From the Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Received for publication, September 19, 2000, and in revised form, December 28, 2000
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ABSTRACT |
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Neurite outgrowth of PC12 cells is induced by
nerve growth factor (NGF) but not by epidermal growth factor (EGF).
This differential response has been explained by the duration of
mitogen-activated protein kinase (MAPK) activation; NGF induces
sustained MAPK activation but EGF leads short-lived activation.
However, precise mechanisms have not yet been understood. Here we
demonstrate the difference between NGF and EGF in regulation of Rac1, a
small GTPase involved in neurite outgrowth, in PC12 cells. NGF
phosphoinositide 3-kinase dependently induces transient
activation of Rac1 and accumulation of active Rac1 at protrusion sites
on the cell surface, inducing filamentous actin-rich protrusions and
subsequent neurite formation in a Rac1-dependent manner. On
the other hand, EGF phosphoinositide 3-kinase independently induces
more transient Rac1 activation but neither accumulates active Rac1 nor
forms Rac1- and filamentous actin-rich protrusions. Difference in the
Rac1 localization between NGF and EGF was also observed with the
localization of exogenously expressed green fluorescent protein-tagged
Rac1. The Rac1-mediated protrusion by NGF is independent of MAPK
cascade, but the subsequent neurite extension requires the cascade.
Thus, the differential activation of Rac1 and localization of active
Rac1 contribute to the difference in the ability of NGF and EGF to
induce neurite outgrowth, and we propose that the MAPK
cascade-independent prompt activation of Rac1 and recruitment of active
Rac1 at the protrusion sites trigger the initiation of neurite formation.
Rat pheochromocytoma PC12 cells have been used as a model system
for neuronal differentiation and neurite outgrowth. After stimulation
with nerve growth factor
(NGF),1 they stop
growing and begin to extend neurites. In contrast, epidermal growth
factor (EGF) does not induce neurite outgrowth but stimulates
proliferation of PC12 cells. The receptors for NGF and EGF belong to a
family of tyrosine kinase receptors, and they transduce signals via
similar signal transduction pathways, including a
Ras-dependent mitogen-activated protein kinase (MAPK) cascade (1-3). It has been proposed that the sustained activation of
Ras and MAPK by NGF is involved in neuronal differentiation of PC12
cells (2, 4, 5). However, a previous study reported that the
receptor-mediated sustained activation of MAPK alone is insufficient to
induce neurite outgrowth (6). Thus, an additional signaling pathway is
suggested to be required for the NGF-induced neurite outgrowth.
Morphological analysis of NGF- and EGF-stimulated PC12 cells revealed
that stimulation with NGF, but not with EGF, induces the rapid
formation of filamentous actin (F-actin)-rich protrusions, followed by
the extension of neuritic processes with growth cone-like structures at
their tips (7, 8). Furthermore, the rapid redistribution of F-actin
induced by NGF was reported to be suppressed by an inhibitor of
phosphoinositide 3-kinase (PI3K) (9), and overexpression of a
constitutively active mutant of PI3K was shown to induce neuritic
process formation (10, 11), suggesting the involvement of PI3K in the
NGF-induced neurite outgrowth. However, precise mechanisms involved in
the cytoskeletal reorganization required for the NGF-induced neurite
outgrowth have not yet been understood.
The Rho family of small GTPases, including Rho, Rac, and Cdc42, serves
as molecular switches by cycling between an inactive GDP-bound state
and an active GTP-bound state, and has been implicated in the
reorganization of actin cytoskeleton in various cell types (12, 13). In
PC12 cells, activation of Rho induces the growth cone collapse and the
retraction of neurites (14, 15). In contrast, studies using a dominant
negative Rac1 show that Rac1 is involved in the NGF-induced neurite
outgrowth (16, 17). However, it remains obscure how NGF regulates Rac1,
inducing neurite outgrowth. Here we demonstrate that NGF PI3K
dependently induces Rac1 activation and formation of cell surface
protrusions where active Rac1 and F-actin are accumulated, whereas EGF
PI3K independently activates Rac1 but fails to form Rac1- and
F-actin-rich protrusions. We propose that the differential activation
and localization of Rac1 contribute to the difference in the ability of
growth factors to induce neurite outgrowth.
Materials--
Agents obtained and commercial sources were as
follows: NGF 2.5S, Promega Corporation; EGF, Becton Dickinson Labware;
LY294002, Sigma; wortmannin, Kyowa Medex Co.; PD98059, Calbiochem;
mouse monoclonal anti-Rac1 antibody, Transduction Laboratories; rabbit polyclonal anti-Cdc42 antibody, Santa Cruz Biotechnology, Inc.; rabbit
anti-Akt and anti-phospho-specific Akt (Ser-473) antibodies, Cell
Signaling; rabbit anti-extracellular signal-regulated kinase1 (ERK1)
and anti-ERK2 antibodies, Upstate Biotechnology, Inc.; and
anti-phospho-specific ERK antibody, New England Biolabs, Inc.
Construction of Expression Plasmids--
Mammalian expression
vector pEF-BOS was kindly provided by Dr. S. Nagata (Osaka University).
Human Rac1 was obtained as described previously (18). cDNA for
Ha-Ras was obtained from Health Science Research Resources Bank (Osaka,
Japan). RasN17 and Rac1N17 were generated by
polymerase chain reaction-mediated mutagenesis (19) and fused
in-frame with a sequence encoding an initiating methionine followed by
the Myc epitope tag sequence at the NH2 terminus contained
in pEF-BOS. Green fluorescent protein (GFP)-Rac1 was obtained by
insertion of the coding sequence of wild-type Rac1 into pEGFP-C1
(CLONTECH). The coding sequence for the Cdc42/Rac interacting binding (CRIB) domain (amino acids 70-150) of rat Cell Culture and Transfection--
PC12 cells were cultured in
Dulbecco's modified Eagle's medium containing 5% fetal bovine serum,
10% horse serum, 4 mM glutamine, 100 units/ml penicillin,
and 0.2 mg/ml streptomycin under humidified conditions in 95% air and
5% CO2 at 37 °C. For immunofluorescence analysis, cells
were seeded onto poly-D-lysine (Sigma)-coated glass
coverslips (circular, 13 mm) in 24-well plates at a density of 2.5 × 104 cells/well. Transient transfections were carried out
using LipofectAMINE 2000 (Life Technologies Inc.) according to the
manufacturer's instructions. Transfected cells were fixed 48 h
after transfection.
Measurement of Rac1 and Cdc42 Activities--
Measurement of
Rac1 and Cdc42 activities was performed according to the modified
method of Benard et al. (21). PC12 cells were seeded in
100-mm culture dishes at a density of 1 × 107
cells/dish, cultured for 24 h, and serum-starved in serum-free Dulbecco's modified Eagle's medium for 12 h. Cells were then
stimulated with 50 ng/ml NGF or 200 ng/ml EGF for the indicated times
and lysed for 5 min with the ice-cold cell lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml aprotinin, and 1 µg/ml leupeptin) containing 8 µg of GST-CRIB. Cell lysates were then centrifuged for 5 min at
10,000 × g at 4 °C, and the supernatant was
incubated with glutathione-Sepharose beads for 30 min at 4 °C. After
the beads were washed with the cell lysis buffer, the bound proteins
were eluted in Laemmli sample buffer and separated by 12.5% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. The separated
proteins were electrophoretically transferred onto a polyvinylidene
difluoride membrane (Millipore Corp.). The membrane was blocked with
3% low fat milk in Tris-buffered saline, and then incubated with a
mouse monoclonal anti-Rac1 (1:1000 dilution) or rabbit polyclonal
anti-Cdc42 antibody (1:100 dilution). The Rac1 and Cdc42 antibodies
were detected using horseradish peroxidase-conjugated goat anti-mouse
IgG and goat anti-rabbit IgG antibodies (DAKO), respectively, and the
ECL detection kit (Amersham Pharmacia Biotech). Densitometry analysis
was performed using NIH Image software, and the amounts of
GST-CRIB-bound Rac1 and Cdc42 were normalized to the total amounts of
Rac1 and Cdc42 in cell lysates, respectively.
Immunofluorescence Microscopy--
All steps were carried out at
room temperature, and cells were rinsed with phosphate-buffered saline
(PBS) between each step. At the indicated times, cells on coverslips
were fixed with 3.7% formaldehyde, PBS for 15 min. After
residual formaldehyde had been quenched with 50 mM
NH4Cl, PBS for 10 min, cells were permeabilized in 0.2%
Triton X-100, PBS for 10 min, and incubated with 10% fetal bovine
serum in PBS for 30 min to block nonspecific antibody binding. Endogenous Rac1 was stained with an anti-Rac1 monoclonal antibody in
PBS at a 1:1000 dilution for 1 h followed by the incubation with a
rhodamine-conjugated donkey anti-mouse IgG (Chemicon International Inc.) in PBS at a 1:500 dilution for 1 h. For detection of cells expressing Myc-tagged RasN17 or Rac1N17,
cells were incubated with an anti-Myc monoclonal antibody 9E10 (0.5 µg/ml) in PBS for 1 h followed by the incubation with a
rhodamine-conjugated donkey anti-mouse IgG in PBS for 1 h. In the
case of double stainings with Myc-tagged RasN17 and
endogenous Rac1, expressed Myc-tagged Ras mutants were visualized using
a rabbit polyclonal anti-Myc antibody (MBL) in PBS at a 1:500 dilution
for 1 h followed by a fluorescein isothiocyanate-conjugated donkey
anti-rabbit IgG (Chemicon International Inc.) in PBS at a 1:250
dilution for 1 h. F-actin was stained with Alexa 488- or
rhodamine-conjugated phalloidin (Molecular Probes) in PBS (0.5 units/ml) for 1 h. Cells were mounted in 90% glycerol containing 0.1% p-phenylenediamine dihydrochloride in PBS. Confocal
microscopy was performed using an MRC-1024 laser scanning confocal
imaging system (Bio-Rad Laboratories) equipped with a Nicon Eclipse
E800 microscope and a Nicon Plan Apo 60 × 1.4 oil immersion objective.
To detect active Rac1 in PC12 cells, we made use of the CRIB domain of
rat Activation of Rac1 by NGF and EGF in PC12 Cells--
Consistent
with previous reports (16, 17), expression of dominant negative Rac1
completely inhibited the neurite outgrowth induced by NGF in PC12 cells
(Fig. 1), indicating that the activity of
Rac1 is critical for the NGF-induced neurite outgrowth. To obtain
direct evidence of the activation of endogenous Rac1 by NGF, we
measured the amount of cellular GTP-bound Rac1 using the GST-fused CRIB
domain of rat Redistribution of Rac1 Induced by NGF--
We next followed
time-dependent changes in the subcellular distribution of
Rac1 and F-actin after the stimulation with NGF and EGF.
Immunofluorescence staining of Rac1 using
an anti-Rac1 antibody revealed that in unstimulated cells Rac1 was
present throughout the cytoplasm and at the cell surface (Fig.
3a and Table I). At 1 min
after stimulation with NGF, cells spread with ruffles around the cell
periphery, and Rac1 was co-localized with F-actin in membrane ruffles.
At 3 min after the addition of NGF, corresponding to the time of the
highest Rac1 activity, cells produced two to four cell surface
protrusions, and Rac1 and F-actin were accumulated and co-localized at
these protrusions. Within 15 min, these F-actin-rich protrusions had
begun to extend and form the short processes where Rac1 remained to be
accumulated, and the co-localization of F-actin and Rac1 at the tips of
processes remained unchanged up to 30 min after the NGF stimulation. On the other hand, stimulation with EGF induced ruffles around the cell
periphery at 1 min after the stimulation, and Rac1 and F-actin were
co-localized to the ruffled area (Fig. 3b). In sharp
contrast to the NGF stimulation, the F-actin- and Rac1-rich ruffles
declined within 3 min, and protrusions and process formation did not
occur. To confirm the NGF-induced dynamic change in the localization of
Rac1 observed above, PC12 cells were transfected with
NH2-terminal GFP-tagged Rac1, and the fluorescence of GFP
was monitored after the stimulation with NGF. GFP-Rac1 was also rapidly
accumulated at the F-actin-rich protrusions in response to NGF with
similar time course of the movement of endogenous Rac1 (Fig.
4).
To determine whether the Rac1 accumulated at protrusions was the
GTP-bound active form, we immunostained for active Rac1 using a
Myc-CRIB domain which specifically binds to GTP-bound active Rac1. As
shown in Fig. 5, NGF stimulation induced
the active Rac1 accumulation at the protrusions where the active Rac1
and F-actin were co-localized. On the other hand, EGF induced the
recruitment of active Rac1 to the cell periphery but the active Rac1
accumulation at the protrusions was not observed. The CRIB domain can
bind to GTP-bound active form of Cdc42 as well as that of Rac1, but NGF
did not increase GTP-bound active Cdc42 (Fig. 2e).
Therefore, Rac1 accumulated at the protrusions by NGF is the GTP-bound
active form of Rac1.
We next examined the requirement of Rac1 in the formation of
F-actin-rich protrusions by using a dominant negative mutant of Rac1,
Rac1N17. The transient expression of Rac1N17
completely inhibited the NGF-induced rapid redistribution of F-actin
and the formation of protrusions (Fig.
6). This result indicates that the rapid
cytoskeletal response to NGF, the formation of F-actin-rich protrusions
at the cell surface, required the activation of Rac1.
Involvement of PI3K in the NGF-induced Activation and
Redistribution of Rac1--
To determine whether the activation of
MAPK cascade or PI3K was involved in the activation of Rac1, we
examined the effect of LY294002 (30 µM) or wortmannin (1 µM), specific inhibitors of PI3K, or PD98059 (25 µM), a specific inhibitor of MAPK kinase (also known as
MEK), on the NGF- and EGF-induced Rac1 activation. Pretreatment with
LY294002 or wortmannin markedly inhibited the NGF-induced activation of
Rac1 at 1 and 3 min (Fig. 7, a
and c), whereas the pretreatment did not significantly
affect the EGF-induced Rac1 activation or the inhibition was very weak
(Fig. 7, b and d). To confirm the action of the
PI3K inhibitors, we examined the effects of PI3K inhibitors on the
NGF-induced Akt phosphorylation, a known PI3K effect, by using
anti-phospho-specific Akt antibody. LY294002 and wortmannin completely
inhibited the NGF-induced Akt phosphorylation (Fig. 7f).
These results indicate that the NGF-induced Rac1 activation is fully
dependent on PI3K, whereas the EGF-induced activation is independent of
PI3K. On the other hand, PD98059 had no effect on either the NGF- or
the EGF-induced activation of Rac1 (Fig. 7, a, b, and
c), although it could suppress the NGF- and the EGF-induced
activation of MAPK (Fig. 7g). These results indicate that
the activation of MAPK cascade is not required for the activation of
Rac1. We further examined whether NGF activated GFP-Rac1 in a
PI3K-dependent manner. NGF activated exogenously expressed
GFP-Rac1 and this activation was inhibited by LY294002 (Fig.
7e).
We next examined the involvement of PI3K and MAPK in the rapid
redistribution of F-actin and Rac1 after the stimulation with NGF. A
previous study reported that the rapid F-actin redistribution induced
by NGF was suppressed by wortmannin (9). Consistent with this result,
we found that the NGF-induced rapid F-actin redistribution was
suppressed by LY294002 (Fig.
8a) and wortmannin (data not
shown). In addition to the effect on F-actin redistribution, the
NGF-induced rapid redistribution of Rac1 was also inhibited by LY294002
(Fig. 8a) and wortmannin (data not shown). The effect of
PI3K inhibitors on the rapid redistribution of Rac1 was also examined
in GFP-Rac1-transfected cells, and the NGF-induced redistribution of
GFP-Rac1 was inhibited by pretreatment with LY294002 (Fig. 8b) and wortmannin (data not shown). These results indicate
that the NGF-induced activation and rapid redistribution of Rac1
requires the PI3K activity.
In contrast, when cells were pretreated with PD98059, NGF could
induce the accumulation of F-actin and Rac1 at the cell surface protrusions (Fig. 8a). However, in the presence of PD98059,
cells did not produce even short processes by 15 min (Fig.
8a) or by 30 min (data not shown) after NGF stimulation, and
they did not extend neurites (data not shown). The accumulation of
F-actin and Rac1 at the protrusions in the PD98059-pretreated cells had disappeared within 60 min (data not shown). These results indicate that
the activation of MAPK is not required for the NGF-induced rapid
redistribution of F-actin and Rac1 but is crucial for the subsequent
neurite outgrowth.
Redistribution of Rac1 by NGF Requires Ras--
Small GTPase Ras
is a critical component in the signaling pathway of NGF-induced PC12
cell differentiation (24-26). To examine whether Ras is involved in
the rapid redistribution of F-actin and Rac1 induced by NGF, PC12 cells
were transiently transfected with a dominant negative mutant of Ras,
RasN17. Expression of RasN17 completely
suppressed the rapid redistribution of F-actin (Fig. 9a) and Rac1 (Fig.
9b) induced by NGF. To confirm the effect of RasN17 expression on the localization of Rac1, we
co-transfected cells with RasN17 and GFP-Rac1.
RasN17 completely suppressed the NGF-induced redistribution
of GFP-Rac1 as well (Fig. 9c). We further examined the
effect of the dominant negative Ras on the NGF-induced GFP-Rac1
activation. RasN17 completely suppressed the NGF-induced
GFP-Rac1 activation (Fig. 7e). These results indicate that
the activity of Ras is required for the rapid redistribution of F-actin
and Rac1 induced by NGF in PC12 cells.
PC12 cells differentially respond to NGF and EGF in neuronal
differentiation, but the precise mechanism for this difference remains
to be defined. In this study, we focused our attention on the
regulation of Rac1 activity which is thought to be required for
neuronal morphology (16, 17), and we demonstrate here that NGF but not
EGF PI3K dependently activates Rac1 and recruits active Rac1 to the
protrusion sites, initiating the neurite formation. We suggest that a
spatial determination mechanism of the signaling pathway involved in
the activation of Rac1 plays a critical role in the initiation of the
neurite outgrowth in PC12 cells.
In PC12 cells, NGF induces neurite outgrowth and eventual cessation of
cell division, whereas EGF leads to a proliferative signal without
neurite formation, although their receptors transduce signals via
similar signal transduction pathways, including MAPK cascade. These
differential responses are thought to be determined by the duration of
MAPK activation; NGF induces sustained MAPK activation for several
hours, but EGF leads short-lived activation (2). However, the
receptor-mediated persistent activation of MAPK alone is shown to be
insufficient to induce neurite outgrowth (6). Furthermore, a recent
study showed that although the sustained activation of MAPK is mediated
by small GTPase Rap1, expression of a dominant negative mutant of Rap1
blocks the sustained activation of MAPK but fails to inhibit the
NGF-induced neurite outgrowth (27). These results indicate that the
NGF-induced neurite outgrowth is not merely determined by the sustained
MAPK activation and another mechanism must exist for the neurite
outgrowth in PC12 cells. In this study, we have shown that NGF PI3K
dependently activated and accumulated Rac1 at the F-actin-rich
protrusions on the cell surface, whereas EGF PI3K independently
activated Rac1 but failed to form Rac1- and F-actin-rich protrusions.
Furthermore, we revealed that Rac1 accumulated at the protrusions by
NGF was active Rac1. Considering the inhibition by dominant negative
Rac1 of the NGF-induced formation of F-actin-rich protrusions and
process formation, it is inferred that PI3K-dependent
activation of Rac1 and accumulation of active Rac1 at the specific
sites on the cell surface induces actin reorganization at the sites,
resulting in the formation of the cell surface protrusions and
subsequent extension of neurites. From these results taken together, we
speculate that temporally and spatially regulated Rac1 activation
initiates the neurite formation.
Previous studies have suggested the involvement of PI3K in the
NGF-induced neurite outgrowth in PC12 cells (9, 28). Here, we showed
that inhibition of PI3K activity suppressed the activation and
redistribution of Rac1 induced by NGF. Therefore, one role of PI3K in
neurite outgrowth in PC12 cells is to regulate the activation and the
localization of Rac1 in response to NGF. In this study, we found that
dominant negative Ras could inhibit the NGF-induced redistribution of
Rac1. Accumulated evidence shows that Ras induces the activation of Rac
through PI3K (29-31). Therefore, we suggest that there is a hierarchy
of activation from Ras to Rac1 through PI3K in the NGF signaling
pathway, and that this signaling pathway is important for the
initiation of the neurite outgrowth by NGF. Activation of PI3K leads to
the production of phosphatidylinositol-3,4-P2 and
phosphatidylinositol-3,4,5-P3. These products can bind to
the pleckstrin homology domain of some GEFs for Rac1, such as Sos and
Vav, and stimulate the activity of GEFs (32, 33). Sos is well known to
be located in the downstream signaling pathways of NGF (34), and Sos,
known as a GEF for Ras, activates Rac through its
NH2-terminal Rac GEF domain containing the tandem Dbl
homology and pleckstrin homology domaina, and Ras-mediated PI3K
activation and subsequent binding of PI3K products to the pleckstrin
homology domain appear to be necessary for the activation of Rac by Sos
(29, 32). The PI3K-dependent Sos activation may be involved
in the regulation of the Rac1 activity by NGF in PC12 cells. In
addition to the modulation of the GEF activity, the pleckstrin homology
domain is known to play an important role in the localization of
signaling molecules, including GEFs for Rac1 (35). We showed here that
the NGF-induced redistribution of Rac1 depends on the PI3K activity.
Therefore, it is conceivable that PI3K recruits a Rac1 GEF via
association with the pleckstrin homology domain and induces the
accumulation and activation of Rac1 at the protrusion sites on the cell
surface. In contrast to NGF, EGF activated Rac1 in a PI3K-independent
manner, indicating the existence of at least two distinct mechanisms in
the activation of Rac1, PI3K-dependent and -independent
pathways, in PC12 cells. NGF and EGF may utilize distinct GEFs for
activation of Rac1 in PC12 cells. A previous report indicated the
PI3K-independent, but protein kinase C-dependent activation
of Rac2 in neutrophils (36). Phosphorylation of a GEF for Rac1 by a
protein kinase may be involved in the activation of Rac1 by EGF.
The involvement of MAPK cascade in neuronal differentiation has been
extensively investigated, and the MAPK cascade was shown to be required
for the NGF-induced neurite outgrowth in PC12 cells (4, 5, 37). We
demonstrated here that inhibition of MAPK cascade by a specific
inhibitor of MAPK kinase had no effect on the formation of F-actin-rich
protrusions as well as the activation and the redistribution of Rac1
induced by NGF. However, this inhibitor suppressed the subsequent
induction of short processes and eventual neurite outgrowth. These
results suggest that the MAPK cascade is required for the extension of
neurites but not for the initiation of neurites induced by NGF, and
that the initiation step and the subsequent extension step of the
neurite outgrowth are regulated by different mechanisms.
In conclusion, we demonstrate here that NGF and EGF differentially
activate Rac1 in PI3K-dependent and -independent manners, respectively, and this PI3K-dependent activation of Rac1
and accumulation of active Rac1 to the protrusion sites on the cell
surface initiate the neurite formation in PC12 cells. This initial
marked activation of Rac1 and its recruitment to the protrusion sites
are the initial steps for the formation of neurites and the steps do
not require MAPK cascade. This work takes a close-up of an important
role of Rac1 in the initiation of neurites, and will help to elucidate the molecular mechanism of neurite formation and extension.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PAK
(20) was obtained by reverse transcriptase-polymerase chain reaction
from PC12 cells, using primers 5'-AAGGGATTCAAGGAGCGGCCAGAGATTTCT-3' containing a BamHI site and
5'-GAAGAATTCTAATCTTAAGCTGACTTATCT-3' containing a stop codon followed
by an EcoRI site. The polymerase chain reaction product was
subcloned into the BamHI/EcoRI sites of pGEX-4T-2
(Amersham Pharmacia Biotech) and sequenced. The CRIB domain of
PAK
was then expressed in Escherichia coli as a fusion protein
with glutathione S-transferase (GST), purified on
glutathione-Sepharose beads, and isolated from the beads with 16 mM reduced glutathione. The purified proteins were dialyzed
with 25 mM Tris-HCl, pH 7.5, 1 mM
MgCl2, 0.2 mM dithiothreitol, and 5% glycerol,
and stored at
80 °C.
PAK with Myc epitope tag at the NH2 terminus. Using
cell culture overlay technique described by Meriläinen et
al. (22) and Kulkarni et al. (23), fixed PC12 cells
were incubated with Myc-CRIB domain (100 µg/ml) for 1 h and the
active Rac1 was detected using anti-Myc polyclonal antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PAK (GST-CRIB), which specifically binds to Rac in its
active GTP-bound state (20, 21). NGF induced a rapid increase in the
amount of cellular GTP-bound Rac1, the elevation reaching the maximum
at 3 min (Fig. 2, a and
e). The level decreased gradually but remained above the
basal for over 60 min after the stimulation. In contrast, EGF induced a
more transient activation of Rac1 within 1 min, and then the level quickly returned to the basal level within 5 min after the stimulation (Fig. 2, b and e). Cdc42 has been also reported
to be implicated in neurite outgrowth similar to Rac1 (16). We then
measured the amount of cellular GTP-bound Cdc42 using GST-CRIB.
However, the level of GTP-bound Cdc42 was not significantly affected by either NGF or EGF (Fig. 2, c, d, and e).
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Fig. 1.
Expression of Rac1N17 inhibits
the NGF-induced neurite outgrowth in PC12 cells. a,
PC12 cells were transiently transfected with an expression vector
encoding Myc-tagged Rac1N17, and then treated with 50 ng/ml
NGF for 48 h. Cells were fixed and stained with an anti-Myc
polyclonal antibody (left panel) to identify transfected
cells. The morphology of the cells was visualized by F-actin staining
with Alexa 488-conjugated phalloidin (right panel). The
results shown are representative of three independent experiments. The
bar represents 25 µm. b, quantification of the
effect of Rac1N17 expression on the NGF-induced neurite
outgrowth. 48 h after transfection, cells were stained with an
anti-Myc polyclonal antibody, and positively stained cells were
assessed. Cells with neurites were defined as the cells that possessed
at least one neurite longer than the length of the cell body, and were
scored as a percentage of the total number of transfected cells. At
least 100 cells were assessed in one experiment, and data are the
mean ± S.E. of triplicate experiments.
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Fig. 2.
Activation of Rac1 by NGF and EGF.
a and b, serum-starved PC12 cells were treated
with 50 ng/ml NGF (a and c) or 200 ng/ml EGF
(b and d) for the indicated times. The cell
lysates were incubated with GST-CRIB, and the amounts of GTP-bound Rac1
and GTP-bound Cdc42 were determined by immunoblotting using monoclonal
anti Rac1 and polyclonal anti-Cdc42 antibodies, respectively
(upper panels). Total amounts of Rac1 and Cdc42 in cell
lysates (lower panels) were also shown. e,
quantification of the Rac1 and Cdc42 activities stimulated by NGF ( )
or EGF (
). The Rac1 and Cdc42 activities are indicated by the
amounts of GST-CRIB-bound Rac1 and Cdc42 normalized to the amounts of
Rac1 and Cdc42 in whole cell lysates, respectively, and values of Rac1
and Cdc42 activities are expressed as fold increase over the values of
serum-starved cells at time 0 min. Data are the mean ± S.E. of
triplicate experiments.
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Fig. 3.
Subcellular distribution of endogenous Rac1
and F-actin in NGF- or EGF-stimulated PC12 cells. After
stimulation with NGF (a) or EGF (b) for the
indicated times, cells were fixed and stained with an anti-Rac1
monoclonal antibody (red) and Alexa 488-conjugated
phalloidin (green). Note that red-green overlap leads to
yellow in Merged image. The results shown are representative of three
independent experiments. The bar represents 10 µm.
Quantification of Rac1 or F-actin distribution shown in Figs. 3-6,
8, and 9
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Fig. 4.
Distribution of GFP-Rac1 and F-actin in
GFP-Rac1-expressing cells. PC12 cells were transiently transfected
with an expression vector encoding GFP-Rac1. 36 h after
transfection, they were serum-starved for 12 h, and then
stimulated with NGF for the indicated times. The cells were fixed and
stained with rhodamine-conjugated phalloidin (red).
Localization of GFP-Rac1 was examined by the fluorescence of GFP
(green). The results shown are representative of three
independent experiments. The bar represents 10 µm.
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Fig. 5.
Immunofluorescence staining for active
Rac1. After stimulation with NGF or EGF for 3 min, cells were
fixed and stained with Myc-CRIB domain (red) and Alexa
488-conjugated phalloidin (green), as described under
"Experimental Procedures." The results shown are representative of
three independent experiments. The bar represents 10 µm.
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Fig. 6.
Effect of Rac1N17 expression on
the NGF-induced rapid F-actin redistribution. PC12 cells were
transiently transfected with an expression vector encoding Myc-tagged
Rac1N17. 36 h after transfection, they were
serum-starved for 12 h, and then stimulated with NGF for 3 min.
The cells were fixed and stained with an anti-Myc polyclonal antibody
(left panels) and Alexa 488-conjugated phalloidin
(right panels). The results shown are representative of
three independent experiments. The bar represents 10 µm.
View larger version (39K):
[in a new window]
Fig. 7.
Effects of LY294002, wortmannin, and PD98059
on the activation of Rac1 by NGF or EGF. a and
b, after serum-starved PC12 cells had been pretreated with
vehicle (None), 30 µM LY294002
(+LY), or 25 µM PD98059 (+PD) for
30 min, they were stimulated with NGF (a) or EGF
(b) for the indicated times. The cell lysates were incubated
with GST-CRIB, and the amounts of GTP-bound Rac1 were determined by
immunoblotting using a monoclonal antibody against Rac1 (upper
panels). Total amounts of Rac1 in cell lysates (lower
panels) were also shown. c and d,
quantification of the effects of LY294002, wortmannin, and PD98059 on
the Rac1 activity stimulated by NGF or EGF. After serum-starved PC12
cells had been pretreated with vehicle (None), 30 µM LY294002 (+LY), 1 µM
wortmannin (+Wort), or 25 µM PD98059
(+PD) for 30 min, they were stimulated with NGF
(c) or EGF (d) for the indicated times. The Rac1
activity are indicated by the amount of GST-CRIB-bound Rac1 normalized
to the amount of Rac1 in whole cell lysates, and values of Rac1
activity is expressed as fold increase over the value of serum-starved
cells at time 0 min. Data are the mean ± S.E. of triplicate
experiments. e, activation of GFP-Rac1 by NGF. PC12 cells
were transiently transfected with an empty vector or
Myc-RasN17 along with GFP-Rac1. 36 h after
transfection, the cells serum-starved for 12 h were pretreated
with or without LY294002, and then stimulated with NGF for 3 min. The
cell lysates were incubated with GST-CRIB, and the amounts of GTP-bound
GFP-Rac1 were determined by immunoblotting using a monoclonal anti-Rac1
antibody (upper panel). Total amounts of GFP-Rac1
(middle panel) and Myc-RasN17 (bottom
panel) were also shown. Immunoreactive bands of GFP-Rac1 and
endogenous Rac1 can be determined by different mobility on
SDS-polyacrylamide gel electrophoresis. f, effects of PI3K
inhibitors on the NGF-induced Akt activation. After serum-starved PC12
cells had been pretreated with vehicle (None), 30 µM LY294002 (+LY), or 1 µM
wortmannin (+Wort) for 30 min, they were stimulated with or
without NGF for 5 min. Phosphorylated Akt in cell lysates was
determined by immunoblotting using an anti-phospho-specific Akt
antibody (upper panel). Total amounts of Akt in cell lysates
(lower panel) are also shown. g, effect of
PD98059 on the NGF-induced ERK activation. After serum-starved PC12
cells had been pretreated with vehicle (None) or 25 µM PD98059 (+PD) for 30 min, they were
stimulated with or without NGF for 5 min. Phosphorylated ERK in cell
lysates was determined by immunoblotting using an anti-phospho-specific
ERK antibody (upper panel). Total amounts of ERK1
(middle panel) and ERK2 (bottom panel) are also
shown.
View larger version (17K):
[in a new window]
Fig. 8.
Effects of LY294002 and PD98059 on the
NGF-induced rapid redistribution of Rac1 and F-actin.
a, after serum-starved PC12 cells had been pretreated with
vehicle (None), 30 µM LY294002, or 25 µM PD98059 for 30 min, they were stimulated with NGF for
the indicated times. Cells were fixed and stained with an anti-Rac1
monoclonal antibody (red) and Alexa 488-conjugated
phalloidin (green). b, PC12 cells were
transiently transfected with an expression vector encoding GFP-Rac1.
36 h after transfection, they were serum-starved for 12 h.
Then the cells were pretreated with vehicle, LY294002, or PD98059 for
30 min, and stimulated with NGF for the indicated times. The cells were
fixed and stained with rhodamine-conjugated phalloidin
(red). Localization of GFP-Rac1 was examined by the
fluorescence of GFP (green). The results shown are
representative of three independent experiments. The bar
represents 10 µm.
View larger version (33K):
[in a new window]
Fig. 9.
Effect of RasN17 expression
on the NGF-induced rapid redistribution of Rac1 and F-actin.
a, PC12 cells were transiently transfected with an
expression vector encoding Myc-tagged RasN17. 36 h
after transfection, they were serum-starved for 12 h, and then
stimulated with NGF for 3 min. The cells were fixed and stained with an
anti-Myc polyclonal antibody (left panels) and
Alexa-488-conjugated phalloidin (right panels).
b, after the RasN17-transfected cells had been
stimulated with NGF for 3 min, they were fixed and stained with an
anti-Myc polyclonal antibody (left panels) and an anti-Rac1
monoclonal antibody (right panels). c, PC12 cells
were co-transfected with expression vectors encoding
Myc-RasN17 and GFP-Rac1. 36 h after transfection, the
cells were serum-starved for 12 h, and then stimulated with NGF
for 3 min. The cells were fixed and stained with an anti-Myc polyclonal
antibody (left panels). Localization of GFP-Rac1 was
examined by the fluorescence of GFP (right panels). The
results shown are representative of three independent experiments. The
bar represents 10 µ m.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by Grants-in-aids 10470482, 11780579, 12053244, and 12210078 for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, the Uehara Memorial Foundation, and The Naito Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Contributed equally to the results of this work.
§ To whom correspondence should be addressed: Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Tel.: 81-75-753-4547; Fax: 81-75-753-7688; E-mail: mnegishi@pharm.kyoto-u.ac.jp.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M008546200
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ABBREVIATIONS |
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The abbreviations used are: NGF, nerve growth factor; EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PI3K, phosphoinositide 3-kinase; F-actin, filamentous actin; GFP, green fluorescent protein; CRIB, Cdc42/Rac interacting binding; GST, glutathione S-transferase; PBS, phosphate-buffered saline; GEF, guanine nucleotide exchange factor.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Carter, A. N.,
and Downes, C. P.
(1992)
J. Biol. Chem.
267,
14563-14567 |
2. | Marshall, C. J. (1995) Cell 80, 179-185[Medline] [Order article via Infotrieve] |
3. | Raffioni, S., and Bradshaw, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9121-9125[Abstract] |
4. |
Nguyen, T. T.,
Scimeca, J. C.,
Filloux, C.,
Peraldi, P.,
Carpentier, J. L.,
and Van Obberghen, E.
(1993)
J. Biol. Chem.
268,
9803-9810 |
5. | Traverse, S., Gomez, N., Paterson, H., Marshall, C., and Cohen, P. (1992) Biochem. J. 288, 351-355[Medline] [Order article via Infotrieve] |
6. | Vaillancourt, R. R., Heasley, L. E., Zamarripa, J., Storey, B., Valius, M., Kazlauskas, A., and Johnson, G. L. (1995) Mol. Cell. Biol. 15, 3644-3653[Abstract] |
7. |
Connolly, J. L.,
Greene, L. A.,
Viscarello, R. R.,
and Riley, W. D.
(1979)
J. Cell Biol.
82,
820-827 |
8. | Paves, H., Neuman, T., Metsis, M., and Saarma, M. (1988) FEBS Lett. 235, 141-143[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Jackson, T. R.,
Blader, I. J.,
Hammonds-Odie, L. P.,
Burga, C. R.,
Cooke, F.,
Hawkins, P. T.,
Wolf, A. G.,
Heldman, K. A.,
and Theibert, A. B.
(1996)
J. Cell Sci.
109,
289-300 |
10. |
Kita, Y.,
Kimura, K. D.,
Kobayashi, M.,
Ihara, S.,
Kaibuchi, K.,
Kuroda, S.,
Ui, M.,
Iba, H.,
Konishi, H.,
Kikkawa, U.,
Nagata, S.,
and Fukui, Y.
(1998)
J. Cell Sci.
111,
907-915 |
11. |
Kobayashi, M.,
Nagata, S.,
Kita, Y.,
Nakatsu, N.,
Ihara, S.,
Kaibuchi, K.,
Kuroda, S.,
Ui, M.,
Iba, H.,
Konishi, H.,
Kikkawa, U.,
Saitoh, I.,
and Fukui, Y.
(1997)
J. Biol. Chem.
272,
16089-16092 |
12. |
Hall, A.
(1998)
Science
279,
509-514 |
13. | Kaibuchi, K., Kuroda, S., and Amano, M. (1999) Annu. Rev. Biochem. 68, 459-486[CrossRef][Medline] [Order article via Infotrieve] |
14. | Jalink, K., Corven, E. J., Hengeveld, T., Morii, N., Narumiya, S., and Moolenaar, W. H. (1994) J. Cell Biol. 126, 801-810[Abstract] |
15. |
Katoh, H.,
Aoki, J.,
Ichikawa, A.,
and Negishi, M.
(1998)
J. Biol. Chem.
273,
2489-2492 |
16. |
Chen, X. Q.,
Tan, I.,
Leung, T.,
and Lim, L.
(1999)
J. Biol. Chem.
274,
19901-19905 |
17. |
Lamoureux, P.,
Altun-Gultekin, Z. F.,
Lin, C.,
Wagner, J. A.,
and Heidemann, S. R.
(1997)
J. Cell Sci.
110,
635-641 |
18. |
Hasegawa, H.,
Fujita, H.,
Katoh, H.,
Aoki, J.,
Nakamura, K.,
Ichikawa, A.,
and Negishi, M.
(1999)
J. Biol. Chem.
274,
20982-20988 |
19. | Ito, W., Ishiguro, H., and Kurosawa, K. (1991) Gene 102, 67-70[CrossRef][Medline] [Order article via Infotrieve] |
20. | Manser, E., Leung, T., Salihuddin, H., Zhao, Z. S., and Lim, L. (1994) Nature 367, 40-46[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Benard, V.,
Bohl, B. P.,
and Bokoch, G. M.
(1999)
J. Biol. Chem.
274,
13198-13204 |
22. |
Meriläinen, J.,
Palovuori, R.,
Sormunen, R.,
Wasenius, V.-M.,
and Lehto, V.-P.
(1993)
J. Cell Sci.
105,
647-654 |
23. |
Kulkarni, S. V.,
Gish, G.,
Geer, P. V.-D.,
Henkemeyer, M.,
and Pawson, T.
(2000)
J. Cell Biol.
149,
457-470 |
24. | Bar-Sagi, D., and Feramisco, J. R. (1985) Cell 42, 841-848[Medline] [Order article via Infotrieve] |
25. | Noda, M., Ko, M., Ogura, A., Lim, D. G., Amano, T., Takano, T., and Ikawa, Y. (1985) Nature 318, 73-75[Medline] [Order article via Infotrieve] |
26. | Szeberenyi, J., Cai, H., and Cooper, G. M. (1990) Mol. Cell. Biol. 10, 5324-5332[Medline] [Order article via Infotrieve] |
27. | York, R. D., Yao, H., Dillon, T., Ellig, C. L., Eckert, S. P., McCleskey, E. W., and Stork, P. J. S. (1998) Nature 392, 622-626[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Kimura, K.,
Hattori, S.,
Kabuyama, Y.,
Shizawa, Y.,
Takayanagi, J.,
Nakamura, S.,
Toki, S.,
Matsuda, Y.,
Onodera, K.,
and Fukui, Y.
(1994)
J. Biol. Chem.
269,
18961-18967 |
29. |
Nimnual, A. S.,
Yatsula, B. A.,
and Bar-Sagi, D.
(1998)
Science
279,
560-563 |
30. | Rodriguez-Viciana, P., Warne, P. H., Khwaja, A., Marte, B. M., Pappin, D., Das, P., Waterfield, M. D., Ridley, A., and Downward, J. (1997) Cell 89, 457-467[Medline] [Order article via Infotrieve] |
31. |
Sarner, S.,
Kozma, R.,
Ahmed, S.,
and Lim, L.
(2000)
Mol. Cell. Biol.
20,
158-172 |
32. |
Das, B.,
Shu, X.,
Day, G.-J.,
Han, J.,
Krishna, U. M.,
Falck, J. R.,
and Broek, D.
(2000)
J. Biol. Chem.
275,
15074-15081 |
33. |
Han, J.,
Luby-Phelps, K.,
Das, B.,
Shu, X.,
Xia, Y.,
Mosteller, R. D.,
Krishna, U. R.,
Falck, J. R.,
White, M. A.,
and Broek, D.
(1998)
Science
279,
558-560 |
34. | Friedman, W. J., and Greene, L. A. (1999) Exp. Cell Res. 253, 131-142[CrossRef][Medline] [Order article via Infotrieve] |
35. | Lemmon1, M. A., Ferguson, K. M., and Schlessinger, J. (1996) Cell 85, 621-624[Medline] [Order article via Infotrieve] |
36. |
Akasaki, T.,
Koga, H.,
and Sumimoto, H.
(1999)
J. Biol. Chem.
274,
18055-18059 |
37. |
Pang, L.,
Sawada, T.,
Decker, S. J.,
and Saltiel, A. R.
(1995)
J. Biol. Chem.
270,
13585-13588 |