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INTRODUCTION |
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-
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.
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EXPERIMENTAL PROCEDURES |
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
-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-
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).
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RESULTS |
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.
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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-
1 is definitively phosphorylated and activated by BDNF. Therefore, tyrosine 785 was mutated to phenylalanine to test
whether PLC-
1 is required for MAPK activation by BDNF. In
fibroblasts engineered to express rat TrkBY785F, which abrogates phosphorylation of PLC-
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-
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-
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.
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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.
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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.
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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.
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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.
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DISCUSSION |
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-
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-
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.