From the
Shc is an adaptor protein that contains two
phosphotyrosine-binding domains, a Src homology 2 (SH2) domain and the
newly described phosphotyrosine interaction (PI) domain. Shc interacts
with several tyrosine-phosphorylated proteins and is itself
tyrosine-phosphorylated in cells stimulated with a variety of growth
factors and cytokines. Upon phosphorylation, Shc binds to the
Grb2
Growth factors play an important role in controlling cell growth
and differentiation. Growth factors bind and activate transmembrane
receptors with intrinsic tyrosine kinase activity leading to the
increased tyrosine phosphorylation of many cellular
proteins(1) . Phosphorylated proteins in turn become targets for
a variety of signaling proteins containing SH2(
In the case of other growth factor receptors, such as NGF
receptor (TrkA) or fibroblast growth factor receptor, the Grb2
We have recently identified a
novel domain in Shc, encompassing amino acids 46-209 of the p52
Shc protein, and shown that it binds to activated growth factor
receptors(19) . A similar region of Shc has been described and
demonstrated to bind an unknown 145-kDa tyrosine-phosphorylated
protein(20) . We have called this region the phosphotyrosine
interaction domain (PI domain). We have also identified PI domains in
several other proteins(21) . The nature of the PI domain binding
to phosphotyrosine is not clear at present. We sought to delineate the
role of the Shc SH2 or PI domains in the NGF receptor signaling
pathway, where binding of Shc to the NGF receptor has an important
biological role. Here we report that the PI domain of Shc specifically
binds to the activated NGF receptor, interacting with phosphotyrosine
490 and surrounding amino acids. We also show that the SH2 domain does
not bind to the activated NGF receptor, but interacts with an as yet
unidentified 115-kDa tyrosine-phosphorylated protein. The data
presented here demonstrate that in PC12 cells Shc can interact with
different tyrosine-phosphorylated proteins through its two
phosphotyrosine binding domains.
To study the interaction of Shc with the activated NGF
receptor (TrkA), we used parental PC12 cells and PC12 cells
overexpressing approximately 1.3
To
study the binding site of the Shc PI domain on the NGF receptors, TrkA,
PDGFR-TrkA chimera (PT-R), or PDGFR-TrkA-Y490F mutant (PT-Y490F)
receptors were immunoprecipitated, in vitro autophosphorylated, and incubated with Shc PI-GST protein. The
amounts of Shc-GST fusion protein bound to autophosphorylated receptors
were detected by immunoblotting with anti-GST antibodies. TrkA and PT
receptors bound equal amount of Shc PI domain, while PT-Y490F did not
bind any detectable amount, although the phosphorylation level of all
the receptors was very similar (Fig. 1d). In the
presence of a synthetic phosphopeptide corresponding to Tyr-490 of the
TrkA receptor, the binding of the Shc PI domain to the PT receptor was
completely abolished (Fig. 1d). These results suggested
a direct interaction between the Shc PI domain and Tyr-490 on the TrkA
receptor. In the same assay, Shc SH2 domain did not bind to the
activated Trk receptor (Fig. 1d).
To further analyze
the nature of this interaction, we studied the ability of synthetic
phosphopeptides () to compete for the binding of the PI
domain to the NGF receptor. A peptide containing the Shc binding site
of TrkA receptor (TrkA 490-10Y7) was able to compete the binding
of PI domain to the activated TrkA receptors (Figs. 1d and
2a). In addition, two peptides corresponding to the
autophosphorylation sites on the EGFR were tested. A peptide
corresponding to Tyr-1148 (E-pY1148-7Y4), but not peptide
E-pY1068-4Y9, efficiently competed the binding of the PI domain
to the NGF receptor. Phosphotyrosine alone (30 mM) was only
able to partially inhibit the binding of the PI domain (Fig. 2a). Both peptides, TrkA 490-10Y7 and
E-pY1148-7Y4, have the NPXpY motif, suggesting that
various peptides with NPXpY motifs are able to compete binding
of the Shc PI domain to the NGF receptor. To delineate the contribution
of several conserved amino acids amino-terminal of phosphotyrosine, we
used mutant pY1148 phosphopeptides and tested their ability to compete
the binding of the PI domain to the NGF receptor ().
Changing the asparagine to alanine (E-pY1148-7Y4 NA) and leucine
to glycine (E-pY1148-7Y4 LG) severely impaired ability of the
peptides to compete for binding, whereas alteration of the proline to
alanine (E-pY1148-7Y4 PA) had only a minor effect (Fig. 2b). Unphosphorylated E-Y1148-7Y4 peptide
was unable to compete the binding of the PI domain to the NGF receptor (Fig. 2b). In addition, a peptide encompassing only four
amino acids amino-terminal to phosphotyrosine E-pY1148-4Y10 was
not able to compete the binding of the PI domain, confirming that other
residues amino-terminal to NPXpY can play an important role
(data not shown).
We have analyzed the role of the PI domain or the SH2 domain
of Shc in binding to the activated NGF receptor (TrkA) in PC12 cells.
Using the phosphopeptide competition assay, we have demonstrated that
the Shc PI domain, not the SH2 domain, binds to an NPXpY motif
on the NGF receptor. We used two different NPXpY-containing
peptides, one corresponding to tyrosine 490 in TrkA receptor and a
second one corresponding to tyrosine 1148 in EGFR. Both peptides were
equally efficient in competing the PI domain binding to the NGF
receptor. However, phosphopeptides that contained only four amino acids
amino-terminal of tyrosine could not compete. This indicated that other
amino acids amino-terminal of NPXpY may have an important
role. The alignment of Shc binding sites have revealed the conserved
hydrophobic residues at the -5 position, asparagine at -3,
and proline at -2 to the phosphotyrosine (). In
addition, this is supported by studies of Campbell et
al.(18) , who identified the conserved hydrophobic residue
in the -5 position of Shc binding sites on middle T antigen and
some receptor tyrosine kinases. By changing specific residues
amino-terminal of phosphotyrosine in the EGFR Y1148 peptide, we have
delineated their contribution in mediating binding to the Shc PI
domain. The mutation of asparagine to alanine or leucine to glycine had
a very strong effect on the ability of peptides to compete for the PI
domain binding, whereas mutation of proline to alanine had a weaker
effect. This is consistent with our results showing that the the PI
domain of Shc binds to LXNPXpY motif surrounding the
tyrosine 1148 on EGFR.
In contrast, the SH2 domain of
Shc did not bind to the activated NGF receptor in any of our assays,
suggesting that the primary interaction domain of Shc with the NGF
receptor is the PI domain. Nonetheless, studies have indicated that the
specificity of Shc binding to some tyrosine-phosphorylated proteins is
determined by the SH2 domain(9, 10) . In the case of
EGFR, Shc SH2 domain directly binds to pY1173 and the PI domain binds
to pY1148. However, in living cells, Shc binding to the EGFR is
probably mediated by the cooperative binding of both the PI and the SH2
domains to two different tyrosine autophosphorylation sites.
It has been demonstrated that the
association of Shc with the activated NGF receptor is required for
neuronal differentiation of PC12 cells and contributes to the
activation of the Ras-MAPK pathway(15, 16) . The data
presented here suggest a model for Shc interactions with the NGF
receptor (Fig. 4). Our data indicate that the PI domain mediates
Shc interaction with the NGF receptor by binding to the
IXNPXpY motif present at tyrosine 490. This is in
agreement with data indicating that mutation of Tyr-490 to
phenylalanine impairs the ability of the NGF receptor to bind and
phosphorylate Shc(14, 15, 16) . This Y490F
mutation also leads to defects in NGF-induced association of Grb2 with
Shc and impairs neuronal differentiation of PC12
cells(15, 16) . Thus we propose that the binding of Shc
to Tyr-490 via its PI domain leads to Shc phosphorylation and the
binding of the Grb2
We thank Kiki Nelson for phosphopeptide synthesis, Ron
Beavis for mass spectroscopy analysis of the peptide, Mikhail L.
Gishicky for the expression plasmids containing Shc-HA-tagged
construct, and Valsan Mandiyan for the Shc 1-209 construct in
pGEX-2T.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Sos complex leading to the activation of the Ras signaling
pathway. Mutational analysis of the nerve growth factor (NGF) receptor
(TrkA) suggested that the binding of Shc to the activated receptor is
required for NGF-induced neuronal differentiation of PC12 cells. Here
we report that the PI domain of Shc directly binds to tyrosine 490 on
the autophosphorylated NGF receptor. The PI domain specifically
recognizes an I/LXNPXpY motif (where p indicates
phosphorylation) as determined by phosphopeptide competition assay. In
addition, the PI domain is able to efficiently compete for binding of
full-length Shc proteins to the NGF receptor. In PC12 cells, the Shc
SH2 domain interacts with an unidentified tyrosine-phosphorylated
protein of 115 kDa but not with the activated NGF receptor. The ability
of Shc to interact with different tyrosine-phosphorylated proteins via
its PI and SH2 domains may allow Shc to play a unique role in tyrosine
kinase signal transduction pathways.
)
domains or other domains able to interact with phosphotyrosine
residues(2, 3, 4) . This creates a network of
specific protein-protein interactions involved in the signal
transduction from cell surface receptors to the nucleus. For example,
Grb2 is an adaptor protein that binds to activated growth factor
receptors, such as EGFR, via its SH2 domain and is bound to Sos, a Ras
guanine nucleotide releasing factor, via its two SH3 domains. It is
thought that the translocation of Grb2
Sos complex to the membrane
regulates the conversion of Ras from an inactive GDP-bound form to an
active GTP-bound form (reviewed in Ref. 5). Activated Ras then
initiates a mitogen-activated protein kinase (MAPK) cascade that relays
signals from the cell membrane to the nucleus (Ref. 6, and references
therein).
Sos
complex does not associate directly with the activated receptors, but
binds to other tyrosine-phosphorylated proteins, such as Shc. Shc is an
SH2 domain-containing adaptor protein that can bind to several growth
factor receptors, including NGF receptor and EGFR, and is also
tyrosine-phosphorylated in cells stimulated with a variety of growth
factors and
cytokines(7, 8, 9, 10, 11, 12, 13, 14) .
Shc binding to tyrosine 490 of the NGF receptor has been shown to be
required for the activation of the Ras signaling pathway and neuronal
differentiation in PC12 cells(15, 16) . Shc binds to an
NPXpY motif on several tyrosine-phosphorylated
proteins(10, 11, 13, 14, 15, 16, 17, 18) .
This is an unusual motif for SH2 domain binding, since SH2 domains
binding specificity is dictated by residues carboxyl-terminal to the
phosphotyrosine moiety(2, 3, 4) . This suggests
that another domain, not the SH2 domain, might be responsible for Shc
binding to the NPXpY motif.
Cell Lines, Antibodies, Immunoprecipitation, and
Immunoblotting
A G418-resistant PC12 cell line expressing
approximately 1.3 10
TrkA receptors (PC12-Trk) was
established using high titer retroviruses generated in GPE-86 producer
cell lines, as described previously(15) . GPE-86 stable cell
lines expressing various chimeric receptors containing the
extracellular domain of the PDGF receptor and the intracellular part of
the TrkA receptor (PT) were grown in Dulbecco's modified
Eagle's medium (DMEM) containing 10% fetal calf serum
(FCS)(15) . PC12-Trk cells were maintained in Dulbecco's
modified Eagle's medium (DMEM) containing 7% FCS and 7% horse
serum. Prior to stimulation, cells were starved for 36 h in 0.1% horse
serum, 0.1% FCS, DMEM. Cells were then stimulated with 100 ng/ml NGF
for 5 min at 37 °C, washed with ice-cold PBS, and lysed in lysis
buffer (50 mM HEPES (pH 7.5), 10% glycerol, 150 mM NaCl, 1% Triton X-100, 1.5 mM MgCl
, 1 mM EGTA, 25 mM NaF, 50 µM ZnCl
, 500
µM sodium orthovanadate, 1 mM phenylmethylsulfonylflouride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin). Lysate protein content was normalized using the Bio-Rad
protein assay(22) . Cell lysis, immunoprecipitation, and
immunoblotting with HRP-protein A/chemiluminescence method were
performed as described previously(23) . The following antibodies
were used for immunoprecipitation: polyclonal rabbit anti-Shc and
anti-Grb2 antibodies that were covalently cross-linked to protein
A-agarose(9) , and affinity-purified polyclonal anti-Trk
antibodies against a COOH-terminal peptide of Trk receptor (Oncogene
Science). Polyclonal anti-phosphotyrosine, anti-GST(9) ,
anti-Grb2, and anti-Shc antibodies in Tris-buffered saline, 5% bovine
serum albumin were used for immunoblotting, as described
previously(23) .
Peptide Synthesis
A Fmoc-based strategy for
peptide synthesis was used in conjunction with standard side
chain-protecting groups as described previously(24) .
Fmoc-Tyr(POMe
)-OH (Bachem Bioscience) was used
for incorporation of phosphotyrosine. Peptides were purified by ether
precipitation, and preparative reverse-phase high performance liquid
chromatography. Analytical high performance liquid chromatography
demonstrated that the products were purified to homogeneity; mass
spectroscopy was used to confirm the accuracy of synthesis. Several
phosphopeptides corresponding to the autophosphorylation sites of Trk
receptor or EGFR were synthesized ().
Fusion Proteins and Binding Assay
GST fusion
proteins of Shc used in this study have been described
previously(19) . Proteins were either used for binding assays on
glutathione-agarose beads or eluted with 15 mM glutathione in
buffer A plus 10% glycerol (pH 8.0), concentrated and stored at
-70 °C in the presence of 20% glycerol, and used for peptide
competition assay(9) . For binding assays, 3 µg of Shc
PI-GST proteins or 7 µg of Shc SH2-GST were bound to
glutathione-agarose beads. The beads were mixed with 2 mg of lysates
from unstimulated or NGF-stimulated PC12-Trk cells for 1 h at 4 °C,
washed four times with lysis buffer, and boiled in 20 µl of 2
sample buffer(23) . Bound proteins were separated on 9%
SDS-polyacrylamide gel electrophoresis gels, transferred to
nitrocellulose, immunoblotted with appropriate antibodies, and detected
using HRP-protein A/chemiluminescence method.
Phosphopeptide and Shc PI Domain Competition
Assays
TrkA receptors were immunoprecipitated from lysates of
starved PC12-Trk cells or GPE-86 cells expressing PDGFR-Trk (PT)
chimeric receptors, washed four times with lysis buffer, and subjected
to in vitro autophosphorylation in the presence of 0.2 mM ATP and 10 mM MnCl for 5 min at room
temperature. After washing, the autophosphorylated receptors were
divided into aliquots and incubated with either Shc PI-GST or
Shc-SH2-GST (human, residues 366-473) in the absence or presence
of 2.5 µM of the various phosphopeptides in a volume of
100 µl for 1 h at 4 °C. After three washes with lysis buffer
the samples were run on SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose and then immunoblotted using
anti-phosphotyrosine or anti-GST antibodies and detected using
HRP-protein A/chemiluminescence as described above. To study the
ability of Shc PI-GST to compete full-length Shc binding to activated
TrkA receptor, we used 293T cells for transient expression of
full-length Shc with an HA tag subcloned in pCGN mammalian expression
vector (kindly provided by Mikhail Gishicky). Eight micrograms of
pCGN-p52 Shc-HA tag were transfected into 293T cells using the calcium
phosphate precipitation method(25) . Trk receptor was
immunoprecipitated from starved PC12-Trk cells, and after in vitro autophosphorylation, 120 µg of 293T cell lysate or 293T cell
lysate expressing p52 Shc-HA tag protein were added to the receptors in
absence or presence of increasing concentration of Shc PI-GST.
Incubation and analysis was as described above using
anti-phosphotyrosine, anti-Shc, or anti GST antibodies.
10
Trk
receptors/cell (PC12-Trk). Unstimulated or NGF-stimulated cells were
lysed, immunoprecipitated with anti-Shc, anti-Grb2, and anti-Trk
antibodies, and subsequently immunoblotted with the indicated
antibodies. In PC12-Trk cells Shc was found in a complex with two
tyrosine-phosphorylated proteins: p140, corresponding to the activated
TrkA receptor, and an as yet unidentified protein of approximately 115
kDa, called p115 (Fig. 1a). In addition,
tyrosine-phosphorylated Shc and p115 proteins were coimmunoprecipitated
with Grb2 in a ligand-dependent manner (Fig. 1a). In the
same cell line, anti-TrkA antibodies coimmunoprecipitated
tyrosine-phosphorylated p46 and p52 Shc proteins, a previously
described tyrosine-phosphorylated protein of 38 kDa, and an unknown
tyrosine-phosphorylated protein of 110-115 kDa that migrates in
SDS gel close to the lower molecular mass form of TrkA (Fig. 1a and Ref. 26). The presence of the lower
molecular mass form of TrkA in anti-Trk immunoprecipitates makes it
difficult to determine if the p115 protein found in anti-Shc and
anti-Grb2 immunoprecipitates is also present in anti-Trk
immunoprecipitates. In these coimmunoprecipitation studies, it was
difficult to simultaneously demonstrate complex formation of all four
proteins: Trk, Shc, Grb2, and tyrosine-phosphorylated p115. This may be
due to the transient nature of protein-protein interactions involved in
complex formation and because of the limited sensitivity of these
assays. In parental PC12 cells, Shc is also found in a complex with
Grb2 and tyrosine-phosphorylated p115 (Fig. 1a). The
coimmunoprecipitation of TrkA receptors with Shc in parental PC12 cells
was below the detection limit in our assay (Fig. 1a).
Taken together, these results suggest that Shc can form a complex with
the activated NGF receptor, tyrosine-phosphorylated p115, and Grb2 upon
NGF stimulation in PC12-Trk cells.
Figure 1:
Intact Shc
PI domain binds to the activated NGF receptor but not to mutant
receptor TrkA Y490F. Parental PC12 cells (PC12) or PC12 cells
overexpressing NGF receptors (PC12-Trk) cells were treated with
(+) or without (-) 100 ng/ml NGF for 5 min, lysed, and
subjected to immunoprecipitation with anti-Shc, anti-Grb2, and anti-Trk
antibodies (a) or and precipitated with indicated GST fusion
proteins or GST alone (b and c). Western blot
analysis using indicated antibodies was performed as described under
``Materials and Methods.'' d, TrkA
receptor, PDGFR-TrkA receptor (PT-R), or PDGFR-TrkA Y490F mutant
receptor (PT-Y490F) were immunoprecipitated with anti-Trk antibodies
from starved PC12-Trk cells or GPE-86 cells expressing various
receptors, autophosphorylated in vitro, and then incubated
with purified Shc PI-GST (0.5 µM) or Shc SH2-GST (1
µM) proteins. In addition, 10 µM peptide
corresponding to the Shc binding site on the TrkA receptor (TrkA
490-10Y7) was added to the autophosphorylated NGF receptor and
Shc PI-GST.
To determine which domain of Shc
interacts with the activated NGF receptor and tyrosine-phosphorylated
p115, we used GST fusion proteins, encoding various constructs of the
Shc PI domain or the Shc SH2 domain, to precipitate proteins from
unstimulated or NGF-stimulated lysates of PC12-Trk cells.
Anti-phosphotyrosine blotting revealed that the Shc PI domain
specifically precipitated activated TrkA receptor and the Shc SH2
domain precipitated tyrosine-phosphorylated p115 (Fig. 1b). None of the proteins was precipitated with
GST alone (Fig. 1b). In these studies, we were not able
to use full-length GST-Shc protein since it was rapidly degraded by
proteolysis during bacterial expression. The binding of the Shc PI
domain to the tyrosine-phosphorylated TrkA receptor required the intact
PI domain, since Shc PI-GSTs with amino-terminal deletion of 85 amino
acids or an internal deletion of amino acids 107-116 (19) were unable to precipitate activated TrkA receptors in the
same binding assay (Fig. 1c). Anti-Trk immunoblotting
showed that this interaction is dependent on activation and tyrosine
phosphorylation of TrkA receptors since no TrkA receptors were
precipitated from non-stimulated cell lysates (data not shown).
Figure 2:
Inhibition of Shc PI domain binding to the
NGF receptors with synthetic phosphopeptides. NGF receptor was
immunoprecipitated from starved PC12-Trk cells, autophosphorylated in vitro, and then incubated without (-) or with Shc
PI-GST (0.2 µM) in the absence or presence of 2.5
µM of the indicated phosphopeptides or 30 mM phosphotyrosine, respectively (a), and with Shc PI-GST
(0.2 µM) in the absence or presence of 2.5 µM of the indicated peptides, respectively (b).
We next compared the ability of Shc PI-GST to
compete binding of full-length Shc to the TrkA receptor. Equal amounts
of 293T cells lysate or 293T cells lysate expressing p52-Shc-HA tag
were incubated with the autophosphorylated TrkA receptors in the
absence or presence of increasing concentration of the PI domain as
indicated. The PI domain competed the binding of full-length Shc to the
NGF receptor in a concentration-dependent manner (Fig. 3). This
indicates that the PI domain of Shc is sufficient to mediate
interaction with the activated Trk receptors in the context of
full-length Shc molecules.
Figure 3:
Inhibition of Shc binding to the NGF
receptor by the PI domain. NGF receptor was immunoprecipitated from
starved PC12-Trk cells, autophosphorylated in vitro, and then
incubated with 293 cell lysates (-) or 293 cell lysates
expressing p52-Shc-HA tag protein in the absence or presence of
increasing concentrations of Shc PI-GST, as
indicated.
(
)It seems very likely
that the interaction of Shc with other tyrosine-phosphorylated proteins
containing the I/L/FNPXpY motif () will be
mediated via the PI domain of Shc.
In NGF-stimulated PC12 cells, it appears that the SH2 domain does
not cooperate with the PI domain but rather binds to an unknown
tyrosine-phosphorylated protein of 115 kDa. Similarly, two other
tyrosine-phosphorylated proteins of 120 and 180 kDa were identified as
the direct targets of the SH2 domain of Shc in fibroblast growth
factor- and PDGF-stimulated cells(20) . Whether the 115-kDa
protein we observe is similar to the 120-kDa protein seen in these
cells remains to be determined.
Sos complex. This complex, localized at the
plasma membrane, activates Ras, eventually leading to MAP kinase
activation. Further complexity is added, however, by the binding of the
Shc SH2 domain to the highly tyrosine-phosphorylated 115-kDa protein.
We have observed Shc interactions with tyrosine-phosphorylated p115 as
early as 3 min and continuing for at least 20 min after NGF stimulation
(data not shown). We have detected p115 in Grb2 immunoprecipitates,
suggesting a complex is formed between Shc, Grb2, and p115. However, we
have not been able to find TrkA in this complex, perhaps due to the
lack of sensitivity of the immunoprecipitation analysis and the
transient nature of the complex. It is also possible that Shc cannot
bind to Trk and p115 at the same time. Further work will be necessary
to address this issue.
Figure 4:
Schematic diagram of the Shc interacting
proteins in NGF-stimulated PC12 cells. The model depicts the diversity
of the Shc interactions with a variety of proteins. The PI domain of
Shc binds to the activated NGF receptor recognizing phosphotyrosine 490
in the context of IXNPXpY motif. Once phosphorylated
on tyrosine 317, Shc serves as a binding site for the SH2 domain of
Grb2 leading to Ras activation. The Shc SH2 domain can interact with
other tyrosine-phosphorylated proteins such as p115. This suggests a
role for Shc in mediating signals responsible for neuronal
differentiation of PC12 cells more complex than that previously
described. Whether Shc can simultaneously interact with both NGF
receptor and p115, as illustrated here, remains to be
determined.
It is possible that SH2 domain binding of Shc
to p115 may provide the mechanism for signal amplification and
diversification during neuronal differentiation. Particularly,
different ways of Shc binding to the NGF or EGF receptors may reflect
their ability to activate the Ras-MAPK pathway with different kinetics
in PC12 cells. It has been shown that the activation of NGF receptor
leads to the prolonged activation of Ras-MAPK pathway and neuronal
differentiation of PC12 cells(27) . In contrast, stimulation of
EGFR in PC12 cells leads to a short and transient MAPK activation,
which is associated with the cell proliferation(28) . Although,
we still do not fully understand the role of Shc in mediating neuronal
differentiation of PC12 cells, the ability of two
phosphotyrosine-binding domains of Shc to select different targets
suggests that it may be more complex and diverse than initially
envisioned.
Table: Sequences of phosphopeptides used in a
competition assay
Table: Alignment of the Shc binding sites with the
NPXY motifs
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.