From the Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622
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ABSTRACT |
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Nerve growth factor (NGF) is essential for the
development and survival of sympathetic and sensory neurons. NGF binds
to TrkA, activates the intrinsic kinase activity of TrkA, and promotes the differentiation of pheochromocytoma (PC12) cells into
sympathetic-like neurons. Several signaling molecules and pathways are
known to be activated by NGF, including phospholipase C PC12 cells, a rat adrenal pheochromocytoma cell line, are a widely
used paradigm for studying
NGF-induced1 neuronal
differentiation. NGF stimulates PC12 cells to differentiate into
sympathetic-like neurons by activating the NGF receptor TrkA, a member
of the Trk family of receptor tyrosine kinases (1-3). Upon NGF
binding, TrkA dimerizes (4) and autophosphorylates multiple tyrosines
within its cytoplasmic domain (5). Signaling molecules containing Src
homology 2 (SH2) or phosphotyrosine-binding domains, such as
phospholipase C SH2-B Materials--
Murine NGF and EGF were from Collaborative
Biomedical Products. Sodium orthovanadate was from Sigma. Recombinant
protein A-agarose was from Repligen. Antibody to rat SH2-B Plasmids and Transfection--
cDNA encoding rat SH2-B Immunoprecipitation and Immunoblotting--
Precedures for
preparing cell lysates, immunoprecipitation, and immunoblotting were
described previously (13). Cell Imaging--
Cells were plated at a density of 0.2 × 105 and cultured in standard medium supplemented with 1 mg/ml G418 on 60-mm collagen-coated culture dishes (Collaborative
Biomedical Products). The next day, NGF was added directly to the
medium at the indicated concentrations. Neurite outgrowths were
monitored and photographed every 12 h using phase contrast
microscopy. NGF was added every 2 days without changing the culture medium.
NGF Stimulates the Association of SH2-B
To determine whether SH2-B NGF Stimulates Tyrosyl Phosphorylation of SH2-B
Interestingly, 1 min of NGF stimulation of PC12 cells overexpressing
TrkA caused significant tyrosyl phosphorylation of SH2-B
In contrast to NGF, EGF did not stimulate tyrosyl phosphorylation of
SH2-B SH2-B
We first examined whether GFP-SH2-B
To study the role of SH2-B
Consistent with SH2-B NGF-induced Tyrosyl Phosphorylation of TrkA, Shc, and PLC
Because the kinetics of the activation of ERK1/2 have been hypothesized
to play an important role in NGF-induced neuronal differentiation of
PC12 cells, we examined the time course of activation of ERK1/2 by NGF
by immunoblotting cell lysates with antibody recognizing only activated
ERK1/2 that is phosphorylated on both tyrosine and threonine. Neither
the extent nor the duration of activation of ERK1/2 by NGF was affected
by overexpression of SH2-B In this study, we show that NGF promotes the association of
SH2-B SH2-B SH2-B Interestingly, overexpression of neither GFP-SH2-B In conclusion, we show that the putative adapter protein SH2-B,
phosphatidylinositol-3 kinase, and the mitogen-activated protein kinase
cascade. However, the mechanism of NGF-induced neuronal differentiation
remains unclear. In this study, we examined whether SH2-B
, a
recently identified pleckstrin homology and SH2 domain-containing
signaling protein, is a critical signaling protein for NGF. TrkA bound
to glutathione S-transferase fusion proteins containing
SH2-B
, and NGF stimulation dramatically increased that binding. In
contrast, NGF was unable to stimulate the association of TrkA with a
glutathione S-transferase fusion protein containing a
mutant SH2-B
(R555E) with a defective SH2 domain. When overexpressed
in PC12 cells, SH2-B
co-immunoprecipitated with TrkA in response to
NGF. NGF stimulated tyrosyl phosphorylation of endogenous SH2-B
as
well as exogenously expressed GFP-SH2-B
but not GFP-SH2-B
(R555E). Overexpression of SH2-B
(R555E) blocked NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of wild type SH2-B
enhanced NGF-induced neurite outgrowth. Overexpression of either wild type or
mutant SH2-B
(R555E) did not alter tyrosyl phosphorylation of TrkA,
Shc, or phospholipase C
in response to NGF or NGF-induced activation
of ERK1/2, suggesting that SH2-B
may initiate a previously unknown
pathway(s) that is essential for NGF-induced neurite outgrowth. Taken
together, these data indicate that SH2-B
is a novel signaling molecule required for NGF-induced neuronal differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(PLC
), phosphatidylinositol 3-kinase, and Shc
proteins, interact with tyrosyl phosphorylated TrkA and transmit NGF
signals (5, 6). Phosphatidylinositol 3-kinase is essential for NGF
protection of PC12 cells from apoptosis (7) but is not required for
NGF-induced neuronal differentiation (8). In contrast, the
Shc/Ras/MEK/ERK pathway appears to be essential for NGF-induced
neuronal differentiation of PC12 cells (5, 8-10). ERK1/2 can be
activated by both NGF and epidermal growth factor (EGF), but the
biological responses to NGF and EGF are opposite: NGF promotes
differentiation, whereas EGF stimulates proliferation of PC12 cells
(11). It was thought that sustained activation of ERK1/2 by NGF
compared with transient activation by EGF contributes to the
specificity of the NGF differentiation signal (11). Prolonged
activation of ERK1/2 by NGF has been shown to be mediated by Rap1 and
required for the expression of neuronal specific genes (12).
Surprisingly, sustained activation of ERK1/2 is not required for
NGF-induced neurite outgrowth (12), demonstrating that morphological
differentiation of PC12 cells is mediated by other as yet unidentified
signaling proteins/pathway(s).
, a predicted adapter protein with multiple potential
protein-protein interaction domains/motifs (e.g. pleckstrin
homology, SH2, and proline-rich), has recently been shown to be
regulated by a variety of ligands that activate receptor tyrosine
kinases or receptor-associated tyrosine kinases (13-15). Three
alternatively spliced isoforms of SH2-B (
,
, and
) have been
described (13, 16, 17)2 that
differ in their C termini downstream of the SH2 domain. No cellular
function had been ascribed to any of these isoforms. In this study, we
show that NGF stimulates association of SH2-B
with TrkA via the SH2
domain of SH2-B
and tyrosyl phosphorylation of SH2-B
. The
mutation of the conserved Arg to Glu within the FLVR motif within the
SH2 domain of SH2-B
abolishes the association of the mutant SH2-B
with TrkA and tyrosyl phosphorylation of the mutant SH2-B
in
response to NGF. Furthermore, this mutant SH2-B
acts as a dominant
negative SH2-B
to block NGF-induced neurite outgrowth when
overexpressed in PC12 cells. These results suggest that SH2-B
is an
essential component of a signaling pathway(s) that is vital for
NGF-induced neurite outgrowth.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(
SH2-B) was raised against a GST fusion protein containing the
C-terminal portion of SH2-B
(13). Monoclonal anti-phosphotyrosine
antibody 4G10 (
PY) and monoclonal antibody to PLC
(
PLC
)
were from Upstate Biotechnology Inc. Polyclonal antibody to Shc
(
Shc) was from Transduction Labs. Polyclonal antibody against the
extracellular domain of TrkA (
TrkA) (18) was kindly provided by Dr.
Louis F. Reichardt (University of California, San Francisco).
Polyclonal antibody against the cytoplasmic domain of TrkA was
purchased from Santa Cruz Biotechnology, Inc. (C-14). Monoclonal
antibody to GFP (
GFP) was from CLONTECH.
Anti-active MAPK (
active MAPK) was from Promega.
was subcloned in-frame at BglII/EcoRI sites into
pEGFP-C1 (CLONTECH), which encodes a red-shifted variant of GFP. Arg-555 in SH2-B
was mutated to Glu, using
QuickChangeTM site-directed mutagenesis kit (Stratagene)
with the primer 5'-GTCTTCTTGGTAGAACAGAGTGAGACAAGA-3'. PC12 cells were
transfected with pEGFP-C1 encoding GFP, GFP-SH2-B
, or GFP-SH2-B
(R555E), using LipofectAMINE Plus (Life Technologies, Inc.). After
72 h at 37 °C in 5% CO2 in standard medium
(Dulbecco's modified Eagle's medium supplemented with 1 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin, 10% heat-inactivated
horse serum, and 5% fetal bovine serum), the transfectants were
cultured for 40 additional days in medium supplemented with 1 mg/ml
G418. The G418-resistant transfectants were pooled, and the top 2% of
cells in terms of expression of GFP fusion proteins were selected by
flow cytometry (Elite, ESP).
PLC
was used at 1:2000, and
active-MAPK was used at 1:20,000 for immunoblotting.
TrkA and
Shc were used at 1:100 for immunoprecipitation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
with TrkA via the SH2
Domain of SH2-B
--
SH2-B in PC12 cells is thought to be the
isoform because after dephosphorylation by alkaline phosphatase, it
comigrates with dephosphorylated SH2-B
expressed ectopically in COS
cells (data not shown). To determine whether SH2-B
plays a role in NGF signaling, we first examined whether SH2-B
interacts with TrkA.
Wild type or mutant SH2-B
was fused to GST. PC12 cells overexpressing TrkA (19) were treated with NGF, and the cell extracts
were incubated with the indicated GST fusion proteins immobilized on
agarose beads. GST fusion proteins containing either wild type (GST-WT)
or the C-terminal 20% of SH2-B
with the entire SH2 domain (GST-SH2)
bound weakly to TrkA from control cells (Fig. 1a). NGF treatment
dramatically increased the binding of TrkA to both GST-SH2-B
and
GST-SH2 (Fig. 1a). Densitometric analysis revealed that NGF
stimulated the association of TrkA with GST-SH2-B
by more than
5-fold (n = 3) and with GST-SH2 by more than 4.5-fold (n = 3). In contrast, only a residual amount of TrkA
bound to GST fusion proteins containing mutant SH2-B
(R555E) in which
Glu replaced the critical Arg within the FLVR motif of the SH2 domain of SH2-B
(GST-RE) (Fig. 1a). NGF treatment did not
increase the binding of TrkA to GST-RE (Fig. 1a). GST alone
did not bind to TrkA, even when cells had been treated with NGF (Fig.
1a). These results suggest that the SH2 domain of SH2-B
is sufficient and necessary for NGF-promoted association of TrkA with
SH2-B
. In addition, the weak binding of GST-WT or GST-SH2 to TrkA
from control cells suggests that the SH2 domain of SH2-B
may have a
low affinity for nontyrosyl phosphorylated, inactive TrkA.
Alternatively, there may be other binding site(s) in the C-terminal
portion of SH2-B
that bind with a low affinity to nontyrosyl
phosphorylated TrkA.
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Fig. 1.
NGF promotes the interaction of
SH2-B with TrkA. a, PC12 cells
overexpressing TrkA were stimulated with 100 ng/ml NGF for 10 min. Cell
lysates were incubated with GST fusion proteins containing wild type
(GST-WT), the SH2 domain of (GST-SH2), or R555E
mutant (GST-RE) SH2-B
. Proteins bound to GST fusion
proteins were immunoblotted (IB) with
TrkA (from Dr.
Louis F. Reichardt, 1:500 dilution). b, PC12 cells stably
overexpressing GFP-SH2-B
were stimulated with 100 ng/ml NGF for 6 min. Proteins in the cell lysates were immunoprecipitated
(IP) with
TrkA (from Dr. Louis F. Reichardt, 1:100
dilution) and immunoblotted sequentially with
SH2-B and
GFP as
indicated. The same blot was stripped and reprobed with
TrkA.
associates with TrkA in cells in response
to NGF, PC12 cells overexpressing GFP-tagged SH2-B
were treated with
NGF, and proteins in the cell lysates were immunoprecipitated with
TrkA. Immunoprecipitated proteins were immunoblotted with either
SH2-B or
GFP (Fig. 1b). NGF significantly promoted the association of TrkA with GFP-SH2-B
(Fig. 1b). When
normalized to the level of TrkA, NGF stimulated the binding of TrkA to
GFP-SH2-B
by 4-fold. In contrast, NGF did not stimulate the
interaction of TrkA with GFP-SH2-B
(R555E) that has a defective SH2
domain (data not shown). Consistent with activated TrkA binding to
SH2-B
, endogenous SH2-B
from NGF-treated cells
co-immunoprecipitated with a tyrosyl phosphorylated protein of a size
appropriate for TrkA (Fig. 2b,
upper panel). Taken together, our results suggest that NGF
stimulates the binding of SH2-B
to TrkA via the SH2 domain of
SH2-B
.
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Fig. 2.
NGF stimulates tyrosyl phosphorylation of
SH2-B . a, PC12 cells were
pretreated with 100 µM Na3VO4 for
60 min prior to 100 ng/ml NGF for 10 min. SH2-B
was
immunoprecipitated (IP) with
SH2-B and immunoblotted
(IB) with
PY (upper panel, 1:7,500 dilution).
The same blot was reprobed with
SH2-B (lower panel).
b, PC12 cells overexpressing TrkA were treated with 100 ng/ml NGF for 1 min, and SH2-B
was immunoprecipitated with
SH2-B
and immunoblotted with
PY (upper panel). The same blot
was reprobed with
SH2-B (lower panel). c, PC12
cells were pretreated with 100 µM
Na3VO4 for 60 min prior to 100 ng/ml NGF or 125 ng/ml EGF for 10 min. SH2-B
was immunoprecipitated with
SH2-B and
immunoblotted with
PY.
in PC12
Cells--
To examine whether NGF stimulates tyrosyl phosphorylation
of SH2-B
, PC12 cells overexpressing either TrkA (Fig. 2b)
or SH2-B
tagged with GFP (Fig.
3b) were stimulated with NGF.
Proteins in the cell lysates were immunoprecipitated with
SH2-B and
immunoblotted with
PY. NGF stimulated tyrosyl phosphorylation of
both endogenous SH2-B
(Fig. 2b, upper panel)
and GFP-SH2-B
(Fig. 3b, upper panel). In
addition, when normal PC12 cells were pretreated for 60 min with 100 µM sodium vanadate (a phosphatase inhibitor) prior to NGF
stimulation, significant NGF-stimulated tyrosyl phosphorylation of
SH2-B
was detected (Fig. 2a, upper panel).
NGF-induced tyrosyl phosphorylation of SH2-B
was difficult to detect
in normal PC12 cells without pretreatment with phosphatase inhibitor
(data not shown), suggesting that NGF induces phosphorylation of
tyrosines within SH2-B
that then undergo rapid dephosphorylation.
NGF also stimulated a significant shift in the mobility of SH2-B
(Fig. 2a, lower panel), which is largely due to
NGF-promoted phosphorylation of SH2-B
on
serines/threonines.3
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Fig. 3.
NGF regulation of stably expressed GFP-tagged
wild type (GFP-WT) or R555E mutant
(GFP-RE) SH2-B .
a, Equal amounts of lysates of PC12 cells overexpressing
GFP, GFP-WT, and GFP-RE were immunoblotted (IB) with
SH2-B. b, PC12 cells overexpressing GFP-SH2-B
(GFP-WT) and GFP-SH2-B
(R555E) (GFP-RE) were
stimulated with 100 ng/ml NGF for 10 min. Proteins in lysates were
immunoprecipitated with
SH2-B and immunoblotted with
PY
(upper panel). The same blots were reprobed with
SH2-B
(lower panel). Because of the difference in the level of
expression of GFP-SH2-B
and GFP-SH2-B
(R555E), the exposure time
for GFP-SH2-B
(R555E) is approximately three times that for
GFP-SH2-B
during ECL.
without a
dramatic change in the mobility of SH2-B
(Fig. 2b, lower panel). This is in contrast to the large mobility
change observed in endogenous SH2-B
in PC12 cells treated with NGF
for 10 min (Fig. 2a, lower panel). This
difference in mobility shift in the two figures is likely due to the
different times of incubation with NGF, because a large mobility change
of SH2-B
was observed when PC12 cells overexpressing TrkA were
treated with NGF for 10 min (data not shown). These data support the
hypothesis that the shift in the mobility of SH2-B
is caused by
phosphorylation of SH2-B
on serines/threonines rather than on
tyrosines. The results also suggest that NGF-stimulated tyrosyl
phosphorylation of SH2-B
may precede serine/threonine
phosphorylation of SH2-B
.
in PC12 cells even in the presence of phosphatase inhibitor
(Fig. 2c). Because EGF stimulates proliferation while NGF
promotes neuronal differentiation of PC12 cells, this differential response of SH2-B
to these two growth factors might contribute to
the specificity of biological response following the activation of
receptors for these two growth factors. Taken together, the results
raise the possibility that NGF-stimulated association of SH2-B
with
TrkA and/or the subsequent tyrosyl phosphorylation of SH2-B
initiates one or more novel signaling pathways that may be specific for
NGF and required for NGF-induced neuronal differentiation.
Is Required for NGF-induced Neurite Outgrowth of PC12
Cells--
To examine the function of SH2-B
in cellular responses
to NGF, we generated PC12 cells that stably overexpress GFP, GFP-tagged SH2-B
, or GFP-SH2-B
(R555E) that lacks a functional SH2 domain. To
eliminate differences due to clonal variation of PC12 cells, we pooled
all G418-resistant clones. The GFP tag enabled us to use flow cytometry
to isolate cells expressing high levels of SH2-B
. GFP-SH2-B
and
GFP-SH2-B
(R555E) are present at ~60 and 20 times, respectively,
the level of endogenous SH2-B
(Fig. 3a).
, like endogenous SH2-B
, is
tyrosyl phosphorylated in response to NGF. PC12 cells overexpressing GFP-SH2-B
(designated as GFP-WT) were stimulated with NGF. Proteins in the cell lysates were immunoprecipitated with
SH2-B and then immunoblotted with
PY. Significant tyrosyl phosphorylation of GFP-SH2-B
was detected (Fig. 3b, upper panel).
Reprobing the same blot with
SH2-B showed that NGF also stimulated a
shift in the mobility of GFP-SH2-B
(Fig. 3b, lower
panel).4 In contrast,
NGF did not stimulate tyrosyl phosphorylation of GFP-SH2-B
(R555E)
(Fig. 3b, upper panel) as anticipated. These results suggest that like endogenous SH2-B
, GFP-tagged SH2-B
binds to activated and tyrosyl phosphorylated TrkA and is tyrosyl phosphorylated by TrkA in response to NGF.
in NGF-induced neuronal differentiation
of PC12 cells, we examined whether SH2-B
is required for NGF-induced
neurite outgrowth, a hallmark of neuronal differentiation. In the
presence of NGF, cells overexpressing GFP alone developed neurite
outgrowths in a manner similar to parental PC12 cells (Fig.
4, a and b, and
data not shown). Strikingly, overexpression of GFP-SH2-B
(R555E)
blocked neurite outgrowth induced by NGF (Fig. 4, c and
d). Even treatment of cells with a supramaximal concentration of NGF (100 ng/ml) for 8 days or longer did not induce
neurite outgrowth (data not shown). This observation suggests that
SH2-B
is required for NGF-induced morphological differentiation of
PC12 cells.
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Fig. 4.
Requirement for SH2-B
for NGF-induced neurite outgrowth. PC12 cells overexpressing
GFP (a, b, e, and f),
GFP-RE (c and d), or GFP-WT (g and
h) were plated on 60-mm culture dishes coated with collagen
and cultured for 5 days with 100 ng/ml NGF (b and
d) or 4 days with 25 ng/ml NGF (f and
h). Cells were visualized using phase contrast microscopy.
The scale bar in panel h represents 20 µm.
playing an essential role in NGF-induced
differentiation of PC12 cells, overexpression of GFP-SH2-B
enhanced
NGF-induced neurite outgrowth when NGF was tested at a submaximal
concentration. For example, 25 ng/ml NGF for 4 days promoted
differentiation of the majority of cells expressing GFP-SH2-B
, whereas only a very few of the cells expressing GFP alone
differentiated in response to NGF (Fig. 4, e-h). Neurite
outgrowth (twice cell diameter) was observed in about 60% of cells
expressing GFP-SH2-B
after 2 days of incubation with 15 ng/ml NGF,
whereas fewer than 1% of the cells expressing GFP had neurites. By day
5, approximately 95% of the cells expressing GFP-SH2-B
had neurite
outgrowth, whereas only about 40% of cells expressing GFP had neurite
outgrowth. In the absence of NGF, overexpression of GFP-SH2-B
did
not induce neuronal differentiation (Fig. 4g), suggesting
that SH2-B
must be activated in some way by NGF, presumably by being
phosphorylated, to mediate neurite outgrowth.
and
Activation of ERK1/2 Are Not Affected by Overexpression of
SH2-B
(R555E)--
To explore the mechanism of action of SH2-B
,
we asked whether overexpression of SH2-B
or SH2-B
(R555E) affects
tyrosyl phosphorylation of TrkA, Shc, or PLC
, all of which are known
to play important roles in NGF signaling (20). Cells stably
overexpressing GFP, GFP-SH2-B
, or GFP-SH2-B
(R555E) were treated
with NGF, and proteins in the cell lysates were immunoprecipitated with
the antibodies against TrkA, Shc, or PLC
and immunoblotted with
anti-phosphotyrosine antibody. NGF induced tyrosyl phosphorylation of
TrkA (Fig. 5a), PLC
(Fig.
5c), and all three isoforms of Shc (Fig. 5b).
Levels of induction were unchanged in cells overexpressing SH2-B
or SH2-B
(R555E) (Fig. 5), suggesting that TrkA activation and signaling events immediately downstream of TrkA are not compromised by
overexpression of SH2-B
(R555E).
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Fig. 5.
Effect of overexpression of GFP-RE and GFP-WT
on NGF-induced tyrosyl phosphorylation of TrkA, Shc and
PLC . Cells overexpressing GFP alone
(GFP), GFP-SH2-B
(GFP-WT), or
GFP-SH2-B
(R555E) (GFP-RE) were stimulated with 100 ng/ml
NGF for 10 min. a, TrkA was immunoprecipitated
(IP) with
TrkA (C-14 from Santa Cruz, 1:100 dilution) and
immunoblotted (IB) with
PY (upper panel). The
same blot was reprobed with
TrkA (C-14, 1:500 dilution).
b, Shc was immunoprecipitated with
Shc (1:100 dilution)
and immunoblotted with
PY (upper panel). The same blot
was reprobed with
Shc (lower panel, 1:250 dilution).
c, PLC
was immunoprecipitated with
PLC
(1:100
dilution) and immunoblotted with
PY (upper panel). The
same blot was stripped and immunoblotted with
PLC
(lower
panel, 1:2000 dilution).
or SH2-B
(R555E) (Fig.
6), consistent with the previous observation that sustained activation of ERK1/2 induced by NGF is not
sufficient for neuronal differentiation of PC12 cells (21, 22).
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Fig. 6.
Effect of overexpression of GFP-RE and GFP-WT
on the activation of ERK1/2 by NGF. Cells overexpressing GFP alone
(GFP), GFP-SH2-B (GFP-WT), or
GFP-SH2-B
(R555E) (GFP-RE) were stimulated with 100 ng/ml
NGF for the indicated times. An equal amount of protein in the lysates
was immunoblotted (IB) with anti-active MAPK antibody
(1:20,000 dilution). The same blots were stripped and reprobed with
ERK2 that recognizes both ERK1 and ERK2 (1:1000 dilution).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
with activated TrkA and stimulates tyrosyl phosphorylation of
SH2-B
. Mutating the SH2 domain of SH2-B
abolishes its ability to
bind activated TrkA and to be phosphorylated in response to NGF,
suggesting that SH2-B
binds via its SH2 domain to tyrosyl phosphorylated TrkA and that this interaction is required for subsequent phosphorylation of SH2-B
. Overexpression of a GFP-tagged SH2-B
(R555E) lacking a functional SH2 domain blocks NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of GFP-tagged wild type SH2-B
enhances NGF-induced neurite outgrowth. SH2-B
represents only the second signaling molecule, the first being Shc,
known to bind to activated TrkA, to be phosphorylated, and to be
required for NGF-induced neuronal differentiation.
has been shown to associate with receptors for insulin,
insulin-like growth factor-1 (14), platelet-derived growth factor (15),
and EGF (data not shown), in addition to TrkA. Of the hormones and
growth factors that bind to these receptors, only NGF and
platelet-derived growth factor have been shown to stimulate tyrosyl
phosphorylation of SH2-B
(14, 15). In PC12 cells, both NGF and
platelet-derived growth factor (23) promote neurite outgrowth and
neuronal differentiation. In contrast, EGF, insulin, and insulin-like
growth factor-1 stimulate cell proliferation (11, 24). This correlation
raises the possibility that ligand-dependent tyrosyl
phosphorylation of SH2-B
plays a critical role in promoting neurite
outgrowth of PC12 cells. Consistent with this idea, NGF stimulates
robust tyrosyl phosphorylation of GFP-SH2-B
(Fig. 3b),
whose expression enhances NGF-induced neurite outgrowth. However, NGF
is unable to stimulate the tyrosyl phosphorylation of
GFP-SH2-B
(R555E) (Fig. 3b), which acts as a dominant
negative SH2-B
to block NGF-induced neurite outgrowth. Because
SH2-B
but not SH2-B
(R555E) binds to TrkA (Fig. 1a), it
is likely that binding of SH2-B
to TrkA is required for its
phosphorylation and that SH2-B
is tyrosyl phosphorylated directly by
activated TrkA.
(R555E) neither binds to TrkA nor interferes with
phosphorylation of endogenous SH2-B
(data not shown) in response to
NGF. Therefore, it seems likely that SH2-B
(R555E) functions as a
dominant negative mutant to interfere with the action of endogenous
SH2-B
by competing with endogenous SH2-B
for downstream effector(s) and sequestering these putative effector(s) in an inactive
state. Phosphorylation of SH2-B
does not appear to be required for
sequestration of effectors, because GFP-SH2-B
(R555E) is not
significantly phosphorylated in response to NGF (Fig. 2b). However, phosphorylation of SH2-B
may be important for activation of
downstream effector(s). Alternatively, the downstream effectors of
SH2-B
may be activated when recruited to TrkA-containing complexes by the interaction of SH2-B
with TrkA.
(R555E) nor
GFP-SH2-B
affect NGF-induced tyrosyl phosphorylation of TrkA, PLC
, three isoforms of Shc and activation of ERK1/2, indicating that
the signaling events immediately downstream of TrkA and the Shc/Ras/MEK/ERKs cascade are not compromised by overexpression of wild
type or dominant negative mutant SH2-B
. This observation demonstrates that the dominant negative effect of SH2-B
(R555E) on
NGF-induced neurite outgrowth is not secondary to a defect in the
activation of TrkA, the ability of TrkA to tyrosyl phosphorylate its
substrates, or the activation of the Shc/Ras/MEK/ERK cascade by NGF.
Thus, SH2-B
is likely to initiate a novel pathway required for
NGF-induced neurite outgrowth.
binds
via its SH2 domain to activated TrkA and is tyrosyl phosphorylated in
response to NGF. We also demonstrate that overexpression of SH2-B
enhances NGF-induced neuronal differentiation, whereas overexpression
of a dominant negative SH2-B
blocks that differentiation. These
findings provide the first insight into the cellular function of SH2-B.
They also suggest that SH2-B
may be one of the hypothesized "missing links" parallel to or downstream of ERKs that are thought to be required for NGF-induced differentiation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Louis F. Reichardt for providing us with antibody to TrkA. We thank Drs. L. S. Argetsinger, J. A. VanderKuur, and M. Stofega for discussions, X. Wang for technical assistance, Drs. B. Margolis and D. Meyer for the gift of PC12 cells, and Dr. K. S. O'Shea for help with phase contrast microscopy.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK 34171 (to C. C.-S.) and National Research Service Award F32-DK-09756 (to J. H.). Oligonucleotides were synthesized by the Biomedical Research Core Facilities, University of Michigan, work that was supported in part by National Institutes of Health Grant P60-DK-20572 to the Cancer Center, Michigan Diabetic Research and Training Center and National Institutes of Health Grant P60-AR20557 to University of Michigan Multipurpose Arthritis and Musculoskeletal Diseases Center. This work was also supported in part by National Institutes of Health Grants AR20557 and CA46592 to the University of Michigan Core Flow Cytometry Facility.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.
Recipient of a Predoctoral Fellowship from the Rackham School of
Graduate Studies, University of Michigan.
§ To whom correspondence should be addressed: Dept. of Physiology, University of Michigan Medical School, Ann Arbor, MI 48109-0622. Tel.: 734-763-2561; Fax: 734-647-9523; E-mail: cartersu{at}umich.edu.
2 K. Nelms, personal communication.
3 Rui, L, J. Herrington and C. Carter-Su, manuscript in preparation.
4
The apparent difference in the amount of
GFP-SH2-B in control and NGF-treated cells in Fig. 3b
(lower panel) is an artifact of this mobility shift and the
large amount of GFP-SH2-B
. Experiments in which a smaller amount of
GFP-SH2-B
was electrophoresed revealed similar amounts of
GFP-SH2-B
from control and NGF-stimulated cells.
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ABBREVIATIONS |
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The abbreviations used are:
NGF, nerve growth
factor;
EGF, epidermal growth factor;
SH, Src homology;
PLC, phospholipase C
;
GST, glutathione S-transferase;
GFP, green fluorescent protein;
MAPK, mitogen-activated protein
kinase.
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REFERENCES |
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