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INTRODUCTION |
The MAP1 kinase cascade
is one of the principal intracellular signaling pathways linking
activation of cell surface receptors to cytoplasmic and nuclear
effectors. The MAP kinases (MAPKs), ERK1 and ERK2, have been shown to
be essential for cellular proliferation as well as for acquisition and
maintenance of a differentiated phenotype (1). One of the best studied
models employed to examine how the MAPKs act to regulate cellular
phenotypes is the rat pheochromocytoma cell line, PC12 cells. These
cells respond to epidermal growth factor (EGF) treatment by an increase
in mitotic rates (2). In contrast, nerve growth factor (NGF)
stimulation of the PC12 cells results in their differentiation into a
sympathetic neuron-like phenotype (3). There is compelling evidence
that the longevity of MAPK activation governs whether these cells are
stimulated either to proliferate or to withdraw from the cell cycle and
differentiate into a neuronal phenotype. EGF and other mitogens provoke
the evanescent activation of the MAPKs, whereas NGF treatment results in the sustained activation of this signaling pathway (4-6). The
question of how different growth factors elicit distinct biological outcomes through common signal transduction elements has provoked considerable interest resulting in a complex and controversial literature.
The MAPK cascade transduces signals from receptor tyrosine kinases to
two members of the Ras family of small G-proteins, Ras and Rap1, which
then stimulates the sequential activation of Raf serine/threonine
kinases (c-Raf and B-Raf), MEK, and the MAPKs (ERK1 and ERK2) (7, 8).
Recent studies of NGF-stimulated PC12 cells suggest that Ras functions
principally to activate c-Raf, whereas Rap1 stimulates B-Raf activity
(9). It is now widely appreciated that the regulation and activation of
the small G-proteins, Ras and Rap1, is a pivotal step governing the
kinetics of MAPK activation. The activation of the Ras family of small G-proteins is mediated through a complex of adaptor proteins and guanine nucleotide exchange factors (GEF). Ras is regulated through its
association with an adaptor complex containing Grb2 and the GEF, SOS
(10). SOS is a GEF of Ras whose activity is positively regulated by its
recruitment to a membrane-associated signaling complex and negatively
regulated in part by an auto-regulatory loop through the MAPK pathway
resulting in its serine/threonine phosphorylation (11, 12). Similarly,
Rap1 activation is accomplished by assembly of a complex including the
adaptor molecule Crk and the GEF, C3G (13, 14). The mechanisms
regulating activation of C3G are presently unclear. It is thought that
C3G is activated by binding to the Crk protein complex. It also has
been suggested that the tyrosine phosphorylation of C3G is important
for its activation (15).
The EGF receptor and the NGF receptor, TrkA, both recruit a variety of
signaling molecules to their receptor complexes upon growth factor
stimulation. Some of these effectors are shared by the two receptor
tyrosine kinases including phosphatidylinositol 3-kinase, phospholipase
C
, and the adaptor proteins Shc and Grb2, whereas others are
specific to the individual receptors (8). TrkA associates with
additional signaling molecules, most prominently FRS2 (16). FRS2 is a
lipid-anchored docking protein that is highly tyrosyl-phosphorylated
upon neurotrophin or FGF stimulation (17). FRS2 has been shown to
associate directly with TrkA and FGF receptors through its
phosphotyrosine binding domain and participates in MAPK activation
(16). FRS2 has four Grb2- and two SHP-2 (a tyrosine
phosphatase)-binding epitopes and recruits both Grb2 and SHP-2 upon
stimulation, forming a protein complex in response to receptor
stimulation. The formation of these FRS2-associated protein complexes
has been postulated to play an important role in the sustained MAPK
activation elicited by NGF and FGF and thus PC12 cell differentiation
(17, 18).
The mechanisms of EGF receptor signaling have been well described. A
unique feature of EGF signaling is that upon ligand binding the EGF
receptor becomes associated with c-Cbl (19). c-Cbl acts both as an
adaptor protein whose phosphorylation leads to formation of
activation-dependent complexes with Crk and
phosphatidylinositol 3-kinase, but also possesses a ubiquitin ligase
activity (19). Following EGF binding, c-Cbl becomes associated with the
receptor and ubiquitinates it, thus triggering its proteasomal
degradation resulting in the down-regulation of EGF signaling
(20-22).
The mechanisms governing MAPK activation in this model system have been
the subject of substantial controversy. Stork and colleagues (9) have
advanced a model in which both NGF and EGF activate Ras and c-Raf
resulting in the transient activation of the MAPKs. They have argued
that NGF elicits the sustained stimulation of the MAPKs through
stimulation of Rap1 and B-Raf, whereas EGF is reported not to activate
Rap1 (9). This model has been challenged by Bos and colleagues (23, 24)
who failed to detect Rap1 activation upon NGF treatment of PC12 cells,
suggesting that persistent ERK activation is mediated principally
through Ras. Much of the controversy over the mechanisms regulating
MAPK activation has arisen over the interpretation of experiments in which elements of the MAPK cascade have been overexpressed.
The aim of the present study was to investigate the molecular
mechanisms subserving the differential regulation of the MAPKs by NGF
and EGF through examination of the interactions and activity of the
endogenous signaling molecules in a well characterized line of PC12
cells. We have arrived at different conclusions regarding the
regulatory mechanisms governing the activation of the MAPK pathway. We
report that the activation of the MAPKs in response to both NGF and EGF
is due almost exclusively to the action of B-Raf, with c-Raf
contributing less than 10% of the signal flux through this pathway. We
found that both NGF and EGF activated Rap1 through formation of a
receptor-linked signaling complex composed of Crk and C3G. The
stability of this complex was the critical factor governing the
longevity of MAPK activation. The Crk-C3G complex was formed through
direct interactions with the EGF receptor and dissociated rapidly
following ligand binding, concomitant with the ubiquitination of the
receptor and its targeting for degradation. In contrast, NGF catalyzed
the assembly of a long lived complex of Crk and C3G with the docking
protein FRS2. FRS2 becomes associated with, and is
tyrosine-phosphorylated by, the NGF receptor TrkA following NGF
treatment and acts to scaffold this complex leading to MAPK activation.
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EXPERIMENTAL PROCEDURES |
Antibodies--
Anti-Ras was from Calbiochem (La Jolla, CA).
Anti-TrkA, anti-EGF receptor, anti-SOS, anti-C3G, anti-c-Cbl,
anti-Rap1, anti-FRS2, and anti-ERK2 antibodies were from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-c-Crk, anti-p130Cas,
and anti-Sin/Efs were from Transduction Laboratories (Lexington, KY).
Anti-phospho-ERK was from New England Biolabs, Inc. (Beverly, MA).
Anti-phosphotyrosine, 4G10 was from Upstate Biotechnology Inc. (Lake
Placid, NY). Anti-B-Raf and anti-c-Raf antibodies were described
previously (25). The anti-ubiquitin antibody was from Stressgen
Biotechnologies (Collegeville, PA).
Cell Lines--
PC12 cells were cultured at 10% CO2
in Dulbecco's minimal essential medium (DMEM) supplemented with 10%
donor horse serum and 5% fetal bovine serum (FBS). PC12 cells stably
expressing wild type FRS2, FRS2-4F mutant, FRS2-1F mutant, or pLXSN
were gifts from Dr. J. Schlessinger (New York University Medical
Center) and were grown at 5% CO2 in DMEM containing 10%
donor horse serum and 10% FBS.
Growth Factor Stimulation and Cell Lysate Preparation--
PC12
cells were starved overnight in DMEM containing 0.5% FBS and then
stimulated for the indicated time at 37 °C with 100 ng/ml EGF or 100 ng/ml NGF (Austral Biological, San Ramon, CA) in DMEM supplemented with
5 mM HEPES, pH 7.4, and 0.1% bovine serum albumin. After
stimulation, cells were washed once with cold phosphate-buffered
saline, solubilized with lysis buffer as described below, and
pulse-sonicated for 5 s twice. Lysates were clarified by
centrifugation at 13,000 × g for 10 min at 4 °C.
Protein concentration was determined by the method of Bradford (26),
and equal amounts of protein were loaded in each experiment.
Generation of GST-B-Raf and GST-c-Raf Ras Binding Domain Fusion
Proteins--
Glutathione S-transferase (GST)-B-Raf RBD
(BRBD) and GST-c-Raf RBD (CRBD) fusion proteins, containing the Ras
binding domain (RBD) of the B-Raf (amino acids 1-272) and c-Raf (amino
acids 1-149), respectively, were constructed by polymerase chain
reaction and cloned into the bacterial expression vector, pGEX-KG. BRBD and CRBD fusion proteins were purified and immobilized on
glutathione-Sepharose 4B (Amersham Pharmacia Biotech). Fusion
protein-bound beads were stored at
80 °C in phosphate-buffered
saline containing 30% ethylene glycol.
Ras Binding Domain Affinity Binding Assay--
PC12 cells were
lysed in RBD assay buffer containing 50 mM Tris, pH 7.5, 10 mM MgCl2, 1% Triton, 0.25% sodium
deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
aprotinin, and leupeptin. The lysates (3 mg of protein) were incubated
with 30 µg of BRBD or CRBD at 4 °C for 45 min. The affinity
precipitates were washed 3 times with lysis buffer and eluted with
Laemmli sample buffer (10% glycerol, 150 mM
-mercaptoethanol, 3% SDS, 150 mM Tris-HCl, pH 6.8).
Proteins were resolved on 12% SDS-PAGE and transferred to
polyvinylidene difluoride membranes.
Immunoprecipitation and Immunoblotting
Analysis--
Immunoprecipitation was performed by lysing the cells in
immunoprecipitation (IP) buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM MgCl2, 1 mM EGTA, 1 mM sodium orthovanadate, 20 mM
-glycerol
phosphate, 20 mM sodium fluoride, 1 mM
phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin and leupeptin).
The lysates (4 mg of protein) were incubated with 1-1.5 µg of the
indicated antibodies for 1 h at 4 °C; protein A-agarose (40 µl, Santa Cruz Biotechnology, Santa Cruz, CA) was added, and the
tubes were rocked at 4 °C for an additional 1.5 h. The FRS2
protein complex was immunoprecipitated with anti-FRS2 (2 µg)
antibodies conjugated to the protein A-agarose (40 µl) using
dimethylpimelimidate as a covalent linker. The antibody-conjugated beads were then incubated with cell lysates for 2.5 h. The
immunoprecipitates were washed three times with lysis buffer and
resuspended in Laemmli sample buffer. Proteins were resolved using
SDS-polyacrylamide gels (SDS-PAGE) and then transferred to
polyvinylidene difluoride membranes. The membranes were blocked in TBST
(10 mM Tris, pH 7.6, 137 mM NaCl, 0.1% Tween
20) containing 3% bovine serum albumin. The blots were first probed
with indicated primary antibodies in 3% bovine serum albumin/TBST and
then with horseradish peroxidase-conjugated secondary antibodies
(Amersham Pharmacia Biotech) in 5% dried milk/TBST. The bound proteins
were visualized by enhanced chemiluminescence (Pierce).
Raf Kinase Assays--
Raf kinase activities were purified by
MonoQ and gel filtration chromatography and then measured in a coupled
kinase assay as described previous (27). Briefly, PC12 cell lysates
were resolved by applying clarified cell lysates to a Mono Q HR 5/5 column (Amersham Pharmacia Biotech), and the ability of the resulting fractions to activate MEK was measured. The MEK kinase-containing fractions were collected and applied to a Superose 12 HR 10/30 column
(Amersham Pharmacia Biotech) to separate B-Raf and c-Raf proteins, and
the resulting fractions were assayed. For immune kinase assays, cell
lysates were prepared by lysis in kinase lysis buffer containing 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 1 mM sodium orthovanadate, 50 mM
-glycerol
phosphate, 1% Triton X-100, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, and protease inhibitors, 1 µg/ml
aprotinin and leupeptin. Raf proteins were isolated by immunoprecipitation using anti-B-Raf or c-Raf antibodies.
Immunoprecipitates were washed twice with kinase lysis buffer and twice
with assay dilution buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 75 mM NaCl, and 5 mM EGTA). Kinase buffer (25 µl; 0.5 µg of MEK, 1.1 µg
of ERK2, 10 mM MgCl2, 160 µM ATP
in assay dilution buffer) was added to initiate the kinase reaction for
20 min at room temperature. A 10-µl aliquot of the reaction mixture
was diluted with 40 µl of assay dilution buffer supplemented with 1 mM sodium orthovanadate and 1 mM
dithiothreitol. An aliquot of the diluted mixture (10 µl) was then
incubated for 20 min at room temperature with 40 µl of kinase buffer
containing [
-32P]ATP, 2 µg of myelin basic
protein, 2 mM MgCl2, 62.5 µM ATP, 22 dpm/fmol [
-32P]ATP, 1 mM sodium
orthovanadate, and 1 mM dithiothreitol in assay dilution
buffer. An aliquot of the final reaction mixture (40 µl) was spotted
onto Whatman P81 filter papers. The papers were washed 3 times with 75 mM phosphoric acid and dried, and incorporated radioactivity was measured by scintillation counting.
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RESULTS |
Differences in the Activation Kinetics of B-Raf and c-Raf by NGF
and EGF Stimulation--
We have examined the activation kinetics of
B-Raf and c-Raf in response to NGF and EGF stimulation. In PC12 cells,
both EGF and NGF stimulated transient c-Raf activation with similar
kinetics. The maximum c-Raf activities were detected after 2-5 min of
stimulation and then declined to basal levels after 30 min (Fig.
1A). However, the pattern of
B-Raf activation was quite different in response to NGF or EGF
stimulation (Fig. 1B). EGF-stimulated B-Raf activity was
transient, decreasing to basal level within 1 h of stimulation. In
contrast, NGF stimulation produced the sustained activation of B-Raf,
which was maximally activated within 5 min and was maintained for
longer than 1 h. Both EGF and NGF stimulate a 3-5-fold increase in the kinase activities of B-Raf and c-Raf. These findings are consistent with the other report in the literature (28).

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Fig. 1.
Differences in the activation kinetics of
B-Raf and c-Raf between NGF and EGF signaling. Serum-starved PC12
cells were stimulated with 100 ng/ml EGF or NGF for the indicated
periods. c-Raf (A) and B-Raf (B) activation
kinetics were measured by a coupled immune kinase assay using
MEK/ERK/myelin basic protein as sequential substrates. Relative kinase
activity was calculated by normalizing kinase activity from growth
factor-stimulated cells to the untreated control cells. The results
presented are the mean of four separate experiments (±S.E.).
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Growth Factor Stimulation of the MAP Kinases Is Due Principally to
B-Raf--
The degree of activation of both B-Raf and c-Raf by NGF was
similar; however, this widely employed type of analysis does not reveal
the dramatic differences in MEK kinase activity exhibited by the two
Raf isoforms. B-Raf exhibits a much higher intrinsic kinase activity
toward MEK. We quantified the proportional contribution of B-Raf and
c-Raf to total MEK kinase activity elicited by NGF treatment of PC12
cells (Fig. 2). B-Raf and c-Raf proteins
were isolated by MonoQ and gel filtration chromatography from
NGF-stimulated PC12 cells. Quantitative analysis of NGF-stimulated Raf
activities revealed that after 5 min of NGF treatment, ~90% of MEK
kinase activity was due to the activity of B-Raf, whereas less than
10% was found to be associated with c-Raf. A similar analysis of B-Raf and c-Raf activity measured in immune kinase assays yielded essentially identical results. We conclude that greater than 90% of MAPK
activation in response to NGF is mediated by B-Raf. c-Raf plays a minor
role in MAPK induction, contributing less than 10% of total MEK kinase activity at its maximum level of activation. Identical results were
obtained from EGF-stimulated cells (data not shown). These data
demonstrate that B-Raf is the principal MEK kinase stimulated by these
growth factors.

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Fig. 2.
Nerve growth factor stimulation of the MAP
kinases is due principally to B-Raf. B-Raf and c-Raf from
NGF-stimulated PC12 cells (100 ng/ml for 5 min) were isolated by either
MonoQ/Gel filtration chromatography or immunoprecipitation. Kinase
activities were measured in a coupled immune kinase assay. The
percentage of MEK kinase activity is calculated as B-Raf or c-Raf
activity/Total (B-Raf + c-Raf) activities × 100%. The data shown
represent the average (±S.E.) of three independent experiments.
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Kinetic Analysis of Ras and Rap1 Activation--
To determine if
differential regulations of B-Raf and c-Raf were mediated by selective
interactions with their small G-protein regulators, Ras and Rap1, we
have investigated the dynamic interactions between Ras, Rap1, and Raf
isoforms. We have used the RBDs of c-Raf and B-Raf to capture the
activated, GTP-bound, forms of Ras and Rap1. Since these RBDs only bind
to active form of Ras and Rap1, the activation kinetics of the
individual small G-proteins can be monitored. Thus, GST fusion proteins
of Ras binding domains (RBDs) of the B-Raf and c-Raf were used to bind
the active forms of these Ras family members following stimulation by
NGF and EGF at different times (29, 30).
The RBDs of B-Raf and c-Raf were able to selectively capture the
activated forms of both Ras and Rap1 (Fig.
3). In EGF-stimulated cells, the
activation kinetics of both Ras and Rap1 were transient with maximum
activation detected at 2 min (Fig. 3A). The amount of
activated Ras and Rap1 declined rapidly to base-line levels after 30 min of EGF stimulation. This transient Ras and Rap1 activation correlated well with the transient MAPK activation as detected by ERK
phosphorylation (Fig. 3C). In contrast, NGF stimulated a
transient activation of Ras but sustained Rap1 activation (Fig. 3B). Substantial levels of the active forms of Rap1 could
still be detected after an hour of NGF stimulation. In addition, Ras activation was more rapid, starting at 2 min, whereas Rap1 activation was slightly delayed until 5 min. Furthermore, by comparing Ras and
Rap1 activation kinetics to MAPK activation, we found that in
EGF-stimulated cells transient MAPK activation is supported by both
transient Ras and Rap1 activities. Importantly, in NGF-treated cells,
the early phase of MAPK activity is correlated well with Ras activity,
whereas that of the later phase is correlated with Rap1 activity,
consistent with findings of York et al. (9).

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Fig. 3.
Kinetic analysis of Ras and Rap1
activation. A and B, cell lysates were
generated from EGF- or NGF-stimulated PC12 cells treated for the
indicated periods. Active forms of Ras and Rap1 were isolated by
incubating cell lysates with GST-B-Raf RBD or GST-c-Raf RBD.
GST-RBD-bound proteins were then subjected to Western blot analysis
using anti-Ras and anti-Rap1 antibodies. C, corresponding
MAPK activation kinetics were evaluated by probing Western blots of
cell lysates with anti-phospho-ERK antibodies. Protein loading was
examined using anti-ERK1/2 antibodies. Representative data from four
independent experiments are shown. AP, affinity
precipitate.
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Growth Factor Regulation of Guanine Nucleotide Exchange
Factors--
The differential activation of the GEFs by NGF and EGF
plays a pivotal role in governing the longevity of the signals driven by Ras and Rap1. The inactivation of Ras occurs concurrently with the
dissociation of the Grb2-SOS protein complex following the phosphorylation of SOS by ERKs and pp90RSK2, providing
feedback inhibition of this pathway (11, 12). The phosphorylation of
SOS after growth factor stimulation can be detected by its reduced
electrophoretic mobility on SDS-PAGE. The mobility shift pattern of SOS
revealed that the activation and inactivation of SOS were identical in
the NGF- and EGF-treated cells (Fig.
4A). SOS exhibited its maximum
mobility shift after 5 min of growth factor treatment. These data are
consistent with the transient activation of Ras in response to both
stimuli.

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Fig. 4.
Growth factor regulation of guanine
nucleotide exchange factors. Cell lysates were generated from EGF-
or NGF-stimulated PC12 cells treated for the indicated periods.
A, mobility shift of SOS was evaluated by probing a Western
blot of the cell lysates with anti-SOS antibodies. B, the
association of C3G with Crk was evaluated by probing the
Crk-immunoprecipitated (IP) protein complex with anti-C3G
antibodies. The blots were then reprobed with anti-Crk antibody to
verify protein loading. In order to show clear changes in C3G signals,
the C3G blot of EGF-stimulated samples was exposed longer than that of
NGF. Representative data from three independent experiments are
shown.
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The inactivation of Rap1 is a consequence of the
dissociation of the Crk-C3G complex (31, 32). We analyzed the
interaction of C3G and Crk by immunoprecipitation of Crk (Fig.
4B). There is constitutive association of C3G with Crk in
unstimulated cells. However, upon stimulation of the cells with EGF,
this complex dissociates after 5 min. In contrast, upon NGF treatment,
the levels of the Crk-C3G complex were enhanced and then remained stably associated for longer than 1 h, suggesting that the
stabilized Crk-C3G complex is responsible for the sustained Rap1
activation and persistent activation of B-Raf observed upon NGF
stimulation. The data shown in Fig. 4B were obtained from
the same experiment, but different exposures have been provided to show
clearly the changes in the Crk-C3G complexes.
NGF and EGF Stimulate the Formation of Molecularly Distinct
Complexes with Crk--
By having observed that EGF and NGF
differentially regulated the amount and stability of the Crk-C3G
protein complex, we proceeded to investigate the possible mechanisms
governing Crk function. PC12 cells express three isoforms of Crk
proteins, with c-CrkII and CrkL predominating (9). Crk possesses an SH2
and two SH3 domains and thus can participate in the linkage of a
complex arrays of proteins to the receptors (33). Crk undergoes
tyrosine phosphorylation after growth factor stimulation (34, 35).
Examination of Crk tyrosyl phosphorylation following EGF stimulation
revealed only a brief period of phosphorylation, whereas the time
course of NGF-stimulated tyrosyl phosphorylation was significantly more protracted (Fig. 5A).

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Fig. 5.
NGF and EGF stimulate the formation of
molecularly distinct complexes with Crk. Crk protein complexes
were immunoprecipitated (IP) from EGF- or NGF-stimulated
PC12 cells treated for the indicated periods. A and
B, Crk-associated proteins were analyzed by probing Western
blots with the anti-phosphotyrosine (4G10) antibody. A and
B were obtained from the same blot. However, A
represents a longer exposure, allowing visualization of phospho-Crk.
The two Western blots shown in A were obtained from films
with the same exposure periods. The blots were then reprobed with an
anti-Crk antibody to verify protein loading. C and
D, Crk immunoprecipitates were analyzed by Western analysis
and probed with antibodies to p130Cas, Sin/Efs, and Cbl.
The blots were then reprobed with anti-Crk antibody to verify protein
loading. Data shown in this figure are representative of three
experiments.
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Importantly, we found that different tyrosyl-phosphorylated proteins
associated with Crk in response to EGF or NGF, detected using the
anti-phosphotyrosine antibody, 4G10 (Fig. 5B). EGF
stimulation of the cells results in the transient dissociation of the
130-kDa (pp130) and 97-kDa proteins (pp97), concurrent with the
association of the complex with the 180-kDa (pp180) and 120-kDa (pp120)
proteins. The cohort of Crk-associated proteins in NGF-stimulated cells is less complex. NGF treatment resulted in an increase in the amount of
pp130 and pp97 associated with Crk. We have determined that pp130 in
the EGF- and NGF-stimulated Crk protein complexes is
p130Cas (Fig. 5C). The pp97 protein has been
identified as Sin/Efs (Fig. 5D). The pp180 and pp120 from
EGF-stimulated cells were identified as the EGF receptor and c-Cbl,
respectively (Fig. 6A and
5C). Identical immunoprecipitation results were obtained
using antibodies either to c-Crk or CrkL (data not shown). We verified
that the association of Sin and p130Cas with Crk was
differentially regulated by EGF and NGF (Fig. 5D). Examination of the Crk immunoprecipitates using an anti-Sin or anti-p130Cas antibody demonstrated that EGF treatment
resulted in displacement of Sin and p130Cas from Crk,
whereas in NGF-stimulated cells there was a dramatic increase in the
association of Sin and p130Cas with Crk. Interestingly, the
increased association of p130Cas and Sin with Crk could
only be detected in adherent cells treated with NGF. When the cells
were stimulated with NGF in suspension, a dissociation of the
Crk-p130Cas and Crk-Sin protein complexes was observed
(data not shown), suggesting that these complexes are regulated by
cytoskeletal organization.

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Fig. 6.
EGF receptor, but not TrkA, is present in a
Crk protein complex. Crk protein complexes were immunoprecipitated
from a time course of EGF- (A) or NGF
(B)-stimulated PC12 cells using anti-Crk antibody. The
immunoprecipitates (IP) were analyzed by probing Western
blots with anti-EGF receptor (A) or anti-TrkA antibodies
(B). The blots were then reprobed with anti-Crk antibody to
verify protein loading. Representative data from three independent
experiments are shown.
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The EGF Receptor but Not TrkA Is Present in the Crk Protein
Complex--
We found that Crk is stably associated with the EGF
receptor in the unstimulated cells. Upon EGF stimulation, the EGF
receptor becomes highly tyrosine-phosphorylated within 2 min (Fig.
7A). However, the level of EGF
receptor in the Crk immunoprecipitates fell rapidly (Fig.
6A). This diminution in the amount of EGF receptor associated with Crk is also readily observed in the phosphotyrosine immunoblots of Crk immunoprecipitates (Fig. 5B). In
contrast, even though TrkA is highly tyrosine-phosphorylated after NGF
stimulation, we failed to detect a tyrosyl-phosphorylated protein
corresponding to TrkA in the Crk complex (Fig. 5B and
6B), nor could we detect any TrkA protein in the Crk
immunoprecipitates (Fig. 6A). These data demonstrate that
Crk and TrkA do not form stable complexes.

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Fig. 7.
Decreased EGF receptor-Crk interaction after
EGF stimulation is due to ubiquitination of EGF receptor. EGF
receptor and TrkA were isolated from EGF- or NGF-stimulated PC12 cells
treated for the indicated periods by immunoprecipitation using anti-EGF
receptor (A) or anti-TrkA antibodies (B). The
tyrosine phosphorylation of the receptors was detected by Western
blotting analysis using anti-phosphotyrosine antibody (4G10). The
ubiquitination of the receptors was detected by probing Western blots
of immunoprecipitates with an anti-ubiquitin antibody. The blots were
then reprobed with either anti-EGF receptor or anti-TrkA antibody to
verify protein expression levels in the cells. The data shown are
representative of three experiments.
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Decreased EGF Receptor-Crk Interaction after EGF Stimulation Is Due
to Ubiquitination and Degradation of the EGF Receptor--
Upon ligand
binding, the EGF receptor undergoes tyrosyl phosphorylation and
recruits c-Cbl to the receptor complex (36, 37). c-Cbl possesses a
ubiquitin ligase activity that extensively modifies the receptor
resulting in its removal from the cell surface and targeting it to the
proteasome for degradation (20, 22). We found both the EGF receptor and
c-Cbl in Crk immune complexes after EGF stimulation, suggesting that
the complex can be down-regulated upon targeting of the EGF receptor to
the ubiquitin-proteasome degradation pathway (Fig. 5B).
Therefore, in order to determine why the levels of the EGF receptor-Crk
complex decreased after EGF treatment, we examined the ubiquitination
levels of the EGF receptor. Interestingly, we found that the EGF
receptor was rapidly and robustly ubiquitinated within 2 min of EGF
stimulation (Fig. 7A). The receptor is actively
de-ubiquitinated over the next few minutes, reflecting the acute
regulation of this modification that is linked to endocytosis and
degradation of the EGF receptor. After 1 h of EGF treatment, no
ubiquitination could be detected concomitant with decreased EGF
receptor protein levels in these cells. Therefore, the ubiquitination
of the EGF receptor provides a mechanism for down-regulating the Crk
signaling complex and thus explains the transient nature of Rap1
activation following EGF stimulation. In contrast, no ubiquitination of
TrkA was detected, and we were unable to detect any changes in the
protein levels of TrkA in response to NGF treatment (Fig.
7B).
Crk Is Recruited to a FRS2 Protein Complex after NGF
Treatment--
A number of signaling proteins have been shown to
interact with Crk in different cell types and in response to different
stimuli (33). Several of these proteins are docking or adaptor
proteins, such as p130Cas, Gab1, c-Cbl, and IRS-1 (33). Crk
is able to form a stable complex with the docking molecule IRS-1 in
response to insulin stimulation (38, 39). FRS2 is considered to be
analogous to IRS-1 in NGF signaling. FRS2 interacts with TrkA, becomes
tyrosine-phosphorylated, and then serves to scaffold a number of
signaling molecules including Grb2 and SHP-2 upon NGF stimulation (17,
18, 40). Phosphorylation of FRS2 is only stimulated by neuritogenic
growth factors, neurotrophins and FGF, but not by the mitogenic growth
factor EGF (17). We investigated the association between Crk and FRS2
following NGF treatment of PC12 cells. In the unstimulated cells, there
was a modest basal level of association of Crk with FRS2. NGF treatment resulted in an increase in FRS2 association with Crk (Fig.
8A). A reverse
immunoprecipitation using anti-FRS2 antibodies was performed, verifying
that NGF stimulated the interaction between Crk and FRS2 (Fig.
8B). In addition, the association of FRS and Crk paralleled the enhanced tyrosine phosphorylation of FRS2 induced by NGF. Therefore, FRS2 is likely to act as a scaffold, recruiting Crk protein
to a complex linked to the TrkA receptor.

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Fig. 8.
Crk is recruited to FRS2 protein complex
after NGF treatment. Serum-starved PC12 cells were stimulated with
100 ng/ml NGF for the indicated periods. A, The Crk protein
complex was immunoprecipitated (IP) using anti-Crk antibody.
The immunoprecipitates were analyzed by probing a Western blot with
anti-FRS2 antibodies. The blot was then reprobed with anti-Crk antibody
to verify protein loading. B, FRS2-associated Crk protein
was examined by immunoprecipitating FRS2 from the cell lysates using
anti-FRS2 antibodies. The immunoprecipitates were then examined by
Western blotting analysis using anti-phosphotyrosine (4G10) or anti-Crk
antibodies. The blot was reprobed with anti-FRS2 antibodies to verify
protein loading. The experiments were repeated twice with similar
results.
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Crk Interacts with the SHP-2-binding Site on FRS2,
Tyr-436--
Previous in vitro studies have shown that
interactions between Crk and FRS2 are mediated by the SH2 domain of Crk
and phosphorylated tyrosine residue(s) of FRS2 (40). FRS2 contains 15 tyrosine residues, with 4 YXN (Tyr-192, Tyr-306, Tyr-349,
and Tyr-392) motifs required for Grb2 binding and 2 YIXV
(Tyr-436 and Tyr-471) motifs essential for SHP-2 binding (41). To
determine which tyrosine residues are important for Crk association, we
analyzed the interaction of Crk with FRS2 from PC12 cells
overexpressing wild type or mutant FRS2 protein (18). Two mutant
FRS2-overexpressing cell lines were studied, one in which all of the
Grb2-binding sites have been mutated (4F mutant FRS2, Y192F, Y306F,
Y349F, and Y392F) and the other containing a tyrosine to phenylalanine mutation within the SHP-2 binding site (1F mutant FRS2, Y436F). Upon
NGF stimulation, we were able to detect considerable amounts of FRS2
proteins in Crk complexes from cells overexpressing wild type and 4F
mutant FRS2. The association of Crk and FRS2 in these cells was induced
by NGF and persisted for up to an hour (Fig. 9). In contrast, 1F FRS2 proteins were
not immunoprecipitated with the Crk complex. The protein level of 1F
FRS2 that is associated with Crk is close to that of the endogenous
FRS2 isolated from cells transfected with the control pLXSN plasmid.
These findings indicate that Tyr-436 of FRS2 is the site of Crk
binding.

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Fig. 9.
Crk interacts with the SHP-2-binding site on
FRS2, Tyr-436. Cells were starved overnight and stimulated with
100 ng/ml NGF for the indicated periods. Cell lysates generated from
PC12 cells expressing pLXSN vector alone or overexpressing wild type
FRS2 or mutant FRS2 (4F-FRS2 or 1F-FRS2) were subjected to
immunoprecipitation using anti-Crk antibodies. The immunoprecipitates
were analyzed by Western blot analysis using anti-FRS2 antibodies. The
blots were reprobed with anti-Crk antibody to confirm protein loading.
Data shown are representative of three experiments.
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FRS2 Mutated at Tyr-436 Has Impaired Rap1 Activation--
To
establish the functional importance of FRS2 in MAPK activation, we
tested whether Rap1 activity was regulated by the FRS2-Crk protein
complex, and we examined the activation of Rap1 by the RBD capture
assay from PC12 cells expressing mutant FRS2 proteins (Fig.
10). NGF stimulated the delayed and
sustained Rap1 activation from cells transfected with the pLXSN plasmid
vector or cells expressing wild type or 4F FRS2 proteins. However, Rap1
was not activated in PC12 cells expressing 1F FRS2 proteins. PC12 cells expressing wild type or 4F FRS2 proteins had enhanced and sustained ERK
activation, whereas cells expressing 1F FRS2 proteins exhibited modest
activation of ERK. We also analyzed the activation kinetics of Ras in
these cells, and we found that Ras was transiently activated by NGF in
all of the cell lines. These experiments demonstrate that the FRS2-Crk
complex functions to regulate Rap1 activation in NGF signaling.

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Fig. 10.
FRS2 mutated at Tyr-436 has impaired Rap1
activation. Cells were starved overnight and stimulated with 100 ng/ml NGF for the indicated periods. Cell lysates generated from PC12
cells expressing pLXSN vector alone or overexpressing wild type FRS2 or
mutant FRS2 (4F-FRS2 or 1F-FRS2) were subjected to RBD affinity binding
assay using GST-c-Raf RBD as the probe. GST-RBD-bound proteins were
then subjected to Western blot analysis using anti-Ras and anti-Rap1
antibodies. Corresponding MAPK activation kinetics were evaluated by
probing Western blots of cell lysates with anti-phospho-ERK antibodies.
Protein loading was examined using anti-ERK2 antibodies. The
experiments were repeated three times with similar results.
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DISCUSSION |
The recognition that the differential regulation of MAPK
activation governs cellular proliferation and differentiation has served to focus attention on the signaling pathways linking growth factor receptors to the MAPKs themselves. The present study documents the central importance of Rap1 and B-Raf in MAPK activation in PC12
cells. Significantly, a conclusion of this study is that signaling
through the c-Raf pathway is a very minor contributor to growth
factor-stimulated MAPK activation in this cell line. The involvement of
Ras in the regulation of sustained MAPK activation in PC12 cells is
controversial. Many of the previous studies were performed by
overexpressing oncogenic v-Ras (RasVal-12) or the dominant
negative RasAsn-17 mutant. Introducing v-Ras into PC12
cells led to prolonged Ras and MAPK activation, whereas
RasAsn-17 inhibited these effects (4, 5, 42, 43). It is now
evident that the interpretation of these studies has been confounded by the interference of high levels of Ras with other small G-protein signaling. Recent studies demonstrate that overexpressing one small
G-protein will interfere with the others binding to their shared
effectors. Specifically, Okada and co-workers (32) have shown that by
overexpressing Rap1 in Chinese hamster ovary cells, the direct
association of Ras and Raf-1 induced by insulin is decreased. Thus,
studies in which elements of the cascade have been expressed at
supraphysiological levels have been misleading as to the importance of
Ras in stimulating MAPK activation.
We have investigated the activation kinetics of Ras and Rap1 in
response to NGF and EGF stimulation. In PC12 cells, by using Raf RBDs
as affinity probes, we observed that NGF stimulated transient Ras, but
sustained Rap1 activation, whereas EGF induced both Ras and Rap1
activity transiently (Fig. 3). The observation that Ras is transiently
activated by both growth factors has been observed previously by other
investigators (9, 44). There is, however, considerable disagreement
over whether and how Rap1 is regulated. We and York et al.
(9) have found a robust activation of Rap1 by NGF. This finding
conflicts with data reported by Zwartkruis and colleagues (24) who
failed to detect Rap1 stimulation following NGF treatment. Conversely,
Zwartkruis et al. (24) found EGF treatment of PC12 cells
resulted in Rap1 activation. This finding is similar to our
observations (Fig. 3A). Stork and colleagues (45) have
argued that EGF does not provoke Rap1 activation. There is no clear
explanation for the different experimental outcomes.
In much of the literature investigating MAPK regulation, there has been
the implicit assumption that B-Raf and c-Raf contribute equally to the
magnitude of MAPK activation in NGF signaling in PC12 cells. Most of
the previous studies report only the relative kinase activity of the
enzymes instead of the absolute levels of kinase activity. When the
total kinase activity of B-Raf and c-Raf derived from equal amount of
cell lysates was measured by two different methods, B-Raf was found to
contribute >90% of MEK kinase activity, whereas c-Raf activity
accounted for <10% of the total activity (Fig. 2) (46). These data
demonstrate that B-Raf is the principal MEK kinase stimulated by NGF,
and c-Raf plays a very minor role (Fig. 2). Consistent with these
findings, Moodie et al. (47) reported that immunodepletion
of B-Raf from rat brain extract reduced MEK kinase activity by 96%.
Papin et al. (48) have shown that B-Raf is a much more
efficient MEK kinase than c-Raf, exhibiting ~10-fold higher specific
activities toward MEK, as a consequence of the more avid binding of
B-Raf to MEK than c-Raf. Therefore, we suggest that the involvement of
c-Raf in NGF- and EGF-dependent MAPK activation in PC12
cells is quantitatively insignificant, and we argue that it is the
magnitude and longevity of the signals derived from the B-Raf pathway
that explains the kinetics of MAPK activation in this cell line. The activation of B-Raf is likely regulated by both Ras and Rap1 (9, 49).
Both we and York et al. (9) found that B-Raf associates with
activated Ras, a finding that is consistent with previous observations
(46, 47, 50, 51). In EGF-stimulated PC12 cells, both the transient
activations of Ras and Rap1 contribute to the transient B-Raf
activities, whereas B-Raf activation by NGF is supported initially by
Ras and then by persistent Rap1 activation.
The longevity of the signals driven by Ras and Rap1 are regulated by
their GEFs, SOS and C3G, respectively. We have shown that EGF and NGF
both stimulated SOS activation with the same kinetics (Fig. 4). The
mechanisms regulating the Crk-C3G complex are less well understood.
Tyrosine phosphorylation of C3G on Tyr-504 has been suggested to
positively regulate its activity (15). We were unable to detect any
tyrosine phosphorylation or mobility changes of the C3G protein upon
EGF or NGF stimulation (data not shown). C3G appears to be regulated
through its association with the adaptor Crk, as the dissociation of
the Crk-C3G complex accompanies the inactivation of Rap1 (31, 32). We
demonstrated that EGF induced a dissociation of the Crk-C3G complex,
consistent with the transient Rap1 activation. In contrast, NGF
stimulated the formation of a stable and sustained Crk-C3G complex,
supporting the prolonged Rap1 activation (Fig. 4). These data provide
support for the view that the stability of the Crk-C3G complex
critically regulates Rap1 activation.
Previous studies have shown that Crk is tyrosine-phosphorylated upon
NGF and EGF stimulation (34, 35). Tyrosine phosphorylation of Crk on
Tyr-222 positioned between the two SH3 domains is thought to function
as a substrate switch such that when Crk is not phosphorylated its SH2
domain is able to interact with the cytoskeletal protein paxillin. When
Crk is phosphorylated on Tyr-222, paxillin is dissociated from the Crk
complex, and the SH2 domain of Crk is free to interact with other
signaling proteins, such as p130Cas (52). This quick
turnover of the Crk protein complex is thought to be important for
regulating cytoskeletal dynamics. In the present study, we found that
EGF stimulated the transient tyrosine phosphorylation of Crk
accompanied by the dissociation of p130Cas and Sin from the
Crk complex. In contrast, NGF induced the sustained tyrosine
phosphorylation of Crk, concurrent with the sustained association of
p130Cas and Sin with Crk (Fig. 5). It is of particular
interest that the NGF-stimulated formation of a Crk complex with
p130Cas and Sin was observed only in attached cells, and we
are currently investigating the biological significance of this
phenomenon. Xing et al. (53) have recently reported that Sin
and p130Cas were involved in regulating Crk/C3G/Rap1
activation by c-Src. Indeed, we have found that the NGF-stimulated
tyrosine phosphorylation of Sin could be inhibited by treating cells
with the Src kinase inhibitor, PP1 (data not shown).
The nature of the molecular linkage between Crk and growth factor
receptors remains unclear. Some studies suggest that c-Cbl and
Shc are candidate molecules linking Crk to upstream receptors, whereas
others indicate that Crk is able to bind to TrkA and the EGF receptor
directly (19, 34, 54-58). The interaction of Crk with the EGF receptor
is likely to be direct as there is a high basal level of association in
the absence of Shc or Cbl. Studies by Ribon and Saltiel (35) reported
that Tyr-992 of the EGF receptor composed a Crk-binding site, and
mutation of Tyr-992 interfered with the binding of the Crk SH2 domain
to the EGF receptor, demonstrating the direct interactions between Crk
and the EGF receptor. We were unable to detect TrkA stably associated
with Crk in growth factor-stimulated cells, likely due to the
relatively low expression levels of the endogenous proteins. The
detection of these interactions in the previous studies was limited to
studies in which TrkA was overexpressed. These data indicate that in
wild type PC12 cells, if Crk and TrkA do exist in the same protein
complex, their association must be unstable or separated by additional
adaptor proteins.
We found it interesting that EGF induced the loss of the EGF receptor
from the Crk protein complex. This is likely to be a result of
recruitment of the ubiquitin ligase c-Cbl to the EGF receptor complex
and extensive ubiquitination of the receptor (Fig. 5) (36). The
ubiquitination of the EGF receptor targets this molecule to the
proteasomes. Indeed, protein levels of the EGF receptor fell below
basal levels after an hour of EGF treatment (Fig. 7). We were surprised
by the observation that the ubiquitination of the EGF receptor occurred
rapidly and robustly within 2 min of EGF stimulation and that ~80%
of the ubiquitin was lost after 5 min of EGF stimulation. Since the
protein levels of the EGF receptor did not drop accordingly, these
findings suggest the regulated de-ubiquitination of the EGF receptor.
This fast turnover of ubiquitination and de-ubiquitination has been
suggested to be involved in the trafficking and endocytosis of the
receptor (59). This type of regulation was restricted to the EGF
receptor, as we did not find c-Cbl in the NGF-stimulated Crk complex.
We did not detect the ubiquitination or degradation of TrkA. Our data
are consistent with the previous findings that c-Cbl does not bind to
TrkA, and TrkA is not rapidly degraded after NGF stimulation (60,
61).
Many signaling proteins have been implicated in promoting neuronal
differentiation in PC12 cells. Both the docking protein FRS2 and the
tyrosine phosphatase SHP-2 have been implicated in the sustained MAPK
activation in response to NGF and appear to be required for inducing
neurite outgrowth in these cells (18). Upon NGF stimulation,
tyrosyl-phosphorylated TrkA recruits FRS2 to its receptor complex
through binding to the phosphotyrosine binding domain of FRS2. FRS2 is
then phosphorylated on multiple tyrosine residues (16, 62). These
tyrosyl-phosphorylated residues serve as docking sites for Grb2 and
SHP-2, transducing signals into the MAPK pathway. Studies by
Schlessinger and co-workers (17, 18) have demonstrated that the
incorporation of SHP-2 in the FRS2 complex is more important than that
of Grb2 in eliciting sustained MAPK activation and neurite outgrowth.
In the present study, we have provided evidence showing that Crk was
recruited into an FRS2 protein complex in response to NGF stimulation
(Fig. 8). Furthermore, the recruitment of Crk and the activation of Rap1 were impaired in PC12 cells overexpressing FRS2 mutated at the
SHP-2-binding motif (Figs. 9 and 10). Since there is no apparent Crk
consensus binding motif (YXXP) on FRS2 and SHP-2, the
interaction between FRS2 and Crk is likely to be indirect and possibly
mediated through SHP-2 and other adaptor molecule(s).
The principal goal of the present study was to provide a mechanistic
explanation for how EGF and NGF could elicit their specific effects on
cellular phenotype. We argue that the growth factor-mediated activation
of the MAPKs in PC12 cells is due principally to their activation
through Rap1 and B-Raf. The experiments in this report indicate that
the intrinsic differences in the composition and stability between the
protein complexes associated with the TrkA and the EGF
receptor are responsible for differential regulation of MAPK activation
(Fig. 11).

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Fig. 11.
Model of EGF- and NGF-stimulated MAPK
activation. MAPK activation in PC12 cells is due principally to
the activation of the Rap1/B-Raf pathway. NGF-stimulated the sustained
MAPK activation through formation of a stable protein complex
consisting of TrkA FRS2, Crk, and C3G. Transient MAPK activation upon
EGF treatment is a result of short lived EGF receptor-Crk-C3G
complex.
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