytosis remains unclear. N-WASP activates the Arp2/3 complex and is
essential for regulation of actin polymerization. Endosomes, pinosomes, and clathrin-coated and secretory vesicles are associated with actin
comet tails in the cytoplasm as have endosomes and lysosomes in
vitro (42, 48). Actin comet formation is dependent on N-WASP (49,
50); therefore, it is possible that N-WASP plays a role in vesicle
transport. In addition, N-WASP interacts with proteins such as
Pacsin, which binds to dynamin through the SH3 domain (51). These
findings suggest that N-WASP could play various roles at different
steps in endocytosis.
In the present study, we showed that N-WASP is recruited to rafts in an
EGF-dependent manner (Fig. 1B). Because N-WASP
was not detected in rafts in serum-starved cells, it is unlikely that N-WASP is involved in receptor-independent endocytosis. Upon EGF stimulation, N-WASP was quickly recruited to rafts and then
internalized into early endosomes together with clathrin-coated
vesicles containing activated EGF receptors.
Stimulation of glucose uptake by insulin in muscle and adipose tissue
requires translocation of the glucose transporter protein GLUT4 from
intracellular storage sites to lipid rafts (52). It was recently
reported that activation of a small GTP-binding protein TC10 and
rearrangement of the peripheral actin cytoskeleton are essential for
insulin-stimulated glucose uptake and GLUT4 translocation (53-55).
TC10 was found to interact with N-WASP, and it was suggested that
recruitment of TC10 is regulated by N-WASP (56). These findings suggest
that N-WASP may be recycled in an EGF-dependent manner
and recruit some proteins to lipids rafts.
EA May Regulate EGF-induced Recruitment of N-WASP to
Rafts--
Immunofluorescence studies with anti-N-WASP antibody
revealed that N-WASP, which is localized in a dot-like pattern at the perinuclear region under resting conditions, spreads throughout the
cytoplasm and is recruited to the plasma membrane after EGF stimulation
(Fig. 1C). In contrast, N-WASP remained in the perinuclear region in CDX-treated cells and did not move to the plasma membrane after EGF stimulation (Fig. 1C). These data suggest that EGF
induces recruitment of N-WASP to rafts in the plasma membrane.
EGF induces activation of a tyrosine kinase leading to
association of Ash/Grb2 and AP2 with the intracellular domain of EGF receptors. Clathrin-coated pits are then formed via AP2 bound to the
activated receptors (11). At this step, it is possible that Epsin
binds to AP2 through the DPW motifs of Epsin (46). In cells
overexpressing ENTH domain-deleted Epsin (Epsin
ENTH), the
activation of tyrosine kinase and recruitment of both AP2 and clathrin
may occur normally, but clathrin-coated pit formation is inhibited
(51). N-WASP was originally identified as Ash/Grb2-binding protein and
was assumed to form a complex with the EGF receptor via Ash/Grb2 (31).
However, EGF-induced recruitment of N-WASP was inhibited in cells
overexpressing a deletion mutant of Epsin where Ash/Grb2 may be
recruited normally to the EGF receptors. Therefore, EGF-induced
recruitment of N-WASP to rafts appears to require clathrin-coated pit
formation. Moreover, we have shown that overexpression of the EA SH3
domain inhibits EGF-induced recruitment of N-WASP and that a mutant of
N-WASP lacking the proline-rich domain, which cannot associate with EA,
does not move to rafts (Fig. 6). These data suggest that EGF-induced
recruitment of N-WASP to rafts may be regulated by EA through the
proline-rich domain of N-WASP.
N-WASP Associates with EA in an EGF-dependent
Manner--
Recently, it was reported that N-WASP associates with
several proteins such as Pacsin and Intersectin-1, and those may be involved in the endocytic machinery (42, 43). These proteins have SH3
domains that interact with the proline-rich domain of dynamin, and
internalization of clathrin-coated vesicles is inhibited in cells
overexpressing these SH3 domains (44). Overexpression of full-length
Pacsin affected cortical actin organization, inducing formation of
filopodia, suggesting that Pacsin activates N-WASP. However,
overexpression of the SH3 domain alone had no effect on the actin
cytoskeleton, indicating that Pacsin-induced cytoskeletal rearrangements are not due directly to Pacsin-N-WASP interactions (57).
Intersectin-1 is a modular scaffolding protein that interacts with
N-WASP through an SH3 domain and with Cdc42 through a Dbl homology
domain (58). Recent studies (59) showed that Intersectin-1 binds to
N-WASP and the activated Cdc42 through its action as a guanine
nucleotide exchange factor.
In the present study, we show that N-WASP forms a complex together with
EA and dynamin in an EGF-dependent manner (Fig. 3). EA and
dynamin regulate the fission step of clathrin-mediated endocytosis
(45), and thus, N-WASP may have some role in clathrin-mediated endocytosis.
EA Enhances N-WASP Activation of Arp2/3 Complex--
N-WASP is
regulated by multiple activators. Individually most of these molecules,
such as Cdc42 and PIP2, yield weak activation, and
stimulation of N-WASP is accomplished by the proper combination of
upstream signals. Thus, N-WASP integrates multiple signals to target
actin polymerization precisely and regulate the actin cytoskeleton.
We confirmed that EA enhances N-WASP activation of the Arp2/3 complex
in vitro (Fig. 4). Despite weak activation of polymerization by EA alone, EA in combination with PA activates N-WASP more strongly than PIP2 (Fig. 4F). Thus, it is possible that
EGF-induced N-WASP regulates the actin cytoskeleton at lipid rafts.
Immunofluorescence studies of COS7 cells overexpressing EA revealed
that actin accumulates at plasma membrane sites where EA and N-WASP
co-localize in an EGF-dependent manner (Fig. 5).
In summary, we have shown that N-WASP is recruited to rafts via an
EA-dependent pathway in response to EGF and associates with
EA. Because EA activates N-WASP in the presence of PA, it seems
reasonable to assume that N-WASP may have some role in EGF-induced endocytosis by binding to EA. Thus our results indicate that N-WASP interacts with EA to regulate the fission step of endocytosis.