Brain-derived Neurotrophic Factor Induces Phosphorylation of Fibroblast Growth Factor Receptor Substrate 2*

John B. Easton, Norma M. Moody, Xiaoyan Zhu, and David S. MiddlemasDagger

From the Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-2794

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival. Gaining an understanding of how BDNF, via the tropomyosin-related kinase B (TRKB) receptor, elicits specific cellular responses is of contemporary interest. Expression of mutant TrkB in fibroblasts, where tyrosine 484 was changed to phenylalanine, abrogated Shc association with TrkB, but only attenuated and did not block BDNF-induced phosphorylation of mitogen-activated protein kinase (MAPK). This suggests there is another BDNF-induced signaling mechanism for activating MAPK, which compelled a search for other TrkB substrates. BDNF induces phosphorylation of fibroblast growth factor receptor substrate 2 (FRS2) in both fibroblasts engineered to express TrkB and human neuroblastoma (NB) cells that naturally express TrkB. Additionally, BDNF induces phosphorylation of FRS2 in primary cultures of cortical neurons, thus showing that FRS2 is a physiologically relevant substrate of TrkB. Data are presented demonstrating that BDNF induces association of FRS2 with growth factor receptor-binding protein 2 (GRB2) in cortical neurons, fibroblasts, and NB cells, which in turn could activate the RAS/MAPK pathway. This is not dependent on Shc, since BDNF does not induce association of Shc and FRS2. Finally, the experiments suggest that FRS2 and suc-associated neurotrophic factor-induced tyrosine-phosphorylated target are the same protein.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Brain-derived neurotrophic factor (BDNF)1 promotes neuronal survival (1-3), axonal growth cone guidance (4), and axonal growth or regeneration (5, 6). In addition, BDNF may have a role in synaptic function underlying long term potentiation in the hippocampus (7, 8). How BDNF achieves specific cellular responses, even though it activates signaling pathways shared by other growth factors, remains a contemporary problem. BDNF is a ligand for TRKB (9-12), which is the second member of the TRK family of protein-tyrosine kinases to be identified. NGF, the archetypal member of the neurotrophin family, associates with and activates TRK (13-15).

One well known example involving PC12 cells as an in vitro model for neuronal differentiation and survival defines a problem. NGF treatment of PC12 cells induces differentiation (16), whereas EGF treatment induces mitogenesis (17). Paradoxically, both NGF and EGF activate shared signaling pathways requiring RAS and PLC-gamma 1. Activation of a growth factor-specific pathway by NGF is a possible explanation for this quandary. Indeed, there is evidence that NGF may activate a neurotrophin-specific pathway requiring SNT (18, 19). SNT is a protein that is phosphorylated on tyrosine in response to NGF treatment of PC12 cells. Another explanation for this dilemma could be that NGF induces a more prolonged activation of the MAPK pathway than EGF in PC12 cells (20). Sustained activation of MAPKs by NGF, compared with EGF, in PC12 cells results in increased MAPK nuclear localization (20). Prolonged activation and consequential nuclear localization of MAPK could result in the sustained phosphorylation of transcription factors including cyclic AMP response element-binding protein (21, 22). Thus, NGF could activate both immediate early genes and delayed response genes, whereas EGF may activate only a subset of these genes, in PC12 cells. NGF induces phosphorylation of tyrosine 490 of Trk, which forms a binding site for the PTB domain (23) of Shc (24, 25). Trk association with and phosphorylation of Shc leads to RAS activation by NGF. Trk could promote prolonged RAS/MAPK pathway activation by prolonged association with Shc. There is some persuasive evidence that the NGF-induced phosphorylated state of the phosphotyrosine motif that Shc binds to in human TRK, Y490, is long-lived (26). This might explain sustained MAPK activation by NGF. However, this explanation still needs to be reconciled with other data indicating that TRK is internalized rapidly following ligand binding (27). An alternative possibility is that neurotrophins activate other pathways leading to MAPK activation. Combined stimulation by several pathways might result in an increased temporal duration of MAPK activation.

In this report, we describe our finding that mutation of Tyr-484 of TrkB abrogated BDNF-dependent Shc association with TrkB, but only attenuated and did not block MAPK phosphorylation. These data show that there must be another pathway activated by BDNF leading to MAPK phosphorylation. These findings compelled a search for other BDNF signaling pathways. FRS2, which is a new substrate for the FGF receptor, was recently cloned (28). FGF induces phosphorylation of FRS2 and the formation of a complex containing FRS2, Grb2, and Sos. This leads to activation of the RAS/MAPK pathway. In this research, we have addressed whether FRS2 is a substrate for TrkB and might be required for a second BDNF signaling pathway leading to MAPK activation. We demonstrate for the first time that, in response to BDNF, FRS2 is phosphorylated on tyrosine. This leads to FRS2 association with Grb2 in both cell lines and, more important, cortical neurons in primary culture. This suggests that there is a physiologically relevant signaling pathway leading to MAPK activation by BDNF that requires FRS2 and is independent of Shc.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Cell Culture-- Rat 2 and NB1643 cells were grown in 10% FBS/RPMI (RPMI 1640 media containing 2 mM glutamine (BioWhittaker) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and 50 units/ml penicillin plus 50 µg/ml streptomycin) (Life Technologies, Inc.) at 37 °C with 5% CO2 in tissue culture plates from Costar or Corning. Cells were trypsinized in trypsin versene mixture (Biowhittaker) for routine counting and splitting. Nuclei were prepared using the method of Butler (29) and counted using a Z2 counter equipped with a 256 channelyzer (Coulter) for cell plating. Human BDNF and NGF were generously supplied by Amgen. 5 mM ATRA (Sigma) stocks were prepared in ethanol and stored for less than 3 months at -80 °C. Cortical neuron cultures were established using methods described by Furshpan and Potter and Xia et al. (30, 31) with modifications. Brains were removed from P0 rats and washed in sterile Gey's balanced salt buffer at 4 °C. Cortex was isolated and cut into small pieces. Tissue was dissociated using the papain dissociation system (Worthington) according to the method provided by the manufacturer. Cultures were grown on polylysine-coated plates in Dulbecco's modified Eagle's medium containing 2 mM glutamine (BioWhittaker) supplemented with 5% rat serum (Harlan) and 50 units/ml penicillin plus 50 µg/ml streptomycin) (Life Technologies, Inc.) at 37 °C. Cytosine-B-D-arabinofuranoside was added to the cortical cultures on the day after plating at 2.5 µM final concentration. Medium was changed every 3 days.

Site-directed Mutagenesis, Vector Construction, and Cell Transfection-- A polymerase chain reaction method (12) was used to construct pTRE.TrkB by amplifying rat TrkB cDNA (32) and introducing EcoRI and BamHI restriction sites for ligation into pTRE (Stratagene). Primers were GCGCGAATTCACCATGGCGCGGCTCTGGG and CCGGGGATCCTAGCCTAGGATGTCCAGG. Sequence analysis, using synthetic primers and automated sequencing (Applied Biosystems), confirmed that there were no mutations introduced into the entire TrkB coding region. pTRE.TrkBY484F and pTrkBY785F were constructed directly in pTRE.TrkB using the Chameleon site-directed mutagenesis kit (Stratagene) according to the manufacturer's suggested protocol using the following primers: ScaI to MluI (Stratagene) and AAAACCCCCAGTTCTTCGGTATCAC for Y484F and a selection primer for XhoI, GCCCTTTCGTCGTCGAGTTTACCACTCC, and GGCGTCGCCCGTCTTCCTGGACATCCTAG for Y785F. Sequence analysis of the kinase domain, using synthetic primers and automated sequencing (Applied Biosystems), confirmed that the mutations were introduce into the kinase domain. Rat 2 Tet-Off cells were constructed by electroporation of the Tet-Off vector into Rat 2 cells, followed by selective growth in media containing G418. Stable Rat 2 Tet-Off cell lines were screened by transient transfection using electroporation of pTRE-Luc. Cells were grown cells in media with or without 5 ng/ml doxycycline, followed by luciferase assays according to the manufacturer's suggested protocol (Stratagene). PTRE, pTRE.TrkB, pTRE.TrkBY484F, and pTRE.TrkBY785F were co-transfected with pTK-Hyg into Rat 2 Tet-Off cells using LipofectAMINE (Life Technologies, Inc.) followed by selection with hygromycin. Stable clonal cell lines were assessed for TrkB expression by immunoblotting with Trk C-14 antibodies (Santa Cruz).

FRS2 Antibodies-- Polyclonal antibodies were generated against an 11-amino acid peptide containing the carboxyl-terminal sequence of FRS2 (KTRHNSTDLPM) (28) that was synthesized using an ABI model 433A synthesizer (Applied Biosystems, Inc.) and coupled to keyhole limpet hemocyanin using glutaraldehyde. Polyclonal antiserum was raised in rabbits immunized with the COOH-terminal peptide-keyhole limpet hemocyanin conjugate (Rockland).

Immunoprecipitation and Immunoblotting Methods-- Rat 2 cells were plated equally at either 0.5 or 1 × 106/10-cm dish, and NB1643 cells were plated equally at 2, 4, or 8 × 106 NB1643 cells/10-cm dish (Corning), treated with 5 µM ATRA (which induces TrkB expression2), and grown for 2-4 days. Cortical neurons were plated at 16 × 106 cells/10-cm dish and grown for 4-7 days. Cells were treated as described in figure legends for each experiment, then lysed at 4 °C in RIPA (33), Nonidet P-40 (34), or Triton X-100 lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta -glycerophosphate, 1 mM Na3VO4, and 1 µg/ml leupeptin) lysis buffers with fresh 1 mM phenylmethylsulfonyl fluoride (diluted from 100-fold stock in methanol) added immediately before lysis. RIPA lysates only were repipetted four times through a 25-gauge needle. Samples were either stored at -80 °C or used directly. Sample lysates, fresh or thawed on ice, were cleared by centrifugation at 10,000 × g for 30 min. For immunoprecipitations, antibodies (see below) were added to the supernatants and incubated on ice for 1 h, then incubated with rotation with 20 µl Protein A-Sepharose (Repligen) or Protein G-agarose (Santa Cruz) at 4 °C. The immune complexes were pelleted in a microcentrifuge and then resuspended in 1 ml of RIPA, Nonidet P-40, or Triton X-100 buffer, vortexed, and pelleted in a microcentrifuge. Washes were repeated three more times, after which remaining buffer was removed. The samples were then taken up in 30 µl of 2× SDS-PAGE sample buffer. After SDS-PAGE, the gels were transferred to Immobilon-P (Millipore) using the Trans-Blot electrophoretic transfer cell (Bio-Rad) containing tris/glycine/methanol transfer buffer (25 mM Tris, 192 mM glycine, and 20% v/v methanol) under a constant voltage of 100 V for 1 h. SDS boiling lysis was accomplished by addition of 400 µl of SDS-PAGE sample buffer previously heated to 100 °C. Cells were scraped off the plate, sheared four times through a 25-gauge needle, transferred to a microcentrifuge tube, and boiled for 5 min, after which 40 µl was used for immunoblot analysis.

Immunoblot analysis with all antibodies, except with anti-phosphotyrosine antibodies, was performed as follows at 22 °C. Membranes were blocked for 1 h in 5% milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20). Blots were washed once for 15 min and twice for 5 min with TBST, then incubated with antibodies in 1% milk/1% BSA in TBST for 1 h. The membranes were then washed as above and incubated with anti-rabbit or anti-mouse horseradish peroxidase in 1% milk/1% BSA in TBST for 1 h. The membranes were then washed for 15 min and then four times more for 5 min in TBST. Protein bands were then visualized using chemiluminescent detection with the ECL or ECL Plus kits (Amersham Pharmacia Biotech) and XAR 5 film (Kodak). Immunoblot analysis to detect phosphotyrosine was performed as follows at 22 °C. The membranes were blocked for 2 h in 5% BSA (Sigma Catalog) in TBST, then fresh 4G10 anti-phosphotyrosine antibodies (1:5000) (Upstate Biotechnology) were added for 1 h with rotation. The blots were washed once for 15 min and twice for 5 min with TBST, then incubated with anti-mouse horseradish peroxidase in 5% BSA (Sigma Catalog) in TBST. The membranes were then washed for 15 min and then four times for 5 min in TBST. Anti-phosphotyrosine containing protein bands were then visualized using chemiluminescent detection with the ECL kit (Amersham Pharmacia Biotech) and XAR 5 film (Kodak).

Specific Conditions for Immunoprecipitation and Immunoblotting Experiments-- For immunoprecipitation experiments, 1 µg of TRK antibodies (Santa Cruz), 5 µg of anti-PLC-gamma antibodies (Upstate Biotechnology), 5 µl of FRS2 antiserum (preimmune and immune), 20 µl of p13suc1-agarose linked (CalBiochem), and 1 µg of anti-Shc antibodies (Transduction Laboratories). For immunoblotting experiments, the following antibody dilutions were used; anti-Shc antibodies at 1:500 (Transduction Laboratories), anti-p42/p44 MAPK antibodies at 1:1000 (New England Biolabs), anti-phospho-specific p42/p44 MAPK antibodies at 1:1000 (New England Biolabs), anti-TRK antibodies at 1:200 (Santa Cruz), 4G10 anti-phosphotyrosine antibodies at 1:5000 (Upstate Biotechnology), and anti-Grb2 antibodies at 1:5000 (Transduction Laboratories).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutation of Tyrosine 484 of TrkB to Phenylalanine Abrogates BDNF-induced Association of Shc with TrkB, but Only Attenuates MAPK Phosphorylation-- Tyrosine 484 of TrkB is required for BDNF-induced association of Shc with TrkB. In Rat 2 fibroblasts expressing TrkBwt, BDNF induces association of Shc with TrkB, whereas in fibroblasts expressing TrkBY484F, BDNF does not induce association of Shc with TrkB (Fig. 1A, top panel). TrkBY484F is autophosphorylated on tyrosines following BDNF treatment, which shows that the kinase activity of the mutant is competent (Fig. 1A, bottom panel). Tyrosine 484 of TrkB is homologous to tyrosine 490 of Trk, which also forms a binding site for Shc (24, 25). The 66- and 52-kDa Shc isoforms bind TrkBwt and not TrkBY484F. The band for the 46-kDa isoform of Shc is obscured by a broad band corresponding to antibodies used in the immunoprecipitation. In order to confirm that clonal selection did not affect the outcome of these experiments, the result that tyrosine 484 is required for Shc association with TrkB was confirmed in a second independently picked clone of Rat 2 fibroblasts expressing TrkBY484F.


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Fig. 1.   Mutation of TrkB tyrosine 484 to phenylalanine blocks BDNF-induced Shc association with TrkB. BDNF-induced phosphorylation of MAPK is attenuated, but not blocked, in fibroblasts expressing TrkBY484F. A, Rat 2 TrkBwt or TrkBY484F fibroblasts were plated and grown for 1 day, then serum-starved for 1 day, treated with BDNF (50 ng/ml) for 5 min, and lysed in Nonidet P-40 buffer. Immunoprecipitation with anti-TRK antibodies was followed by immunoblot analysis with anti-Shc antibodies (top panel) or anti-phosphotyrosine antibodies (bottom panel). The positive control for Shc was from a whole cell lysate from HepG2 cells. B, Rat 2 TrkBwt or TrkBY484F fibroblasts were plated and grown for 2 days, then serum-starved for 1 day, treated with BDNF (50 ng/ml) for 15 min, and lysed in SDS-PAGE sample buffer heated to 100 °C. Immunoblot analysis was performed using anti-p42/p44 MAPK, anti-phospho-specific p42/p44 MAPK, and anti-Trk antibodies.

In these experiments, Rat 2 fibroblasts were engineered to express TrkBwt, TrkBY484F, or TrkBY785F using the Tet-Off system (35). In this system, a hybrid fusion protein containing the Tet repressor and the VP16 activation domain of herpes simplex virus binds to a compound promoter containing the tet-responsive element and the minimal cytomegalovirus promoter in the absence of tetracycline. Removal of tetracycline from the medium activates transcription of, in this case, TrkB.

BDNF-induced phosphorylation of MAPK is only attenuated, and is not blocked, in fibroblasts expressing TrkBY484F compared with TrkBwt (Fig. 1B, top panel). Phosphorylation of MAPK is indicative of MAPK activation (36). MAPK protein levels were similar in all four lanes (Fig. 1B, center panel). Contrasting the phosphorylation levels of MAPK to levels of TrkB expression (Fig. 1B, top and bottom panels) shows that BDNF-induced MAPK phosphorylation was attenuated in cells expressing TrkBY484F compared with cells expressing TrkBwt. MAPK protein levels, as predicted, are not altered in a short temporal experiment lasting 15 min (Fig. 1B, center panel). Since both the phosphospecific antibodies and the MAPK antibodies recognize both p42 and p44 MAPKs, it is likely that p42 is expressed at higher levels than p44 in these cells. TrkB protein levels are similar in the cells expressing TrkBwt and TrkBY484F.

Tyrosine 484 of TrkB is required for prolonged activation of MAPK in fibroblasts. BDNF induces a sustained phosphorylation of MAPK in fibroblasts expressing TrkBwt (Fig. 2). In cells expressing TrkBY484F, BDNF induces a similar magnitude of MAPK phosphorylation. However, this phosphorylation has a much shorter temporal duration (Fig. 2). These temporal data provide compelling evidence that the activation of MAPK is attenuated in cells expressing TrkBY484F compared with cells expressing TrkBwt. This argues that there is likely to be another BDNF signaling pathway resulting in MAPK phosphorylation. PLC-gamma 1 is definitively phosphorylated and activated by BDNF. Therefore, tyrosine 785 was mutated to phenylalanine to test whether PLC-gamma 1 is required for MAPK activation by BDNF. In fibroblasts engineered to express rat TrkBY785F, which abrogates phosphorylation of PLC-gamma 1 (data not shown), BDNF induces a sustained phosphorylation of MAPK that is similar to the phosphorylation of MAPK observed in cells expressing TrkBwt (Fig. 2). Therefore, it seems likely that PLC-gamma 1 is not required for BDNF-induced phosphorylation of MAPK. It seems likely that the observed slight increases in MAPK phosphorylation, both in magnitude and temporal duration, in the fibroblasts expressing TrkBY785F is due to greater levels of TrkBY785F protein when contrasted with the cells expressing TrkBwt. However, since the levels of TrkBY785F are clearly higher than the TrkB protein levels in the cells expressing TrkBwt or TrkBY484F, we cannot completely rule out that PLC-gamma 1 is not required for full MAPK activation. TrkBY785F expressed in these fibroblasts is competent for BDNF-induced kinase activity and autophosphorylation (data not shown). The conclusion drawn from these data is that there must be another BDNF signaling mechanism resulting in phosphorylation of MAPK. This compelled a search for other substrates of the TrkB receptor that might activate the MAPK pathway.


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Fig. 2.   BDNF induces a more transient temporal phosphorylation of MAPK in rat 2 fibroblasts expressing TrkBY484F than TrkBwt or TrkBY785F. Rat 2 TrkBwt, TrkBY484F, or TrkBY785F fibroblasts were plated and grown 2 days and then serum-starved for 1 day. Cells were treated with BDNF (50 ng/ml) and lysed at the various time points subsequent to BDNF addition in SDS-PAGE sample buffer heated to 100 °C. Immunoblotting was performed using anti-p42/p44 MAPK, anti-phospho-specific p42/p44 MAPK, and anti-Trk antibodies.

BDNF Induces Phosphorylation of FRS2-- BDNF induces phosphorylation of FRS2 on tyrosine in response to BDNF in Rat 2 fibroblasts engineered to express TrkBwt (Fig. 3). We raised antibodies using a peptide corresponding to the COOH-terminal sequence of FRS2. Fig. 3 illustrates that a 90-kDa protein is immunoprecipitated by the FRS2 carboxyl-terminal antiserum. The mobility of this 90-kDa protein is similar to the reported mobility for FRS2 (28). This 90-kDa protein is phosphorylated in response to BDNF and is not immunoprecipitated by preimmune serum. Additionally, addition of free peptide to the immunoprecipitation blocks specific precipitation of this 90-kDa band by anti-FRS2 antibodies. Although BDNF clearly induces tyrosine phosphorylation of FRS2 in fibroblasts, we wanted to test whether or not neurotrophins induced FRS2 phosphorylation in a physiologically relevant cell line. BDNF and NGF induce tyrosine phosphorylation of FRS2 in NB1643 cells (Fig. 4, top panel). NB1643 is a human neuroblastoma cell line that expresses both TRKB and TRK following ATRA treatment.2 BDNF and NGF induce autophosphorylation of TRKB and TRK, respectively (Fig. 4, bottom panel). This indicates that both TRKB and TRK are competent for protein-tyrosine kinase activity in these neuroblastoma cells. The minor mobility difference observed between FRS2 isolated from BDNF- and NGF-treated cells is likely to be caused by greater phosphorylation of FRS2 following BDNF treatment. TRKB levels are higher than TRK levels in NB1643 cells, which explains the observation that the autophosphorylation of TRKB is greater than TRK in these neuroblastoma cells (Fig. 4, bottom panel). From these data, we have not concluded that FRS2 is a better substrate for TRKB than TRK.


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Fig. 3.   BDNF induces phosphorylation of FRS2 on tyrosine. Rat 2 TrkBwt fibroblasts were plated and grown for 3 days, serum-starved for 1 day, and treated with or without BDNF (50 ng/ml). Immunoprecipitation with preimmune and immune polyclonal antiserum with or without 100 µM peptide corresponding to the carboxyl-terminal 11 amino acids of FRS2 was followed by anti-phosphotyrosine immunoblotting.


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Fig. 4.   Both BDNF and NGF induce phosphorylation of FRS2 on tyrosine in NB1643 cells and fibroblasts expressing TrkBwt. NB 1643 cells were plated in media containing 5 µM ATRA and grown for 4 days, then treated for 5 min with either 50 ng/ml BDNF or NGF. Cells were then lysed in RIPA buffer and immunoprecipitated with anti-FRS2 (top) or anti-Trk antibodies (bottom) and immunoblotted with anti-phosphotyrosine antibodies.

To summarize, the neurotrophins, BDNF and NGF, induce tyrosine phosphorylation of a 90-kDa protein immunoprecipitated with antibodies raised to the carboxyl-terminal sequence of FRS2. In this neuronal cell line, we noted that BDNF and NGF stimulate tyrosine phosphorylation of other proteins with predicted molecular masses between 60 and 90 kDa. Whether these proteins are associated with FRS2 in a complex and co-immunoprecipitated has not been established. The identity of these proteins is unknown. Kouhara et al. (28) have suggested that FRS2 and SNT are probably the same protein. We also noted that the molecular mass of FRS2, determined by gel mobility, is similar to the molecular weight of SNT, a previously described protein phosphorylated on tyrosine in response to NGF treatment of PC12 cells (18).

FRS2 and SNT Are Phosphorylated in Response to BDNF in Cortical Neurons, Fibroblasts Engineered to Express TrkB, and Neuroblastoma Cells-- BDNF induces phosphorylation of FRS2 in cortical neurons (Fig. 5A). This suggests that BDNF signaling requiring FRS2 is likely to be physiologically relevant. Cortical neurons in primary culture were treated with BDNF, then lysed in nondenaturing detergent buffers. Then FRS2 or SNT was precipitated with antibodies recognizing FRS2 or p13suc1-agarose beads, respectively. Both the anti-FRS2 antibodies and p13suc1-agarose beads precipitate a protein doublet phosphorylated specifically on tyrosine in response to BDNF with masses predicted by mobility of about 90 kDa (Fig. 5A). In both Rat 2 fibroblasts engineered to express TrkBwt and NB1643 cells, BDNF induces tyrosine phosphorylation of the same 90-kDa protein doublet precipitated with either anti-FRS2 antibodies or p13suc1-agarose beads (Fig. 5B). It was not possible to precipitate SNT with p13suc1-agarose beads and followed by immunoblotting with FRS2 antiserum. The FRS2 antiserum does not work for immunoblotting. The data, when taken together, that FRS2 and SNT are phosphorylated on tyrosine in response to BDNF and that they have identical molecular masses lend further support to the assertion by Kouhara et al. (28) that it is likely SNT and FRS2 are the same protein. In the experiment with cortical neurons and NB1643 cells depicted in Fig. 5, immunoprecipitation of FRS2 or SNT also precipitated proteins phosphorylated on tyrosine with molecular masses between 60 and 90 kDa. This is similar to the data observed in the experiment depicted in Fig. 4. There may be other proteins, phosphorylated on tyrosine in response to BDNF, that form a complex with FRS2.


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Fig. 5.   BDNF induces phosphorylation of FRS2 and SNT in cortical neurons, NB1643 cells, and fibroblasts expressing TrkBwt. A, Rat P0 cortical neurons were grown for 4 days and treated for 5 min with or without 50 ng/ml BDNF. Cells were then lysed in either RIPA or Nonidet P-40 buffer and immunoprecipitated with anti-FRS2 or p13suc1 beads, respectively, and then immunoblotted with anti-phosphotyrosine antibodies. B, top, Rat 2 TrkBwt fibroblasts were plated and grown for 3 days. Bottom, NB1643 cells were plated in media containing 5 µM ATRA and grown for 4 days. Cells were then treated for 5 min with or without 50 ng/ml BDNF and then lysed in RIPA or Nonidet P-40 buffer and immunoprecipitated with anti-FRS2 antiserum or p13suc1 beads, respectively, and then immunoblotted with anti-phosphotyrosine antibodies.

FRS2 Does Not Form a Complex with Shc-- Fibroblasts expressing TrkB were treated with BDNF, then lysed in nondenaturing detergent containing buffer. Immunoprecipitation of FRS2, as predicted, precipitated FRS2 as visualized on an anti-phosphotyrosine immunoblot (Fig. 6, top panel). However, Shc was not immunoprecipitated by anti-FRS2 serum, with or without BDNF. This suggests that BDNF does not induce formation of a complex containing FRS2 and Shc. Conversely, immunoprecipitation with anti-Shc antibodies precipitated Shc and did not precipitate FRS2 (Fig. 6, top panel). BDNF clearly does not induce the formation of a complex containing FRS2 and Shc in fibroblasts. In a neuronal background, immunoprecipitation of FRS2 from NB1643 cells treated with BDNF precipitated FRS2, but not Shc (Fig. 6, bottom panel). Conversely, immunoprecipitation of Shc did not precipitate FRS2. Therefore, BDNF does not induce the formation of a complex containing FRS2 and Shc in neuronal cells and fibroblasts. As noted previously, there are other proteins (with molecular masses between 60 and 90 kDa) phosphorylated on tyrosine following BDNF treatment immunoprecipitated along with FRS2. The identity of these proteins is not known.


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Fig. 6.   BDNF does not induce association of FRS2 and Shc. Rat 2 TrkBwt fibroblasts were plated and grown 2 days, and NB1643 cells were plated in media containing 5 µM ATRA and grown for 2 days. Then cells were treated with or without 50 ng/ml BDNF. Cells were lysed in Nonidet P-40 buffer and immunoprecipitated with either anti-FRS2 antiserum or anti-Shc antibodies followed by anti-phosphotyrosine immunoblotting.

BDNF Induces the Formation of a Complex Containing FRS2 and Grb2-- To test whether BDNF also signals through Grb2 similar to FGF (28), we asked whether BDNF induces formation of a complex containing FRS2 and Grb2 in a physiologically relevant culture system. P0 cortical neurons isolated from rat brains were treated with or without BDNF and lysed in a nondenaturing detergent containing buffer. BDNF induces the formation of a complex containing FRS2 and Grb2, since immunoprecipitation of FRS2 from cells treated with BDNF precipitates Grb2 (Fig. 7). Furthermore, BDNF induces formation of a complex containing Grb2 and FRS2 in fibroblasts. Immunoprecipitation of FRS2 from Rat 2 fibroblasts engineered to express TrkB treated with BDNF precipitates Grb2 (Fig. 7). BDNF does not induce formation of a complex containing Grb2 and FRS2 in a control fibroblast cell line that does not express TrkB. Likewise, in a neuroblastoma cell line, NB1643, BDNF induces formation of a complex with GRB2. Therefore, it is likely that BDNF, similar to FGF (28), induces association of FRS2 with Grb2. This in turn induces association of Grb2 with Sos and could activate RAS and the RAS/MAPK pathway. In summation, the data show that BDNF induces a complex containing FRS2 and Grb2 in neuroblastoma cells and fibroblasts. More important, this is the first demonstration that BDNF induces formation of a complex containing FRS2 and Grb2 in cortical neurons in primary culture, which suggests that BDNF signaling through FRS2 via Grb2 is very likely to have physiological relevance.


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Fig. 7.   BDNF does induce association of FRS2 with Grb2 in cortical neurons, fibroblasts, and NB1643 cells. Rat P0 cortical neurons were grown for 7 days. Rat 2 Hyg or TrkBwt fibroblasts were plated and grown for 2 days and serum-starved for 1 day, and NB1643 cells were plated in media containing 5 µM ATRA and grown for 2 days and serum-starved for 1 day. Then cells were treated with or without 50 ng/ml BDNF for 5 min. Cells were lysed in Triton X-100 buffer and immunoprecipitated with anti-FRS2 antiserum followed by Grb2 immunoblotting.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The data reported here provide evidence that BDNF activates at least two signaling pathways, via TrkB, that lead to phosphorylation of MAPK. Multiple neurotrophin signaling pathways leading to MAPK activation may underlie the observed sustained activation of MAPK induced, for example, by NGF (20). This has generated a hypothesis that we are now testing. BDNF may activate two or more pathways that lead to sustained MAPK activation. A previously established pathway requires Shc. The data in this report provide evidence for another pathway requiring FRS2/SNT. Phosphorylation of Shc leads to formation of a ternary complex containing Shc, Grb2, and Sos, which in turn leads to RAS activation (37, 38). FGF induces phosphorylation of FRS2 and formation of a complex with Grb2, which could lead to RAS activation (28, 39). In this report, we have shown that BDNF induces tyrosine phosphorylation and the formation of a complex containing Grb2 and FRS2. This is likely to lead to MAPK activation. The combined activation of these pathways may lead to sustained activation of the MAPK pathway by BDNF. It remains to be determined whether there are kinetic differences in the activation of MAPK by these various pathways. The data presented here indicate that abrogation of Shc interaction with tyrosine 484 of TrkB in fibroblasts results in a rapid and short-lived activation of MAPK, which suggests Shc may be required for a more prolonged activation of MAPK. A putative role for Shc in the prolonged activation of MAPK by neurotrophins is consistent with the observation by Segal et al. (26) that the phosphorylated state of tyrosine 490 of Trk may be long-lived. Tyrosine 490 is the binding site for Shc in Trk and is homologous to tyrosine 484 in TrkB. It will be important to determine whether other signaling pathways, perhaps requiring FRS2/SNT or other adapter proteins, contribute to sustained activation of the MAPK pathway.

There may be other signaling pathways leading to MAPK signaling activated by BDNF. rAPS and SH2-B are substrates of the TRK family receptors, including TrkB, which were identified recently by Qian et al.(40). rAPS is a putative adapter protein containing a pleckstrin homology domain and a Src homology 2 domain. SH2-B is a putative adapter protein containing a Src homology 2 domain. Both rAPS and SH2-B, when transfected into 293 cells along with Trk, enhanced NGF-mediated activation of MAPK. It also seems likely that the cellular background will have a role in MAPK activation by neurotrophins. Perhaps BDNF utilizes various subsets of signaling pathways in different cells. For example, the prolonged activation of MAPK by BDNF observed in fibroblasts in this report does not seem as sustained as the MAPK activation observed in PC12 cells by NGF (16). This suggests the pathways in fibroblasts could be different from PC12 cells.

FRS2 was cloned based on binding to Grb2 in FGF-stimulated cells (28), whereas SNT was identified as a 78-90-kDa protein phosphorylated on tyrosine in response to NGF treatment (18). FRS2 resolved on gels is a doublet with a predicted molecular mass, based on mobility, of 92-95 kDa (28). This is similar to the observations reported here of a doublet with estimated molecular mass by gel mobility of 90 kDa. Kouhara and colleagues suggested (28) that it is probable that FRS2 and SNT are identical. The data presented here support the contention that FRS2 is SNT, since they comigrate. In addition, BDNF induces phosphorylation of FRS2 and SNT. Notably, phosphorylation of SNT was not induced by EGF in PC12 cells (18). EGF also does not stimulate phosphorylation of FRS2 (28).

The cDNA clone for FRS2 encodes on open reading frame of 508 amino acids with a predicted mass of 57 kDa. FRS2 is myristoylated and contains a consensus myristoylation sequence in the amino terminus, followed by a PTB domain. FRS2 is localized to the cell membrane (28). A mutant of FRS2, which is not myristoylated, is localized to the cytoplasm. This demonstrates myristoylation targets FRS2 to the cell membrane (28). However, in cell fractionation experiments, tyrosine-phosphorylated SNT was found predominantly in the nuclear fraction along with, as expected, c-Jun (18). These disparate results have not been resolved yet. FRS2/SNT is a substrate for the FGF receptor, TRK, and TRKB, which is consistent with plasma membrane localization.

In the experiments reported here, FRS2 migrates as a protein doublet on SDS-PAGE gels. Whether these two proteins are derived from the same gene and have different masses because of post-translational modification, such as phosphorylation, or alternative splicing of their mRNAs is not known. Alternatively, it is not known if the proteins are derived from two related genes. There are data suggesting that there are two members of the SNT family (41). However, it has been reported that apparently only SNT-1, which is FRS2, binds to p13suc1-agarose beads. SNT-2 may not bind to p13suc1-agarose beads (41). The proteins comprising the observed FRS2 doublet described in this report are recognized by antibodies raised against the COOH terminus of FRS2 and bind to p13suc1 beads.

In observations similar to the data reported here, mutation of Trk tyrosine 490 to phenylalanine also resulted in attenuation, but not abrogation, of NGF-dependent MAPK phosphorylation (25). It was suggested that activation of PLC-gamma 1 may lead to phosphorylation of MAPK in PC12nnr5 cells engineered to express TrkY490F. The data presented here show that neither the level nor, more important, the time course of MAPK phosphorylation seems altered in fibroblasts expressing TrkBY785F contrasted with TrkBwt. This suggests that an adapter protein, perhaps FRS2, SH2-B, or APS, rather than PLC-gamma 1, may be required for BDNF-dependent MAPK phosphorylation in fibroblasts expressing TrkBY484F.

Expression of a mutant Trk in PC12nnr5 cells, where a tripeptide sequence, KFG, in the juxtamembrane region was deleted did not result in NGF-dependent phosphorylation of SNT (19). The sequence of FRS2 (28) predicts it contains a PTB domain, which binds motifs containing phosphotyrosine. However, several proteins, X11, FE65, and Numb, have PTB domains that bind motifs that do not contain phosphotyrosine (38). Xu et al. (41) have found that the PTB domain of SNT-1, which they conclude is FRS2, is required for association with the FGF receptor in in vitro experiments. Moreover, a juxtamembrane region of the FGF receptor is also required for the association of the PTB domain of SNT-1 in vitro. In additional experiments in NIH3T3 cells, it was found that the PTB domain of SNT-1/FRS2 and the juxtamembrane region of the FGF receptor are required for phosphorylation on tyrosine of SNT-1/FRS2 in vivo in response to FGF. It should prove enlightening to determine if this KFG motif is also required for phosphorylation of FRS2 by TrkB, as is reported for phosphorylation of SNT by TRK (19).

We observed other proteins phosphorylated on tyrosine that are, apparently, immunoprecipitated with FRS2 in NB1643 cells. It remains to be determined if these proteins with apparent molecular masses between 60 and 90 kDa form complexes with FRS2. FGF induces FRS2 association with Shp2 (42). Shp2 is a protein-tyrosine phosphatase. Moreover, binding of Shp2 to FRS2 is required for FGF-induced PC12 differentiation (43). FGF induces the formation of a complex containing FRS2, Shp2, and Grb2 that may also contribute to activation of the RAS/MAPK pathway. In addition, Shp2 phosphatase activity is required for sustained activation of MAPK by FGF (43). In experiments related to this subject, it was found that mice homozygous for a deletion mutant of Shp2 die in mid-gestation (44). In cell cultures established from mutant embryos, it was found that Shp2 was required for full and sustained activation of the MAPK pathway. This raises the intriguing possibility that FRS2 may also stimulate or sustain MAPK activity by activating a protein-tyrosine phosphatase, Shp2.

BDNF has roles in cell survival, axonal growth, and even synaptic plasticity. Delineating the signaling pathways required for these specific cellular effects is of considerable contemporary interest. Data derived from both in vitro experiments and targeted disruption of the BDNF and TrkB genes suggest that BDNF is required for trophic support of some neurons (1-3,45-47). Wortmannin, a pharmacological inhibitor of PI3K among other enzymes, blocked NGF-dependent survival of PC12 cells (48). PDGF promotes survival of PC12 cells engineered to express the PDGF receptor (48). However, expression of a mutant of PDGF receptor incapable of activating PI3K did not prevent apoptosis. The serine/threonine kinase, AKT, may also be an important effector of PI3K for preventing apoptosis (49). Neurotrophins, it seems, require PI3K for promoting cell survival. However, the RAS/MAPK is required for differentiation of PC12 cells (24, 25). BDNF has other physiological roles as diverse as growth cone guidance (4) and synaptic plasticity. BDNF stabilizes synapses (50) and may be required for maintenance of long term potentiation in the hippocampus (7, 8). It is, therefore, important to determine the signaling pathways required by BDNF to achieve signaling specificity. It remains to be determined how BDNF promoted signaling through FRS2 integrates into the scenario of BDNF-activated signaling pathways leading to these diverse cellular effects.

In this paper, we show that association of Shc with phosphorylated tyrosine 484 of TrkB accounts for only part of the observed activation of the RAS/MAPK pathway. These data compelled a search for other BDNF-activated signaling pathways that result in MAPK phosphorylation that are independent of Shc. We have shown that BDNF induces phosphorylation of FRS2, which is likely identical to SNT, and that BDNF induces association of FRS2 with Grb2. This is likely to lead to activation of the RAS/MAPK pathway. Experiments identifying critical regions of TrkB required for phosphorylation of FRS2 will be important. This will allow the design of mutant TrkB receptors that do not signal through FRS2 for testing the requirement for FRS2 in BDNF signaling. Experiments testing whether BDNF induces sustained activation of the MAPK pathway by signaling through independent pathways requiring Shc and FRS2, respectively, will be indispensable for understanding BDNF signaling specificity.

    ACKNOWLEDGEMENTS

BDNF and NGF were generously provided by Dr. Andy Welcher, Amgen, Thousand Oaks, CA. Neuroblastoma cell lines were provided by the Pediatric Oncology Group (POG) courtesy of Dr. Susan Cohn and Susan Roe.

    FOOTNOTES

* This work was supported by the American Lebanese Syrian Associated Charities and by Grants 1 R29 CA 71628 (to D. S. M.) and CA 21765 for Cancer Center Support (CORE) from the National Cancer Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom reprint requests should be addressed: Dept. of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale, P.O. Box 318, Memphis, TN 38101-0318. Tel.: 901-495-2775; Fax: 901-521-1668; E-mail: david.middlemas{at}stjude.org.

2 D. S. Middlemas, B. K. Kihl, and J. Zhou, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: BDNF, brain-derived neurotrophic factor; NB, neuroblastoma; ATRA, all-trans-retinoic acid; NGF, nerve growth factor; PI3K, phosphatidylinositol 3-kinase; PLC-gamma 1, phospholipase C-gamma 1; BSA, bovine serum albumin; SNT, suc-associated neurotrophic factor-induced tyrosine-phosphorylated target; FRS2, fibroblast growth factor receptor substrate 2; TRK, tropomyosin-related kinase; Grb2, growth factor receptor-binding protein 2; Sos, son of sevenless; PC12, pheochromocytoma cells 12; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis; TBST, Tris-buffered saline with Tween; FGF, fibroblast growth factor; PTB, phosphotyrosine binding; RIPA, radioimmune precipitation buffer.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Klein, R., Smeyne, R. J., Wurst, W., Long, L. K., Auerbach, B. A., Joyner, A. L., and Barbacid, M. (1993) Cell 75, 113-122[Medline] [Order article via Infotrieve]
  2. Klein, R. (1994) FASEB J. 8, 738-744[Abstract/Free Full Text]
  3. Minichiello, L., and Klein, R. (1996) Genes Dev. 10, 2849-2858[Abstract]
  4. Song, H. J., Ming, G. L., and Poo, M. M. (1997) Nature 388, 275-279[CrossRef][Medline] [Order article via Infotrieve]
  5. Sawai, H., Clarke, D. B., Kittlerova, P., Bray, G. M., and Aguayo, A. J. (1996) J. Neurosci. 16, 3887-3894[Abstract/Free Full Text]
  6. Novikov, L., Novikova, L., and Kellerth, J. O. (1997) Neuroscience 79, 765-774[CrossRef][Medline] [Order article via Infotrieve]
  7. Patterson, S. L., Abel, T., Deuel, T. A., Martin, K. C., Rose, J. C., and Kandel, E. R. (1996) Neuron 16, 1137-1145[Medline] [Order article via Infotrieve]
  8. Kang, H., Welcher, A. A., Shelton, D., and Schuman, E. M. (1997) Neuron 19, 653-664[Medline] [Order article via Infotrieve]
  9. Soppet, D., Escandon, E., Maragos, J., Middlemas, D. S., Reid, S. W., Blair, J., Burton, L. E., Stanton, B. R., Kaplan, D. R., Hunter, T., Nikolics, K., and Parada, L. F. (1991) Cell 65, 895-903[Medline] [Order article via Infotrieve]
  10. Squinto, S. P., Stitt, T. N., Aldrich, T. H., Davis, S., Bianco, S. M., Radziejewski, C., Glass, D. J., Masiakowski, P., Furth, M. E., Valenzuela, D. M., DiStefano, P. S., and Yancopoulos, G. D. (1991) Cell 65, 885-893[Medline] [Order article via Infotrieve]
  11. Klein, R., Nanduri, V., Jing, S. A., Lamballe, F., Tapley, P., Bryant, S., Cordon-Cardo, C., Jones, K. R., Reichardt, L. F., and Barbacid, M. (1991) Cell 66, 395-403[Medline] [Order article via Infotrieve]
  12. Middlemas, D. S. (1993) Methods Neurosci. 12, 139-155
  13. Kaplan, D. R., Martin-Zanca, D., and Parada, L. F. (1991) Nature 350, 158-160[CrossRef][Medline] [Order article via Infotrieve]
  14. Kaplan, D. R., Hempstead, B. L., Martin-Zanca, D., Chao, M. V., and Parada, L. F. (1991) Science 252, 554-558[Medline] [Order article via Infotrieve]
  15. Klein, R., Jing, S. Q., Nanduri, V., O'Rourke, E., and Barbacid, M. (1991) Cell 65, 189-197[Medline] [Order article via Infotrieve]
  16. Greene, L. A., and Tischler, A. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2424-2428[Abstract]
  17. Huff, K., End, D., and Guroff, G. (1981) J. Cell Biol. 88, 189-198[Abstract/Free Full Text]
  18. Rabin, S. J., Cleghon, V., and Kaplan, D. R. (1993) Mol. Cell. Biol. 13, 2203-2213[Abstract]
  19. Peng, X., Greene, L. A., Kaplan, D. R., and Stephens, R. M. (1995) Neuron 15, 395-406[Medline] [Order article via Infotrieve]
  20. Marshall, C. J. (1995) Cell 80, 179-185[Medline] [Order article via Infotrieve]
  21. Segal, R. A., and Greenberg, M. E. (1996) Annu. Rev. Neurosci. 19, 463-489[CrossRef][Medline] [Order article via Infotrieve]
  22. Finkbeiner, S., Tavazoie, S. F., Maloratsky, A., Jacobs, K. M., Harris, K. M., and Greenberg, M. E. (1997) Neuron 19, 1031-1047[Medline] [Order article via Infotrieve]
  23. van der Geer, P., Wiley, S., Lai, V. K.-M., Olivier, J. P., Gish, G. D., Stephens, R., Kaplan, D., Shoelson, S., and Pawson, T. (1995) Curr. Biol. 5, 404-412[Medline] [Order article via Infotrieve]
  24. Obermeier, A., Bradshaw, R. A., Seedorf, K., Choidas, A., Schlessinger, J., and Ullrich, A. (1994) EMBO J. 13, 1585-1590[Abstract]
  25. Stephens, R. M., Loeb, D. M., Copeland, T. D., Pawson, T., Greene, L. A., and Kaplan, D. R. (1994) Neuron 12, 691-705[Medline] [Order article via Infotrieve]
  26. Segal, R. A., Bhattacharyya, A., Rua, L. A., Alberta, J. A., Stephens, R. M., Kaplan, D. R., and Stiles, C. D. (1996) J. Biol. Chem. 271, 20175-20181[Abstract/Free Full Text]
  27. Jing, S., Tapley, P., and Barbacid, M. (1992) Neuron 9, 1067-1079[Medline] [Order article via Infotrieve]
  28. Kouhara, H., Hadari, Y. R., Spivak-Kroizman, T., Schilling, J., Bar-Sagi, D., Lax, I., and Schlessinger, J. (1997) Cell 89, 693-702[Medline] [Order article via Infotrieve]
  29. Butler, W. B. (1984) Anal. Biochem. 141, 70-73[Medline] [Order article via Infotrieve]
  30. Furshpan, E. J., and Potter, D. D. (1989) Neuron 3, 199-207[Medline] [Order article via Infotrieve]
  31. Xia, Z., Dudek, H., Miranti, C. K., and Greenberg, M. E. (1996) J. Neurosci. 16, 5425-5436[Abstract/Free Full Text]
  32. Middlemas, D. S., Lindberg, R. A., and Hunter, T. (1991) Mol. Cell. Biol. 11, 143-53[Medline] [Order article via Infotrieve]
  33. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  34. Middlemas, D. S., Meisenhelder, J., and Hunter, T. (1994) J. Biol. Chem. 269, 5458-5466[Abstract/Free Full Text]
  35. Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5547-5551[Abstract]
  36. Payne, D. M., Rossomando, A. J., Martino, P., Erickson, A. K., Her, J. H., Shabanowitz, J., Hunt, D. F., Weber, M. J., and Sturgill, T. W. (1991) EMBO J. 10, 885-892[Abstract]
  37. Basu, T., Warne, P. H., and Downward, J. (1994) Oncogene 9, 3483-3491[Medline] [Order article via Infotrieve]
  38. Pawson, T., and Scott, J. D. (1997) Science 278, 2075-2080[Abstract/Free Full Text]
  39. Ong, S. H., Goh, K. C., Lim, Y. P., Low, B. C., Klint, P., Claesson-Welsh, L., Cao, X., Tan, Y. H., and Guy, G. R. (1996) Biochem. Biophys. Res. Commun. 225, 1021-1026[CrossRef][Medline] [Order article via Infotrieve]
  40. Qian, X., Riccoio, A., Zhang, Y., and Ginty, D. D. (1998) Neuron 21, 1017-1029[Medline] [Order article via Infotrieve]
  41. Xu, H., Lee, K. W., and Goldfarb, M. (1998) J. Biol. Chem. 273, 17987-17990[Abstract/Free Full Text]
  42. Ong, S. H., Lim, Y. P., Low, B. C., and Guy, G. R. (1997) Biochem. Biophys. Res. Commun. 238, 261-266[CrossRef][Medline] [Order article via Infotrieve]
  43. Hadari, Y. R., Kouhara, H., Lax, I., and Schlessinger, J. (1998) Mol. Cell. Biol. 18, 3966-3973[Abstract/Free Full Text]
  44. Saxton, T. M., Henkemeyer, M., Gasca, S., Shen, R., Rossi, D. J., Shalaby, F., Feng, G. S., and Pawson, T. (1997) EMBO J. 16, 2352-2364[Abstract/Free Full Text]
  45. Minichiello, L., Piehl, F., Vazquez, E., Schimmang, T., Hokfelt, T., Represa, J., and Klein, R. (1995) Development 121, 4067-4075[Abstract/Free Full Text]
  46. Pinon, L. G., Minichiello, L., Klein, R., and Davies, A. M. (1996) Development 122, 3255-3261[Abstract/Free Full Text]
  47. Schimmang, T., Alvarez-Bolado, G., Minichiello, L., Vazquez, E., Giraldez, F., Klein, R., and Represa, J. (1997) Mech. Dev. 64, 77-85[CrossRef][Medline] [Order article via Infotrieve]
  48. Yao, R., and Cooper, G. M. (1995) Science 267, 2003-2006[Medline] [Order article via Infotrieve]
  49. Dudek, H., Datta, S. R., Franke, T. F., Birnbaum, M. J., Yao, R., Cooper, G. M., Segal, R. A., Kaplan, D. R., and Greenberg, M. E. (1997) Science 275, 661-665[Abstract/Free Full Text]
  50. Kwon, Y. W., and Gurney, M. E. (1996) J. Neurobiol. 29, 503-516[CrossRef][Medline] [Order article via Infotrieve]


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