(Received for publication, May 17, 1995; and in revised form, June 20, 1995)
From the
Nerve growth factor (NGF) and epidermal growth factor (EGF) elicit contrasting actions on PC12 pheochromocytoma cells; NGF causes neuronal differentiation, and EGF induces proliferation. However, ectopic expression of the Src homology 2 (SH2) and SH3-containing oncogenic adaptor protein v-Crk in PC12 cells results in EGF-inducible neuronal differentiation (Hempstead, B. L., Birge, R. B., Fajardo, J. E., Glassman, R., Mahadeo, D., Kraemer, R., and Hanafusa, H.(1994) Mol. Cell. Biol. 14, 1964-1971). Here we show that v-Crk complexes with both the tyrosine-phosphorylated EGF receptor and the Ras guanine nucleotide exchange factor SOS in PC12 cells and is involved in an pathway analogous to that of Grb2. Expression of v-Crk results in an enhanced and sustained activation of Ras and mitogen-activated protein (MAP) kinase following EGF or NGF stimulation, implying that v-Crk can couple divergent tyrosine kinase pathways to Ras. To investigate the causal relationship between EGF receptor binding, MAP kinase activation, and neurite outgrowth, we stably expressed two v-Crk SH2 point mutants, v-Crk(R273N) and v-Crk(H294R) in PC12 cells. Mutations within the SH2 domain of v-Crk block binding of v-Crk to the tyrosine phosphorylated EGF receptor, compromise v-Crk's ability to cause EGF-dependent neurite outgrowth, and act in a dominant negative manner for NGF-induced neurite outgrowth. However, the kinetics of MAP kinase activation in EGF- or NGF-treated v-Crk(R273N)PC12 cells was comparable with that in v-CrkPC12 cells. These data are consistent with a model in which v-Crk regulates the strength of a tyrosine kinase signal leading to prolonged activation of Ras and MAP kinase. However, the experiments with the SH2 mutants suggest that sustained activation, by itself, may not be sufficient to switch the fate of v-CrkPC12 cells from proliferation toward differentiation.
Among the molecular determinants that generate and maintain
diverse cellular morphologies and functions, peptide growth factors,
via their cognate receptors, are known to elicit a wide range of
biological responses that include proliferation and survival as well as
differentiation(1) . Collective efforts to understand the
mechanisms of growth factor actions have revealed that many growth
factor receptors possess intrinsic tyrosine kinase activity (2, 3) and become phosphorylated in the intracellular
domain following ligand binding. A general paradigm for the activation
and signaling of receptor-type tyrosine kinases involves the
recruitment of cytoplasmic molecules that contain Src homology (SH) ()2 domains (for review, see (4, 5, 6, 7, 8) ). A number
of the signal transducing enzymes, such as phospholipase C-
,
phosphatidylinositol 3-kinase, and rasGAP, demonstrate ligand-dependent
interaction with receptor tyrosine kinases(1) . Paradoxically,
cellular responses to each growth factor are distinct and in some
instances, opposite. Thus, the unique signaling mechanisms that impart
growth factor specificity at a postreceptor level are still largely
unknown.
The rat pheochromocytoma PC12 cell line is a
well-characterized model system for studying the various aspects of
neuronal differentiation(9, 10) . PC12 cells respond
to the neurotrophic NGF (for review, see Refs. 11 and 12) by developing
many characteristics of sympathetic neurons, including the cessation of
cell division as well as the outgrowth of long branching neurites. When
exposed to EGF, however, these cells proliferate and resemble their
nonneoplastic adrenal chromaffin counterparts(13) . Recent
studies indicate that the duration and intensity of MAP kinase
activation may correlate with the generation of differentiative events
in PC12 cells(14, 15, 16) . This hypothesis
is supported by several studies that show that overexpression of
signaling molecules that activate MAP kinase such as the NGF receptor
Trk A(17) , insulin receptor(19) , Ras(20) ,
-platelet-derived growth factor receptor(21) ,
Shc(22) , MEK(16) , or Raf (23) result in
spontaneous or augmented neurite outgrowth in PC12 cells. Indeed, the
finding that overexpression of the EGF receptor in PC12 cells results
in EGF-induced neurite outgrowth (18) argues that tyrosine
kinase growth factor receptors are inherently similar and that the
strength of a tyrosine kinase signal may serve as a molecular basis for
the differential responses of PC12 cells toward EGF and NGF.
p47, or v-Crk, is a 47-kDa protein that
is derived from the fusion of viral Gag sequences with the SH2 and the
first SH3 domains of the c-Crk adaptor protein and displays growth
factor-independent transforming activity in chicken embryo fibroblasts (24, 25) . However, when stably expressed in PC12
cells, v-Crk confers differentiative responses, including neurite
outgrowth and expression of neuronal specific markers, following EGF
administration(26) . To examine the mechanism of EGF-induced
neurite outgrowth by v-Crk, we compared the responses of wild-type
v-Crk or SH2-inactive v-Crk mutants with respect to their ability to
bind the EGF receptor, activate MAP kinase, and induce neurite
outgrowth. Our results indicate the importance of the SH2 domain of
v-Crk in transducing EGF receptor signals from the activated EGF
receptor to cause neurite outgrowth. However, sustained activation of
the MAP kinase pathway occurs to a similar extent in cells expressing
either wild-type v-Crk or the v-Crk SH2 mutants, suggesting that
sustained MAP kinase activation, by itself, may be necessary but not
sufficient to cause EGF-induced neurite outgrowth in the v-CrkPC12 cell
model.
PC12 cells, maintained at 70% confluency on rat tail collagen-coated 10-cm tissue culture plates, were transfected using 20 µg of plasmid DNA containing the R273N v-crk or H294R v-crk mutants using lipofectamine according to the manufacturer's protocol. Colonies surviving selection in Geneticin (G418) (500 µg/ml of culture medium) were expanded, and individual clones were analyzed for protein expression by Western blotting with anti-Gag antibodies. Native PC12 cells and v-CrkPC12 lines (clones V1 and V15) were maintained as described previously(26) . To determine the rate of neurite extension, cells were plated at low density in Dulbecco's modified Eagle's medium containing 2% calf serum plus 1% horse serum in the presence or absence of growth factors. Processes greater than two cell body diameters in length (i.e. about 20 µm) were scored as neurites.
Cell lysis for experiments involving
immunoprecipitations were carried out in HNTG buffer (1% Triton X-100
buffer containing 10% glycerol, 150 mM NaCl, and 10
mM Tris-HCl, pH 7.5) containing phosphatase and protease
inhibitors and incubated with 1 µg of either anti-Gag (monoclonal
antibody 3C2), or anti-EGF receptor polyclonal antibody for 3 h prior
to incubation with rabbit anti-mouse passified Protein A or Protein
A-Sepharose beads, respectively, for an additional 1 h. For the in
vitro kinase assay, immunoprecipitates of cellular extracts were
washed and incubated in kinase buffer (HNTG buffer containing 0.1%
Triton X-100, 10 mM MnCl, and 10 µCi of
[
-
P]ATP) for 30 min at room temperature.
Immune complexes were washed again to remove excess nucleotides and
resolved electrophoretically through an 8.5% polyacrylamide gel. After
fixation (50% methanol, 10% acetic acid), the gels were rinsed and
treated with 1 N KOH at 55 °C for 2 h to enrich in
phosphotyrosine protein adducts. The gels were then dried and exposed
to x-ray film for autoradiography.
Among several independent PC12 clones stably expressing the SH2 mutant proteins, two cell lines, the N9-v-Crk (designated v-Crk(R273N) hereafter), and the R1-v-Crk (designated v-Crk(H294R) hereafter), were chosen for further analysis since they contain levels of mutant protein comparable with those in the wild-type v-Crk expressing PC12 clone V1 (designated v-CrkPC12 hereafter) that we have previously characterized ( Fig. 1and Fig. 4C)(26) . The level of expression of the mutant protein exceeds that of endogenous c-Crk by approximately 10-fold. The morphological responses (i.e. neurite outgrowth) to defined growth factors were examined in parental PC12 cells and PC12 cells expressing wild-type or mutant v-Crk ( Fig. 2and 3). In the absence of exogenous growth factors, the v-Crk-expressing cells closely resemble the parental PC12 line (Fig. 3, leftpanels). All transfectant lines and parental PC12 cells extend neurites following NGF treatment, with v-CrkPC12 cells displaying an accelerated rate of NGF-induced neurite outgrowth (Fig. 2)(26) . However, the rate of neurite outgrowth following NGF is significantly slower in cells expressing v-Crk(R273N) or v-Crk(H294R), as compared with the parental PC12 cell line, suggesting that the mutant v-Crk proteins act as dominant negative inhibitors for the NGF signaling pathway ( Fig. 2and 3). For example, after 72 h, approximately 90 and 80% of the v-CrkPC12 and native PC12 cells, respectively, extend neurite processes greater than two cell bodies, whereas the majority of the v-Crk(R273N)PC12 cells have short stubby processes that extended only a short distance from the cell body (Fig. 3d). However, prolonged NGF exposure (greater than 1 week) induces extensive neurite networks in v-Crk(R273N)PC12 and v-Crk(H294R)PC12 cells, indicating that given sufficient time, these cells do have the capacity to fully extend neurite processes. Importantly, EGF treatment of v-Crk(R273N)PC12 or v-Crk(H294R)PC12 cells (up to 2 weeks) does not induce a differentiated phenotype; only the v-CrkPC12 cells are able to extend neurites in the presence of this factor (Fig. 2B). Four additional clonal cell lines expressing similar levels of v-Crk(R273N) and v-Crk(H294R) mutants gave similar results (data not shown). Thus, the SH2 domain of v-Crk is required to mediate an EGF-induced differentiative response in PC12 cells.
Figure 1:
SDS-polyacrylamide gel electrophoresis
immunoblot analysis of v-Crk(R273N) and v-Crk(H294R) transfected cells.
PC12 cells were transfected with 20 µg of R273N v-crk or
H294R v-crk DNA in the expression vector pMEX under the control of the Moloney murine leukemia virus long
terminal repeat. After selection in G418, individual colonies were
picked and expanded (indicated as Clone#). 40 µg
of cellular lysate was probed with anti-Gag monoclonal antibody (anti-3C2). PC12 indicates no DNA was
transfected, and V1 is a wild-type v-Crk-expressing PC12 cell
line.
Figure 4:
R273N-v-Crk proteins are impaired in
tyrosine kinase signaling in response to EGF or NGF. A,
v-Crk(R273N)-expressing PC12 cells are impaired in SH2, but not
SH3-dependent binding to cellular proteins. Parental PC12 (lane1), v-Crk-expressing clones V1 (lanes2-4) or V15 (lanes5-7), or
v-Crk(R273N) cells (lanes8-10) were stimulated
in the presence of 50 ng/ml EGF or NGF for 1 min as indicated. Treated
or nontreated cells were lysed and immunoprecipitated with anti-Gag
antibodies bound to rabbit anti-mouse passified Protein A-Sepharose and
blotted with either anti-P-Tyr antibodies (panelA)
or anti-SOS antibodies (panelB). The migration of
the major tyrosine-phosphorylated proteins are indicated on the left, and the migration of co-electrophoresed molecular weight
standards (10
) are indicated on the right.
The migration of SOS is indicated by an asterisk. C,
v-Crk(R273N) and v-Crk(H294R) are not tyrosine phosphorylated following
NGF or EGF treatment. 40 µg of cellular lysate was resolved
electrophoretically by SDS-polyacrylamide gel electrophoresis and
immunoblotted with anti-3C2 antibody. The shift in migration of
tyrosine phosphorylated v-Crk is indicated on the
left.
Figure 2:
Growth
factor-induced neurite outgrowth in PC12 cells expressing v-Crk or
v-Crk(R273N) or v-Crk(H294R). PC12 cells were treated with 50 ng/ml NGF (panelA) or 50 ng/ml EGF (panelB)
for up to 72 h. Neurite-like processes greater than two cell bodies in
length were scored positive as neurite extensions. , native PC12
cells;
, V1;
, v-Crk(R273N);
, v-Crk(H294R). Errorbars in A denote the average ±
S.E. of three independent experiments.
Figure 3:
Morphologies of native PC12, v-CrkPC12,
and v-Crk(R273N) cells following treatment with NGF. Parental PC12
cells (a and b) and PC12 cells expressing either
v-Crk(R273N) mutant (c and d) or wild-type v-Crk
protein (e and f) were maintained in the presence (b, d, f) or absence (a, c, e) of 50 ng/ml NGF for 72 h. Magnification is
63.
To assess whether tyrosine kinase activity is directly associated with v-Crk or v-Crk(R273N), detergent lysates were immunoprecipitated with anti-Gag antibodies, washed, and subjected to an in vitro kinase assay (Fig. 5A). In the absence of EGF stimulation, a 130-kDa protein was the major tyrosine-phosphorylated substrate in wild-type v-Crk lysates with less prominent tyrosine phosphorylation of a 90-kDa protein. However, p130 and p90 were not detected in the control or v-Crk(R273N) kinase reactions (compare lanes2 and 3 in Fig. 5A). When lysates were prepared from EGF-stimulated cells, the pattern of tyrosine phosphorylation was similar to the unstimulated sample except for the appearance of a 185-kDa ligand-inducible band in wild-type v-Crk-expressing cells but not v-Crk(R273N)PC12 cells (Fig. 5A, compare lanes3 and 6). Conversely, the p140 tyrosine-phosphorylated TrkA receptor band was not detected in immunoprecipitates from NGF-stimulated cells (data not shown), nor was there a tyrosine-phosphorylated band corresponding to TrkA after anti-Gag immunoprecipitation (Fig. 4C). To demonstrate that p185 indeed represents the autophosphorylated EGF receptor, we immunoprecipitated the EGF receptor from lysates of EGF-stimulated cells supernatant in which v-Crk was precleared by anti-Gag antibodies (Fig. 5B). As indicated, the p185 precipitated with anti-Gag antibody in the v-CrkPC12 cells but not v-Crk(R273N)PC12 cells and co-migrated with the bona fide EGF receptor. It is important to note that an active EGF receptor was precipitated from the lysates of v-Crk(R273N)PC12 cells with anti-EGF receptor antibody (lane3). This rules out the possibility that mutation within the EGF receptor kinase was the reason why cells expressing v-Crk(R273N) mutants did not function as wild-type v-Crk with respect to EGF-induced neurite outgrowth.
Figure 5:
Tyrosine kinase activity is associated
with v-Crk in PC12 cells. A, unstimulated PC12,
v-Crk-expressing clone V15, or v-Crk(R273N) mutant were treated exactly
as described in Fig. 4, except that after immunoprecipitation,
the immune complexes were washed in kinase buffer and further incubated
with 10 µCi of [P]ATP and 10 mM
MnCl
for 30 min. Phosphorylated proteins were resolved
electrophoretically, and the gel was fixed and washed for 2 h at 55
°C in 1 N KOH prior to autoradiography with an
intensifying screen. In panelB, lysates of
unstimulated V1 or v-Crk(R273N) cells were prepared as in Fig. 4except that after anti-Gag immunoprecipitation, the
cleared supernatant in A was reprecipitated with an anti-EGF
receptor antibody and collected with Protein A-Sepharose
beads.
Figure 6:
v-Crk
induces sustained p21activation following EGF
or NGF stimulation in PC12 cells. PC12 cells were deprived of serum
overnight and then labeled with 0.2 mCi/ml
[
P]orthophosphate for 18 h in phosphate-free
media. The cells were then treated with 50 ng/ml NGF (solidline) or 50 ng/ml EGF (dashedline) for
up to 20 min. p21
was immunoprecipitated with
anti-Y13-259 antibody, and the bound nucleotides were separated
and quantified. The data are represented as the percent GTP relative to
the total GDP and GTP. Data are the average ± S.E. of at least
three independent experiments.
Figure 7:
Neurite outgrowth in parental or PC12
cells stably expressing v-Crk (clone VI) or v-Crk(R273) (clone N9) after co-transfection with c-ras or
v-ras
genes. Subconfluent cultures were
co-transfected with 18 µg of the indicated ras gene plus
with 1.8 µg of reporter plasmid DNA pSV
-gal DNA using
lipofectamine as a transfection vehicle. 72 h after transfection, the
cells were fixed in 0.25% glutaraldehyde, washed extensively with
phosphate-buffered saline, and stained with 5-bromo-4-chloro-3-indoyl
-D-galactoside to monitor transfected
cells.
Figure 8: Altered kinetics of MAP kinase tyrosine phosphorylation by v-Crk and R273N-v-Crk in response to EGF or NGF stimulation. In panelA, cultures of PC12 or v-CrkPC12 cells were treated with EGF or NGF for up to 2 h, and equivalent amounts of cellular protein were resolved electrophoretically and probed with monoclonal anti-PY20 antibodies. PanelB represents a second independent experiment and compares the tyrosine phosphorylation in PC12, v-CrkPC12, and v-Crk(R273N)PC12 cells. In each experiment, the identities of the 42- and 44-kDa proteins were confirmed by reprobing the blots with anti-Erk1- and anti-Erk2-specific antisera (not shown). The migration of tyrosine-phosphorylated Erk1 and Erk2 is indicated by arrows. Data are representative of at least four separate experiments.
To assess if prolonged tyrosine phosphorylation of p42 and p44 MAP kinases reflected a prolonged enzymatic activation, the activity of MAP kinase was analyzed by an immunoprecipitation/kinase assay (Fig. 9). In native PC12 cells, NGF treatment results in a more sustained activation of MAP kinase than does EGF stimulation (Fig. 9, compare AversusC). In PC12 cells expressing v-Crk, however, EGF causes a persistent MAP kinase activation that is reminiscent of that in NGF-treated PC12 cells (compare panelsA and C). NGF-treated v-CrkPC12 cells also displayed significantly protracted kinetics of MAP kinase deactivation relative to comparably treated native PC12 cells (Fig. 9C). Importantly, v-Crk(R273N)PC12 cells also exhibit a sustained elevation of MAP Kinase activity following EGF treatment such that this response was kinetically similar from that of the wild-type v-Crk expressing cells (compare panelsA and B). Likewise, NGF treatment of v-Crk(R273N)PC12 cells resulted in a more robust and prolonged MAP kinase activation compared with native PC12 cells (panelD). Thus, while wild-type v-Crk's ability to potentiate growth factor-induced neurite outgrowth may correlate with its capacity to modify the timing of tyrosine phosphorylation and activity of MAP kinase compared with native PC12 cells, the v-CrkSH2 mutation suggests that the duration of MAP kinase activation, and the ensuing effects on neurite outgrowth can be dissociated.
Figure 9:
Altered kinetics of MAP kinase activity by
v-Crk and v-Crk(R273N) in response to EGF or NGF stimulation of PC12
cells. Cultures of PC12 cells, v-CrkPC12 cells, or R273N-v-Crk cells
were treated for up to 2 h with EGF (A and B) or NGF (C and D). Equivalent concentrations of total
cellular lysate were immunoprecipitated with anti-MAP kinase
antibodies. Precipitates were assayed for MAP kinase activity using
[P]ATP and myelin basic protein as a substrate.
Kinase activity was determined by Cerenkov counting of myelin basic
protein excised from the dried gels. Vertical bars indicate the
averages of two independent experiments, each performed on duplicate
samples. The data were normalized so that 1.0 equals the count/min
value of MAP kinase in native PC12 cells after 5 min of growth factor
treatment.
We have shown previously that PC12 cells expressing the SH2/SH3-containing v-crk oncogene product respond to EGF by adopting a neuronal phenotype that is characteristic of NGF-treated parental PC12 cells(26) . Here we report that v-Crk can bind stably to both the activated EGF receptor and the guanine nucleotide exchange protein mSOS to result in a sustained activation of Ras and MAP kinase following EGF stimulation. Although sustained MAP kinase activity may be necessary to elicit EGF-dependent neurite outgrowth in PC12 cells expressing wild-type v-Crk, the fact that the SH2 mutants of v-Crk also sustain MAP kinase activity but do not cause neurite outgrowth argue that sustained activation, by itself, may not be sufficient to switch the fate of PC12 cells from proliferation toward differentiation.
v-Crk-mediated neuronal differentiation requires
prior growth factor activation. The notion that v-Crk couples the
signal from activated EGF receptor to Ras in order to promote neurite
outgrowth is supported by several experimental findings. First, we have
found no evidence of constituitive Ras or MAP kinase activation in
v-Crk-expressing PC12 cells in the absence of a ligand signal, although
both Ras and MAP kinase activities were prolonged following EGF
stimulation relative to parental PC12 cells. Second, mutations in the
SH2 domain of Crk abolished binding of v-Crk to the EGF receptor and
failed to induce neurite outgrowth. Finally, and most importantly, only
after transfection with active v-ras DNA, but not
wild-type c-ras
DNA, did we observe neurite
outgrowth in the parental or v-Crk-expressing PC12 cells, indicating
that c-Ras
cannot substitute for a tyrosine kinase signal
in the v-CrkPC12 cells. These data together point to the combined
action of a ligand-stimulated signal and overexpression of an adaptor
protein, v-Crk, in converting the cellular response to a mitogenic
signal into differentiative events, such as neurite outgrowth.
Our findings argue that v-Crk can act in a compensatory or qualitatively analogous manner to Grb2 in transducing an EGF-induced signal to Ras in PC12 cells. Indeed, the SH2 domain of both Crk and Grb2 can bind tyrosine-phosphorylated EGF receptor, and the SH3 domains of both Crk and Grb2 can bind SOS(37, 38, 39, 40, 41) . In addition, the CrkSH3 also stably interacts with an additional Crk-specific guanine nucleotide exchange protein called C3G, whose function, as revealed in yeast complementation studies, might also involve Ras activation(42) .
The Ras signaling pathway is critically important for both NGF-induced differentiation and EGF-induced mitogenesis in native PC12 cells(20, 43) . Expression of a dominant negative Asn-17 ras allele in PC12 cells inhibits EGF-induced mitogenesis(44) . Similarly, microinjection of anti-Ras neutralizing antibodies inhibits NGF-induced neurite outgrowth in nondividing PC12 cells(43) . However, as shown here, and consistent with results of others(14, 45) , the activation of Ras to the GTP bound state following EGF and NGF stimulation of PC12 cells are kinetically distinct. In EGF-stimulated cells, Ras-GTP reached a peak activation by 5 min and returned to base line by 20 min, whereas Ras-GTP was sustained for at least 20 min following NGF stimulation. Therefore, in this model, Ras activity is required to reach a critical threshold in order to be recognized as a differentiative signal. In v-Crk-expressing PC12 cells, the duration of Ras and MAP kinase were sustained such that the signals following EGF or NGF treatment were indistinguishable and thus, both growth factors induced neurite outgrowth.
The findings that v-Crk SH2 point mutants lose their ability to interact with the tyrosine phosphorylated EGF receptor but retain their ability to sustain the kinetics of growth factor-inducible MAP kinase activation suggests that the v-CrkSH3 domain may be solely responsible for activation of the Ras/MAP kinase pathway in this system. This is not only supported by the finding that v-Crk(R273N) retains it capacity to bind SOS, but also by the fact that there occurs a significant band shift on the v-CrkSH3-associated SOS upon EGF stimulation (Fig. 5C). Such a band shift on SOS or CDC25 has been attributed to serine/threonine phosphorylation following translocation to the plasma membrane in response to receptor stimulation(46) . Recent studies by Aronheim et al.(47) demonstrate that constituitive targeting of hSOS to membranes by N-terminal farnesylation is sufficient to activate Ras and MAP kinase. Interestingly, the virally-derived sequences of Gag in v-Crk also appear to localize v-Crk to the plasma membrane(29) . Hence, by analogy, v-Crk(R273N) may partition SOS or other SH3-complexed proteins to the cell surface, which can sustain the activation of Ras and MAP kinase after ligand stimulation. One important distinction in this system is that MAP kinase activation requires prior growth factor stimulation, and hence may require a coordinated recruitment of other signaling proteins into a functional complex prior to the involvement of the v-CrkSH3 domain. However, while these results do suggest that the v-CrkSH2 and SH3 domains need not be functionally coupled to permit EGF-induced sustained MAP kinase activation, SH2-dependent binding of v-Crk to the EGF receptor is absolutely required for EGF-induced neurite outgrowth.
The results
of the v-Crk SH2 mutants also raise the question of whether the timing
of MAP kinase is indeed critical for controlling differentiative versus proliferative signals in PC12 cells. In addition to
these studies involving v-Crk, several other signaling molecules linked
to MAP kinase activation can convert the otherwise mitogenic signals
for PC12 cells into differentiative signals, including overexpression
of the insulin receptor, Src, Shc, and activated forms of Raf and
Ras(19, 20, 22, 48, 49, 50) .
Indeed, the findings that expression of inactive forms of MAPKK1
prevent MAP kinase activation and neurite outgrowth suggest that an
activation threshold for MAP kinase may be a focal point for PC12
differentiation(16) . Traverse et al.(18) have also reported, by overexpressing the EGF
receptor in PC12 cells, that a differential activation of MAP kinase
may form the basis for the differential responses to EGF and NGF. Our
present results are entirely consistent with the idea that sustained
MAP kinase activity can be responsible for v-Crk's ability to
cause EGF to be neuritogenic if one considers that additional critical
target(s) of the SH2 mutants are downstream of MAP kinase. Indeed, the
fact that cells expressing v-Crk(R273N) or v-Crk(H294) mutants also
impose a dominant negative effect on NGF-induced neurite outgrowth
support the idea that such mutants may impair a signaling network
involving neurite elongation or growth cone assembly. However, the
findings that transfection of v-Ras into cells expressing
v-CrkSH2 mutants cause spontaneous neurite outgrowth argue that at
least the sequela of events necessary for v-Ras-induced neurite
outgrowth are not compromised in these cells.
Stable expression of v-Crk in PC12 cells also accelerates the kinetics of NGF-induced neurite outgrowth(26) , and our present results indicate that this may also occur via the ability of v-Crk to further sustain the NGF-induced activation of the Ras/MAP kinase cascade. While we have previously reported that the glutathione S-transferase-CrkSH2 domain alone can bind tyrosine-phosphorylated TrkA in lysates of Trk-overexpressing cells (26) , we have been unable to demonstrate stable association of full-length v-Crk with endogenous TrkA or TrkA kinase activity from cellular lysates. The reason for this apparent discrepancy is unclear, but it may be due to differences in binding affinities of a glutathione S-transferase fusion protein versus its full-length counterpart. However, v-Crk is an excellent substrate for TrkA in vitro, and it also becomes tyrosine phosphorylated within 30 s following NGF stimulation in vivo(26) . It is noteworthy that Grb2 has been shown to bind directly to the activated EGF receptor, but not the TrkA receptor(45, 51, 52) , although recent studies suggest that coupling to Shc provides the the major pathway linking activated EGF receptor to Grb2-SOS(53) . Interestingly, Matsuda and co-workers (54) have shown that the SH2 domain of the proto-oncogene c-Crk also binds tyrosine phosphorylated Shc and may suggest that v-Crk interacts only indirectly with TrkA receptor via its interaction with Shc. However, our results suggest that the differential interactions of v-Crk with the EGF and TrkA receptors, per se, are not the major criteria in determining PC12 cell fate decisions since both growth factors induce differentiation when v-crk is transfected into PC12 cells, but only the EGF receptor interacts directly with v-Crk.
The fact that pools of v-Crk bind simultaneously to the EGF receptor, p130, and paxillin suggest that v-Crk can act coordinately to influence several kinase-initiated pathways. Likewise, the fact that SH2 mutants abolish v-Crk's ability to bind all of these proteins concomitantly also suggests that several pathways may be compromised simultaneously. Although we have made a strong correlation between Ras/MAP kinase activation and PC12 differentiation by wild-type v-Crk, more specific mutations within v-Crk and its targets will be required to distinguish the relative contributions of several tyrosine kinase signaling pathways to Ras/MAP kinase activation and in turn how v-Crk ultimately affects growth factor receptor-mediated neurite outgrowth.