©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
v-Crk Modulation of Growth Factor-induced PC12 Cell Differentiation Involves the Src Homology 2 Domain of v-Crk and Sustained Activation of the Ras/Mitogen-activated Protein Kinase Pathway (*)

(Received for publication, May 17, 1995; and in revised form, June 20, 1995)

Kenneth K. Teng (1) Harry Lander (2) J. Eduardo Fajardo (3)(§) Hidesaburo Hanafusa (3) Barbara L. Hempstead (1)(¶) Raymond B. Birge (3)(**)

From the  (1)Division of Hematology-Oncology, Department of Medicine and the (2)Department of Biochemistry, The New York Hospital, Cornell University Medical College, New York, New York 10021 and the (3)Laboratory of Molecular Oncology, The Rockefeller University, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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) , beta-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.


MATERIALS AND METHODS

Reagents

Mouse 2.5 S NGF and EGF were purchased from Bioproducts for Science, (Indianapolis, IN) and were used at 50 ng/ml. [-P]ATP (6000 Ci/mmol) was obtained from DuPont NEN (Boston, MA), and [P]orthophosphate and enhanced chemiluminesence (ECL) were from Amersham Corp. Lipofectamine was from Life Technologies, Inc. Rabbit polyclonal anti-PY polyclonal antibodies were generated against bacterial lysates prepared from v-Abl-transformed bacteria(27) , and monoclonal antibodies against the viral Gag region (anti-3C2) have been described previously(28) . Commercially available antibodies include peroxidase-conjugated sheep anti-mouse antibody and peroxidase-conjugated donkey anti-rabbit antibody from Boehringer Manheim (Indianapolis, IN), antibodies specific for mouse SOS 1/2 (D-21), murine Erk1 and Erk2, and v-Ha-Ras antibodies coupled to agarose beads (Y13-259) from Santa Cruz Biotechnology, and antibodies to human EGF receptor from UBI.

Generation of Crk SH2 Mutant-expressing PC12 Cell Lines

The v-crk SH2 mutants R273N and H294R were generated by oligonucleotide-directed mutagenesis and subcloned into pBluescript (pCT10)(29) . Histidine at position 294 was changed to arginine (H294R), and arginine at position 273 was changed to asparagine (R273N). These amino acid changes were generated because the arginine at position 273 of the molecule is absolutely conserved in all known SH2 domains, except protein tyrosine phosphatase 1C, and is located within the highly conserved FLVRXS hexapeptide(29) . Likewise, the histidine at position 294 (beta-D4 position) is invariant in most SH2 domains and is located at the C-terminal boundary of the motif. A 1.8-kilobase AlwNI fragment containing the entire coding region of the v-crk SH2 mutant was subcloned from pCT10 ligated into BamHI-linearized pMEX vector, using BamHI adaptors (5`-GATCAAG-3` plus 5`-GATCCCT-3`).

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.

Transient Ras Expression Assay

For the Ras expression studies, we utilized a lipofectamine-based transient transfection with c-ras DNA (pBV1631) and v-ras DNA (pBV1423) under the control of the Moloney murine leukemia virus long terminal repeat (30) (kindly provided by Drs. Alex Papageorge and Douglas Lowy, National Cancer Institute, Bethesda, MD). 18 µg of Ras DNA, 1.8 µg of pSVbeta-Gal vector, and 10 µg of sheared salmon sperm DNA (5` 3`, Inc.) was mixed with 100 µl of lipofectamine and used for transfection per 10-cm dish. pSVbeta-Gal was used as a reporter vector (Promega) to determine in situ transfection efficiency(31, 32) . Following incubation in serum-free Opti-MEM for 12 h, 1.0 ml of Dulbecco's modified Eagle's medium containing 6% calf serum plus 4% horse serum was added for an additional 16 h. The culture was rinsed and incubated with fresh Dulbecco's modified Eagle's medium containing 10% serum for 72 h prior to fixing the cells in 100 mM sodium phosphate buffer, pH 7.0, containing 0.25% glutaraldehyde and 1 mM MgCl(2). Visualization of cells expressing beta-galactosidase was determined colorimetrically by incubating the fixed cells in 5-bromo-4-chloro-3-indoyl beta-D-galactoside as described previously(31, 32) .

Western Immunoblotting and Immunoprecipitations

Immunoblot analysis was performed as described previously(26) . Protein concentrations were determined using a Bio-Rad assay, and equivalent amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis with 10% polyacrylamide. Samples separated by SDS-polyacrylamide gel electrophoresis were electroblotted onto supported nitrocellulose (S & S, Keene, NH). Detection of peroxidase-labeled secondary antibodies was achieved using ECL according to the protocol provided by manufacturer.

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(2), 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.

MAP Kinase Assay

Soluble cellular lysates prepared in RIPA buffer (pH 7.5, containing 10 mM Tris-HCl, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS) plus phosphatase and protease inhibitors were adjusted to equal protein concentrations (approximately 1 mg/ml), precleared with Protein A-Sepharose, and incubated with combined anti-ERK1 and -ERK2 antisera (each used at 1:400 dilution) for 2 h at 4 °C. Protein A-Sepharose was added to each sample, and incubation was continued for an additional 1 h. MAP kinase reactions were performed essentially as described previously using myelin basic protein as a substrate(33) , after which the samples were subjected to SDS-polyacrylamide gel electrophoresis on a 15% polyacrylamide gel. Kinase activity was determined by autoradiography of the dried gel followed by excision of radioactive bands and Cerenkov counting. In each experiment, counts/min from assays performed without immunoprecipitate (background counts) were subtracted from the total counts of each sample to determine the net counts/min incorporation into the myelin basic protein substrate.

Quantitation of p21-associated Nucleotides

The assay used to measure the GTP/GDP ratio of immunoprecipitated p21 was essentially that of Downward et al.(34) . Native or v-CrkPC12 cells were plated on rat tail collagen-coated 60-mm dishes and metabolically labeled in serum-free media containing 0.2 mCi/ml [P]orthophosphate for 18 h prior to stimulation with 50 ng/ml NGF or EGF for up to 20 min. HNTG-extracted detergent lysates were immunoprecipitated with anti-v-Ha-Ras (Y13-259)-agarose, washed extensively, and after elution of guanine nucleotides, 5 µl of sample was spotted onto polyethyliimine thin-layer chromatography plates, run for 3 h in 0.75 M KH(2)PO(4), pH 3.4, and exposed to PhosphorImaging screens overnight. Spots migrating with the same mobility as Ras GDP or GTP standards were quantified using a PhosphorImager (Molecular Dynamics). The percent of GTP bound to p21 was calculated using the following formula, which takes into account the extra phosphate on GTP as compared with GDP: ( GTP)/(GDP + GTP).


RESULTS

Mutations within the SH2 Domain of v-crk Negate v-Crk-dependent Neurite Outgrowth

Our previous studies have demonstrated that stable expression of the v-crk oncogene product in PC12 cells accelerated NGF-promoted neuritogenesis and caused EGF, which is mitogenic for native PC12 cells, to induce a differentiated phenotype(26) . To assess the role of the v-Crk SH2 domain in modulating neurite outgrowth, we have stably expressed two v-Crk SH2 point mutants, v-Crk(R273N) and v-Crk(H294R) in PC12 cells (see ``Materials and Methods''). These residues have been shown by x-ray crystallographic studies to interact with the ring of the phosphotyrosine(35) . Both v-Crk mutants have been shown to be transformation incompetent in fibroblasts when expressed as chicken retroviruses(29) . Moreover, a mutation at the comparable beta-D4 position in the GAP SH2 domain prevents GAP binding to EGF receptor and p62(36) .

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 (10^3) 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.



R273N and H294R v-Crk SH2 Point Mutants Compromise the Interaction of v-Crk with the EGF Receptor

To investigate why v-Crk SH2 mutants do not perform the same function as wild-type v-Crk protein in inducing EGF-dependent neurite outgrowth, we examined the ability of the mutant v-Crk protein to bind cellular proteins known to interact with wild-type v-Crk (Fig. 4). Results in Fig. 4A show that v-Crk mutants fail to bind and co-precipitate tyrosine-phosphorylated p130, p90, and p70 paxillin in PC12 cells (compare lanes2, 5, and 8 in Fig. 4A). After EGF stimulation, v-Crk binds stably to the tyrosine-phosphorylated EGF receptor; however, this was abolished in the v-Crk(R273N) mutant (compare lanes3, 6, and 9 in Fig. 4A). In addition, both v-Crk(R273N) and v-Crk(H294R) failed to become tyrosine-phosphorylated following EGF or NGF stimulation (Fig. 4C), indicating that, unlike wild-type v-Crk, these mutants are not substrates for a ligand-inducible tyrosine kinase. To assess whether point mutants within the SH2 domain of v-Crk impair the binding activity of the v-Crk SH3 domain, we examined the binding of v-Crk proteins to the the Ras guanine nucleotide exchange protein, mSOS, which has been shown previously to bind to the N-terminal SH3 domain of Crk in a ligand-independent manner(37, 38) . A v-CrkbulletSOS complex could be detected by immunoblot analysis in both wild-type v-Crk and v-Crk(R273N) cells. (Fig. 4B). In the absence of EGF stimulation, SOS was detected as three distinct bands ranging from 145 to 170 kDa, but after ligand stimulation, only a single slower migrating species was detectable.

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(2) 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.



Prolonged Temporal Effects of EGF on Ras Activation in v-CrkPC12 Cells

Since PC12 cellular differentiation requires Ras activation (20, 43) , and v-Crk expression in PC12 cells accelerates growth factor-dependent neurite outgrowth, we sought to determine if this phenomenon could be correlated with a change in the kinetics of the Ras pathway. In parental PC12 cells, Ras is activated transiently by EGF, where the percentage of Ras in the GTP-bound state (approximately 18%) is maximal by 5 min and returns to base line (approximately 6%) within 20 min of stimulation (Fig. 6). By contrast, NGF results in more sustained activation of Ras to the GTP-bound form, where the increased level of this complex persists for at least 20 min (Fig. 6, rightpanel). However, treatment of the v-CrkPC12 cells with either NGF or EGF induces a sustained induction of Ras-GTP, such that after 20 min, approximately 18% of the p21 was in the GTP-bound form. These results indicate that v-Crk amplifies EGF-induced Ras activation such that it resembles kinetics observed following NGF treatment. It is important to note that the levels of Ras-GTP prior to growth factor stimulation are similar in the native and v-Crk expressing PC12 cells, with approximately 6% of the Ras in the active GTP-bound form.


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.



c-Ras^Hand v-Crk Cannot Substitute for a Receptor Tyrosine Kinase Signal to Induce Neurite Outgrowth

To address whether v-Crk can induce Ras-GTP in the absence of growth factor receptor activation, c-Ras^H was transiently expressed in native or v-Crk-expressing PC12 cells by transfection with the pBV1631 ras plasmid DNA and the reporter plasmid pSVbeta-gal (molar ratio of 10:1 for c-ras^H DNAbulletpSVbeta-gal). Parallel cultures were transfected with oncogenic v-ras^H plasmid DNA (pBW1623 is isogenic to c-ras^H except for mutations at amino acids 12 and 59(30) ) since expression of oncogenic Ras proteins can induce spontaneous neurite outgrowth in PC12 cells(20) . As shown in Fig. 7and summarized in Table 1, expression of c-Ras^H failed to induce neurite outgrowth in native PC12 cells, nor did it induce neurites in v-Crk expressing PC12 cells without growth factor addition (0% of the doubly transfected cells possessed neurites). Taken together with the findings that p21 was not constitutively activated in v-Crk PC12 cells (Fig. 6), these results indicate that although v-Crk is able to potentiate growth factor signaling, co-expression of v-Crk and c-Ras^H fails to substitute for growth factor receptor tyrosine kinase-dependent PC12 cellular differentiation. However, transient expression of v-Ras^H in native PC12, v-CrkPC12, or v-Crk(R273N)PC12 cells caused spontaneous neurite extension with comparable frequencies (approximately 44-80% of the transfected cells extend neurite processes (Table 1). This observation suggests that at least one of the actions of v-Crk on growth factor signaling lies upstream of Ras activation because expression of the constitutively active Ras protein can overcome the dominant negative effects of the v-Crk SH2 mutation on NGF-induced neurite outgrowth.


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^H or v-ras^H genes. Subconfluent cultures were co-transfected with 18 µg of the indicated ras gene plus with 1.8 µg of reporter plasmid DNA pSVbeta-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 beta-D-galactoside to monitor transfected cells.





EGF Induces Prolonged Tyrosine Phosphorylation and Activation of MAP Kinases in v-CrkPC12 and v-Crk(R273N) Cells

It has been suggested that distinct effects of EGF and NGF on PC12 cell differentiation are explained by differences in the extent and duration of MAP kinase activation(14, 15) . Hence, to relate the effects of v-Crk on growth factor-mediated differentiation, we compared the time courses of tyrosine phosphorylation of MAP kinases in growth factor-treated native PC12, v-CrkPC12 cells, and v-Crk(R273N)PC12 cells (Fig. 8). Consistent with prior studies(18, 19) , tyrosine phosphorylation of MAP kinase proteins in native PC12 cells was much more transient after EGF stimulation than that after NGF stimulation, such that after 30 min, tyrosine phosphorylation was only detectable in the NGF-treated cells (Fig. 8A, compare lanes1-5 in lefttwopanels). In the v-CrkPC12 cells, however, MAP kinase phosphorylation was sustained relative to native PC12 cells upon EGF stimulation, and mimicked the time course of tyrosine phosphorylation observed in NGF-treated native PC12 cells (Fig. 8A, compare time course in upperleftpanel and lowerrightpanel). Interestingly, EGF- or NGF-treated v-Crk(R273N)PC12 cells also displayed a delay in the dephosphorylation of MAP kinase relative to native PC12 cells (Fig. 8B).


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.




DISCUSSION

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^H DNA, but not wild-type c-ras^H DNA, did we observe neurite outgrowth in the parental or v-Crk-expressing PC12 cells, indicating that c-Ras^H 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^H 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.


FOOTNOTES

*
This work was supported in part by Public Health Serice Grants PHS-GM51446 (to R. B. B) and PHS-CA44356 (to H. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health Training Grant CA09673. Present address: Departamento de Bioquimica y Nutricion, Pontifica Universidad Javeriana, Carrera 7 43-82, Santafe de Bogota, D.C. Colombia. efajardo{at}javercol.javeriana.edu.co.

Supported by Public Health Service Grant NS30687, and grants from the American Health Assistance Foundation and Hirsch/Caullier-Weill Trust.

**
To whom correspondence should be addressed: Laboratory of Molecular Oncology, The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-7412; Fax: 212-327-7943; birger{at}rockvax.rockefeller.edu.

(^1)
The abbreviations used are: SH, Src homology; EGF, epidermal growth factor; NGF, nerve growth factor; MAP, mitogen-activated protein.


ACKNOWLEDGEMENTS

We thank Debbie Mahadeo for technical assistance, and Drs. Robert Glassman, Stephan Feller, and Alvaro Monteiro for helpful discussions and critical comments on the manuscript.


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