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(Received for publication, June 5,
1995; and in revised form, January 19, 1996) From the
The cellular homologs of the v-Crk oncogene product consist
primarily of Src homology region 2 (SH2)
Many receptors for growth and differentiation factors possess
intrinsic protein-tyrosine kinase activity, which is increased
following ligand binding, resulting in tyrosine autophosphorylation (1) . These tyrosine-phosphorylated proteins serve as high
affinity binding sites for Src homology 2 (SH2) domain-containing
signaling proteins(2, 3, 4) . SH2 domains
have been identified in a wide range of proteins. Some of these
proteins are enzymes with defined catalytic
activity(5, 6, 7, 8) , while others
serve as adapters or activators for other proteins. SH2 proteins can
also contain src homology 3 (SH3) domains, regions that bind
to proline-rich sequences in other proteins (9, 10) . The Crk proteins, originally isolated as an oncogene product in an
avian sarcoma virus(11) , belong to the adapter-type
SH2-SH3-containing molecules. Other members of this group include Grb2 (12) and Nck(13) . These bifunctional proteins are
thought to couple tyrosine-phosphorylated receptors or their substrates
via the SH2 domain to downstream effectors via SH3 binding. v-Crk
encodes a viral Gag protein fused to one SH2 and one SH3 domain. Three
cellular homologs of v-Crk have been identified, Crk-I, a 21-kDa
protein with only one SH2 and SH3(14) , Crk-II, a 40/42-kDa
protein consisting of one SH2 and two SH3 domains(15) , and
CrkL, a 36-kDa Crk-like protein with one SH2 and two SH3
domains(16) . Despite their lack of tyrosine kinase activity,
expression of v-Crk or Crk-I but not Crk-II leads to cell
transformation and increased tyrosine
phosphorylation(11, 15, 17) . Furthermore,
v-Crk binds directly to the major tyrosine-phosphorylated proteins in
v-Crk-transformed cells, presumably via its SH2 domain(18) .
These phosphoproteins include paxillin, a focal-adhesion-associated
protein(19) , and the newly identified p130 Although
the mechanism by which neurotrophic factors support the survival and
differentiation of sympathetic neurons remains elusive, tyrosine
phosphorylation is known to play an important role in signal
initiation(27, 28) . We report here that both NGF and
EGF rapidly stimulated the tyrosine phosphorylation of endogenous
CRK-II in PC-12 pheochromocytoma cells and induced its association with
a variety of intracellular signaling proteins.
Denaturing
immunoprecipitation of tyrosine-phosphorylated Crk-II was performed by
first immunoprecipitating Crk-II from untreated or treated cell lysates
with anti-Crk-II antibodies as described above. After the immune
complexes had been washed 3 times with lysis buffer, they were
denatured by boiling in 50 µl of denaturing buffer (1% SDS, 50
mM Tris-HCl, pH 7.7) for 5 min. The agarose beads were
pelleted by centrifugation, and the supernatants were saved. To 25
µl of the supernatants were added 1 ml of the lysis buffer and 2
µg of the anti-phosphotyrosine antibodies. The immune complexes
were mixed with protein G/protein A-agarose, and the proteins were
resolved by SDS-PAGE as described above. Autoradiographs were
quantified by computer-assisted video densitometry using the Bio Image
system (Imaging Systems, Millipore Corp., Ann Arbor, MI).
Figure 1:
NGF and EGF induce the tyrosine
phosphorylation of Crk-II in PC-12 cells. PC-12 cells were untreated (C) or stimulated with NGF (100 nM) or EGF (100
ng/ml) for 1 min at 37 °C. Lysates were prepared and mixed with
anti-Crk-II antibodies. The immunoprecipitates were subjected to
SDS-PAGE and immunoblotted with anti-phosphotyrosine antibodies (anti-pY) 4G10 and PY20 (panel A). The same blot was
stripped and reprobed with anti-Crk-II antibodies (panel B).
Lysates from untreated PC-12 cells (C) or cells stimulated
with NGF (100 nM) or EGF (100 ng/ml) for 1 min at 37 °C
were immunoprecipitated with anti-Crk-II antibodies. The
immunoprecipitates were washed with lysis buffer and denatured by
boiling in 1% SDS as described under ``Experimental
Procedures.'' The eluted materials were diluted and
immunoprecipitated with anti-phosphotyrosine antibodies. The
immunoprecipitates were analyzed by immunoblotting with anti-Crk-II
antibodies (panel C).
The proteins in the anti-Crk-II immunoprecipitates
were subjected to second round of immunoprecipitation under denaturing
conditions with anti-phosphotyrosine antibodies, followed by
immunoblotting with anti-Crk-II antibodies (Fig. 1C). A
single band was recognized by the anti-Crk-II antibodies in the
anti-phosphotyrosine immunoprecipitates. This band appears to comigrate
with the 42-kDa form of Crk-II in the control loading. On the basis of
comparison of the Crk-II signals present in the loading control (Fig. 1C, lane 4, 50% of the material used in
the first round) and the anti-phosphotyrosine immunoprecipitates (lanes 2 and 3), we estimated that a significant
proportion of the Crk-II (about 30-40%) is
tyrosine-phosphorylated. Furthermore, it appears that the more slowly
migrating species of Crk-II, which is phosphorylated on tyrosine (Fig. 1A), preferentially bound to the
anti-phosphotyrosine antibodies. These results further established the
fact that Crk-II undergoes an increase in tyrosine phosphorylation upon
NGF or EGF stimulation.
Figure 2:
NGF and EGF induce the tyrosine
phosphorylation of p130
In normal 3Y1 cells, p130
Figure 3:
NGF and EGF promote increased association
of endogenous Crk with p130
We recently reported that insulin,
NGF, and EGF stimulated the tyrosine phosphorylation of two proteins
with molecular masses between 115 and 125 kDa that specifically
immunoprecipitated with anti-Syp antibodies (36) . However, no
p130 The concentration dependence of the effect
of NGF and EGF on the appearance of the Crk-p130
Figure 4:
Dose dependence of NGF and EGF-induced
binding of p130
Figure 5:
Binding of GST-Crk-SH2 to
tyrosine-phosphorylated proteins from PC-12 cells stimulated with NGF
or EGF. PC-12 cells were stimulated with NGF (100 nM) or EGF
(100 ng/ml) for 1 min at 37 °C or left unstimulated (C).
Cell lysates were incubated with GST-Crk-SH2, GST-Grb2-SH2 fusion
proteins, or GST alone immobilized on glutathione-agarose beads for 90
min at 4 °C. Bound proteins were eluted and separated by SDS-PAGE
followed by immunoblotting with anti-phosphotyrosine antibodies (anti-pY) 4G10 and PY20 (panel A). The positions of
p70, p125-135, EGF receptor (EGFR), and Shc are indicated as
appropriate. Molecular mass markers, indicated at the left,
are given in kDa. The blot from panel A was stripped and
reprobed with anti-p130
To test whether the
125-135-kDa phosphotyrosine-containing protein that bound to
GST-Crk-SH2 is p130 Both NGF and EGF
have been shown to stimulate the tyrosine phosphorylation of paxillin
(data not shown and (26) ). The 70-kDa tyrosine-phosphorylated
protein precipitated with GST-Crk-SH2 was confirmed to be paxillin by
reprobing the blot with anti-paxillin antibodies (Fig. 5C). Paxillin failed to bind to GST alone or to
the SH2 domain of Grb2. Recently, studies indicated that the focal
adhesion kinase (p125
Figure 6:
Phosphopeptide competition of GST-Crk-SH2
binding to p130
The SH2 region of
Grb2 is predicted to bind to a consensus sequence
pYXNY(38) . The high affinity binding site for Grb2 on
the EGF receptor has been mapped to the autophosphorylation site
Tyr Cellular overexpression of the oncogenic form of Crk leads to
cell transformation and elevation of intracellular phosphotyrosine
levels of specific proteins(11, 15, 17) .
Moreover, overexpression of v-Crk in PC-12 cells accelerates their
differentiation in response to both NGF and EGF(26) . Although
this protein apparently lacks catalytic activity, both its transforming
and differentiating functions require intact SH2 and SH3
domains(14, 18, 25) . Crk has been shown to
associate with two guanine nucleotide exchange proteins, Sos and C3G,
via its SH3 domain(22, 23, 24) , suggesting a
role in regulating the Ras signaling pathway. Additionally, Crk
proteins can undergo tyrosine phosphorylation in transformed cells or
in response to growth factors (21, 26, 35, 40) . Although
transfection studies with v-Crk have suggested that this protein plays
a major role in tyrosine kinase-initiated signals, little is known
about the interactions of endogenous Crk family members. We report
here that treatment of PC-12 cells with NGF or EGF causes the rapid
tyrosine phosphorylation of the 42-kDa form of endogenous Crk-II. In
addition to Crk, the NGF- and EGF-dependent tyrosine phosphorylation of
another SH2 and SH3 domain-containing adapter, Nck, has been
reported(41, 42) , although Grb2 is not
tyrosine-phosphorylated after exposure to any growth
factor(12) . The role of the tyrosine phosphorylation of Crk
is not clear, but this modification may modulate interactions of the
Crk-SH3 domain. Alternatively, the tyrosine phosphorylation of Crk may
induce its interaction with other SH2-containing signaling proteins, or
perhaps it can produce an intramolecular interaction. In this regard,
the c-Abl kinase binds to the Crk-SH3 domain and
tyrosine-phosphorylates c-Crk on a single tyrosine, which creates a
binding site for Crk-SH2 domain(21, 43) . In
addition to its tyrosine phosphorylation, Crk is also known to
associate with certain tyrosine-phosphorylated proteins via its SH2
domain(17, 18, 19) . The major
phosphotyrosine-containing protein detected in v-Crk-transformed cells
has recently been cloned(20) . This 130-kDa protein, called
Cas, associates directly with v-Crk in these cells (20) . We
report here that treatment of PC-12 cells with NGF or EGF produced
p130 A low but detectable level of
tyrosine-phosphorylated p130 Following NGF or EGF treatment of PC-12 cells,
GST-Crk-SH2 also binds the tyrosine-phosphorylated paxillin, a focal
adhesion-associated protein. Paxillin becomes phosphorylated on
tyrosine in response to a number of stimuli (26, 46, 47) and is thought to be a substrate
of the focal adhesion kinase p125 Despite the relatively high
binding affinity of Crk to phosphorylated paxillin in vitro,
so far we have detected neither paxillin nor p125 The multitude of
interactions described here indicate that Crk is indeed a versatile
signaling molecule that is likely to participate in a number of
pathways. Its interactions with p130
Volume 271,
Number 13,
Issue of March 29, 1996 pp. 7375-7380
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
in PC-12 Cells (*)
and 3 (SH3)
domains. v-Crk overexpression causes cell transformation and elevation
of tyrosine phosphorylation in fibroblasts and accelerates
differentiation of PC-12 cells in response to nerve growth factor
(NGF). To further explore the role of Crk in NGF-induced PC-12 cell
differentiation, we found that both NGF and epidermal growth factor
stimulate the tyrosine phosphorylation of endogenous Crk II. Moreover,
hormone stimulation enhanced the specific association of Crk proteins
with the tyrosine-phosphorylated p130, the major
phosphotyrosine-containing protein in cells transformed with v-Crk.
This interaction is mediated by the SH2 domain of Crk and can be
inhibited with a phosphopeptide containing the Crk-SH2 binding motif.
Furthermore, the Crk-SH2 domain binds tyrosine-phosphorylated paxillin,
a cytoskeletal protein, following treatment of PC-12 cells with NGF or
epidermal growth factor. These data suggest that Crk functions in a
number of signaling processes in PC-12 cells.
, the
predominate tyrosine-phosphorylated protein detected in
v-Crk-expressing cells(20) . p130
has no
catalytic domain, but it contains a cluster of predicted high affinity
Crk-SH2 binding motifs and forms a stable complex in vivo with
v-Crk. The Crk-SH3 domain has been shown to interact with several
proteins including Abl (21) Sos(22) , and more
recently, C3G(22, 23, 24) . Since Sos and C3G
contain a guanine-nucleotide exchange activity, Crk proteins have been
hypothesized to play a role in the regulation of
p21
-GTP formation. In this regard, Crk mutants
with dysfunctional SH2 or SH3 domains inhibited nerve growth factor
(NGF)-induced differentiation(22, 25) . Moreover,
expression of v-Crk in PC-12 cells enhanced differentiation produced by
both NGF and epidermal growth factor (EGF)(26) .
Cell Lines
PC-12 rat pheochromocytoma cells were
grown as described(29) . Before hormonal treatment,
subconfluent cultures of cells grown in 100- or 150-mm dishes were
serum-deprived for 12-24 h. Unless otherwise indicated, 100
nM NGF (Bioproducts for Science) or 100 ng/ml EGF
(Collaborative Research) were added directly to the medium, and the
incubation continued for the indicated times at 37 °C.Immunoprecipitations and Immunoblotting
After
hormonal treatment, cells were washed twice with ice-cold
phosphate-buffered saline before the addition of lysis buffer
containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 10%
glycerol, 1% Triton X-100, 1.5 mM MgCl
, 1 mM EDTA, 10 mM sodium pyrophosphate, 1 mM sodium
orthovanadate, 100 mM NaF, 10 µg/ml each aprotinin and
leupeptin, and 1 mM phenylmethylsulfonyl fluoride and
incubated on ice for 15 min. After centrifugation at 10,000 
g for 15 min at 4 °C, equal protein concentrations of
lysates were immunoprecipitated with 2 µg of anti-Crk-II,
anti-p130, or anti-paxillin antibodies (Transduction
Laboratories, Lexington, KY). After 12 h at 4 °C,
immunoprecipitates were mixed with protein G/protein A-agarose
(Oncogene Science) for 2 h, and the immunocomplexes were solubilized at
100 °C for 5 min in 25 µl of Laemmli sample buffer. Proteins
were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to nitrocellulose membrane. Individual proteins were
detected with the specified antibodies, and phosphotyrosine was
detected using a mixture of the anti-phosphotyrosine antibodies RC20H
(Transduction Laboratories) and 4G10 (Upstate Biotechnology, Inc., Lake
Placid, NY). Bound antibodies were detected with horseradish
peroxidase-linked secondary antibodies followed by the enhanced
chemiluminescence (ECL) system (Amersham Corp.). To reprobe
immunoblots, the nitrocellulose membranes were incubated for 30 min at
60 °C with 62.5 mM Tris-HCl, pH 6.8, containing 2% SDS and
0.7%
-mercaptoethanol and then were washed extensively with 10
mM Tris-HCl, pH 8, and 150 mM NaCl.In Vitro Binding Assays and Phosphopeptide
Competition
The glutathione S-transferase (GST) fusion
protein containing the SH2 domain of Crk was the generous gift of Drs.
R. B. Birge and H. Hanafusa(19) . The GST-Grb2 fusion protein
was described previously (30) , and the generation and
production of GST-Grb2-SH2 fusion protein was as described
elsewhere(31) . For in vitro association experiments,
GST fusion proteins bound to glutathione-agarose beads or GST alone
were incubated for 90 min at 4 °C with 250 µl of cell lysate
(equivalent to 5 10
cells in lysis buffer). After
washing the beads 3 times with lysis buffer, the adsorbed proteins were
eluted and analyzed as described above for immunoprecipitates. The
phosphotyrosine-containing peptides derived from the major
phosphorylation sites of the EGF receptor, DADEpYLIPQQG (pY992),
VPEpYINQSVPK (pY1068), and NPVpYHNQPLN (pY1086) were synthesized,
purified, and characterized as described previously(32) . For
phosphopeptide competition experiments, peptides (final concentration
100 µM) were preincubated with the GST-SH2 domain for 30
min prior to the addition of cell lysates, and the incubation continued
for 90 min. After washing with lysis buffer, the SH2 bound proteins
were separated by SDS-PAGE and identified by immunoblotting with
anti-phosphotyrosine or anti-p130 antibodies.
NGF and EGF Stimulate the Tyrosine Phosphorylation of
Crk in PC-12 Cells
The binding of NGF and EGF to their tyrosine
kinase receptors in PC-12 cells induces their association with a
variety of SH2-containing proteins. In some cases, recruitment of these
proteins to the receptors can result in their tyrosine
phosphorylation(33, 34) . v-Crk overexpression in
PC-12 cells markedly enhanced both NGF- and EGF-induced
differentiation. Moreover, v-Crk is tyrosine-phosphorylated in response
to both NGF and EGF in these cells(26) . Because of the
proposed role for Crk in NGF signaling pathways in PC-12 cells, we
first examined the growth factor-dependent tyrosine phosphorylation of
endogenous Crk proteins in these cells. PC-12 cells were treated with
NGF or EGF for 1 min, and lysates were precipitated with anti-Crk-II
antibodies. The resulting immunoprecipitates were separated by
SDS-polyacrylamide gel electrophoresis and blotted with
anti-phosphotyrosine antibodies (Fig. 1A). Both NGF and EGF
stimulated the tyrosine phosphorylation of an immunoprecipitated
protein migrating with an apparent molecular mass of 42 kDa (Fig. 1A). When the membrane used for the experiment shown
in panel A was stripped of the antibodies and reprobed with
the anti-Crk-II antiserum, we detected predominately the 40-kDa form of
Crk-II with only a trace of the 42-kDa form (Fig. 1B).
This pattern was identical in both unstimulated and stimulated cells,
as was reported(22) . Thus, the growth factors appear to
selectively produce the phosphorylation of the higher molecular weight
form of Crk-II, in agreement with previous studies demonstrating that
the extended 42-kDa form of Crk-II is modestly tyrosine-phosphorylated
by EGF in human carcinoma A431 cells overexpressing this form of
Crk(35) .
NGF and EGF Stimulate the Tyrosine Phosphorylation of
p130
In previous studies, a 130-kDa
tyrosine-phosphorylated protein was detected in fibroblasts transformed
by v-Crk as well as by the nonreceptor tyrosine kinase
v-Src(17, 20) . This protein was subsequently
identified as p130 in PC-12 Cells
(20) . Although it is
apparently devoid of kinase activity, p130
has an SH3
domain and clusters of tyrosine phosphorylation sites with proline in
the +3 position, suggesting that it might specifically interact
with the Crk SH2 domain(9) . Therefore, we were interested to
determine whether stimulation of PC-12 cells with growth factors could
produce the tyrosine phosphorylation of p130
. PC-12 cells
were treated with 100 nM NGF or 100 ng/ml EGF for the
indicated times. Lysates were prepared and incubated with
anti-p130
antibodies, and the precipitated proteins were
analyzed by immunoblotting with anti-phosphotyrosine antibodies (Fig. 2A). When immunoprecipitated from untreated PC-12
cells, p130
exhibited a low level of tyrosine
phosphorylation. Both NGF and EGF induced the tyrosine phosphorylation
of p130
within 1 min of exposure. This phosphorylation
remained stable for 30 min. In addition, the tyrosine phosphorylation
of two proteins with apparent molecular masses values of 42 and 50 kDa
were also increased in these immunoprecipitates in response to NGF and
EGF.
in PC-12 cells. PC-12 cells were
stimulated with NGF (100 nM) or EGF (100 ng/ml) for the
indicated times at 37 °C or left unstimulated (C). The
cell lysates were immunoprecipitated (IP) with
anti-p130
antibodies. The immunoprecipitates were
subjected to SDS-PAGE and immunoblotted with anti-phosphotyrosine
antibodies (anti-pY) 4G10 and PY20 (panel A).
Molecular mass markers, indicated at the left, are given in
kDa. The arrows indicate the positions of 125-135-, 50-,
and 42-kDa phosphotyrosine-containing proteins. The blot from panel
A was stripped and reprobed with anti-p130
antibodies (panel B).
was detected as
multiple bands at
115 and 125 kDa. Cellular transformation by
v-Crk is associated with the appearance of a broad 130-kDa
band(20) . It appears, therefore, that in NGF- and
EGF-stimulated PC-12 cells, p130
is also detected as a
broad band, perhaps due to different phosphorylation states. The blot
was completely stripped of the anti-phosphotyrosine antibodies, and the
region between the 68- and 200-kDa prestained molecular size markers
was excised and reprobed with anti-p130
antibodies (Fig. 2B). p130
was precipitated equally
from all samples and comigrated with the major
phosphotyrosine-containing protein detected in this size range. In
order to identify the 42-kDa phosphotyrosine-containing protein, the
region between the 29- and 68-kDa prestained molecular size markers was
immunoblotted with anti-Crk-II antibodies. Although v-Crk overexpressed
in 3Y1 cells could be immunoprecipitated with anti-p130
antibodies(20) , we were unable to detect Crk-II proteins
by blotting with anti-Crk-II antibodies the anti-p130
immunoprecipitates from NGF- or EGF-treated cells. It appears
that the level of expression of the 42-kDa form of Crk-II is very low
compared with the 40-kDa form (Fig. 1B). It is
therefore possible that the relative amount of this form of Crk-II
coimmunoprecipitated with the anti-p130
antibodies is
substantially lower than that immunoprecipitated with anti-Crk-II
antibodies and cannot be detected under these conditions.
NGF and EGF Augment the Formation of a Crk-p130
To further explore the growth
factor-dependent interaction of endogenous Crk-II with
p130 Complex in PC-12 Cells
, PC-12 cells were treated with NGF or EGF for 1 min,
and lysates were incubated with anti-Crk-II, anti-p130
,
or anti-SH PTP2 (Syp) (8) antibodies prior to SDS-PAGE and
immunoblotted with anti-p130
antiserum (Fig. 3).
The p130
protein was easily detected in the
anti-p130
precipitates from both untreated or treated
cells. Interestingly, treatment of cells with NGF or EGF resulted in
increased association of p130
with Crk-II, as detected in
the anti-Crk-II precipitates.
in PC-12 cells. The lysates
from PC-12 cells unstimulated (C) or stimulated with NGF (100
nM) or EGF (100 ng/ml) for 1 min at 37 °C, were
immunoprecipitated (IP) with anti-Crk-II,
anti-p130
, or anti-Syp antibodies. The resulting
immunoprecipitates were subjected to immunoblotting with
anti-p130
antibodies. The arrow indicates the
position of p130
. Molecular mass markers, indicated at
the left, are given in kDa.
immunoreactive species were detected in anti-Syp
immunoprecipitates, indicating that Syp-associated phosphoproteins are
not related to p130
. Moreover, p130
did not
associate with other SH2/SH3-containing proteins including Nck and Shc
(data not shown), demonstrating a specific interaction between Crk-II
and p130
.
complex
in PC-12 cells was also evaluated (Fig. 4). Increasing
concentrations of NGF or EGF were added to PC-12 cells for 1 min.
Lysates were immunoprecipitated with anti-Crk-II antibodies followed by
immunoblotting with anti-phosphotyrosine, anti-Crk-II, or
anti-p130
antibodies. Immunoblotting with
anti-phosphotyrosine antibodies revealed an increase in
tyrosine-phosphorylated 125-135-kDa protein associating with
endogenous Crk (Fig. 4A). This increase was observed
with as little as 1 nM NGF and 1 ng/ml EGF. The samples
contained equal amounts of Crk-II as detected with anti-Crk-II
antibodies immunoblotting (Fig. 4B). To test whether
the 125-135-kDa phosphoprotein is p130
, the same
blot was stripped and reprobed with anti-p130
antibodies. Fig. 4C shows that p130
comigrated on
SDS-PAGE with the 125-135-kDa phosphotyrosine-containing protein.
to Crk. PC-12 cells were stimulated with
various concentrations of NGF (nM) or EGF (ng/ml) for 1 min at
37 °C or left unstimulated (C). The cells were then lysed
and immunoprecipitated with anti-Crk-II antibodies. The resulting
immunoprecipitates were subjected to immunoblotting with
anti-phosphotyrosine antibodies (anti-pY) 4G10 and PY20 (panel A). The arrow indicates the position of
p130
. Molecular mass markers, indicated at the left, are given in kDa. The blot from panel A was
stripped and reprobed with anti-Crk-II antibodies (panel B) or
anti-p130
antibodies (panel
C).
p130
Experiments with peptide libraries suggested that the SH2
domain of Crk specifically binds to phosphotyrosine residues containing
the primary sequence pYXXP(9) , of which there are 15
in p130 Associates with Crk via Its SH2
Domain
(20) . The interactions of
phosphotyrosine-containing proteins with the SH2 domain of Crk were
further examined in PC-12 cells stimulated with NGF or EGF using the
SH2 domain of Crk expressed as a GST fusion protein immobilized on
glutathione-agarose beads. Following treatment of PC-12 cells with NGF
or EGF for 1 min, lysates were precipitated with GST-fusion proteins of
Crk-SH2 or Grb2-SH2 or GST alone. The precipitates were washed, and the
bound proteins were separated by SDS-PAGE and detected by
immunoblotting with anti-phosphotyrosine antibodies. As shown in Fig. 5A, both GST-Crk-SH2 and GST-Grb2-SH2 but not GST alone
precipitated a 170-kDa tyrosine-phosphorylated protein present in
EGF-treated PC-12 cells that was shown to be the EGF receptor (data not
shown and (26) and (34) ). Additionally, both NGF and
EGF stimulated the interaction of 125-135-kDa and 70-kDa
phosphotyrosine-containing proteins with GST-Crk-SH2 but not with
GST-Grb2-SH2 or GST alone. GST-Grb2-SH2 precipitated a
tyrosine-phosphorylated protein migrating at approximately 52 kDa,
previously identified as Shc(34) .
antibodies (panel B).
The blot from panel B was stripped and reprobed with
anti-paxillin antibodies (panel C). Cell lysates from PC-12
cells unstimulated (C) or stimulated with NGF (100
nM) or EGF (100 ng/ml) for 1 min at 37 °C were incubated
with GST-Crk-SH2 or GST-Grb2-SH2 fusion proteins as described above.
The bound proteins were immunoblotted with anti-p125
antibodies (panel D).
, the same blot was stripped and
reprobed with anti-p130
antibodies. Fig. 5B shows that p130
comigrated on SDS-PAGE with the
125-135-kDa phosphotyrosine-containing protein complexed with
GST-Crk-SH2 but not with GST-Grb2-SH2 or GST alone.
)-dependent tyrosine phosphorylation
of paxillin creates binding sites for Crk(37) . We therefore
tested the ability of the isolated SH2 domain of Crk to precipitate
p125
from PC-12 cells treated with NGF or EGF. Fig. 5D shows an anti-p125
immunoblot of
cell lysates from NGF- or EGF-treated PC-12 cells incubated with
GST-Crk-SH2 or GST-Grb2-SH2. Anti-p125
antibodies were
able to detect p125
only in samples precipitated with
GST-Crk-SH2 from cells treated with NGF or EGF.
Inhibition of GST-Crk-SH2 Interactions by a Specific
Phosphopeptide
To further explore the specificity of GST-Crk-SH2
binding to these phosphoproteins, we assessed the ability of
phosphopeptides derived from three of the major autophosphorylation
sites of the EGF receptor (Tyr, Tyr
, and
Tyr
) to affect GST-Crk-SH2 interactions with
tyrosine-phosphorylated proteins. GST-Crk-SH2 or GST-Grb2-SH2 was
preincubated with the phosphopeptides at 100 µM prior to
the addition of lysates from PC-12 cells that had been treated with NGF
or EGF. As evident from the anti-phosphotyrosine immunoblotting of the
bound proteins (Fig. 6A), binding of the EGF receptor and
p130
to GST-Crk-SH2 was quantitatively inhibited by the
phosphopeptide DADEpYLIPQQG (pY992) which contains proline in the
+3 position relative to tyrosine, indicating a Crk-SH2 binding
motif. The peptide NPVpYHNQPLN (pY1086) only partially inhibited the
binding of GST-Crk-SH2, while the phosphopeptide VPEpYINQSVPK (pY1068)
had no effect. Reprobing the same immunoblot with anti-p130
antibodies revealed a concordant decrease in the amount of
p130
associated with the Crk-SH2 domain in the presence
of pY992 and pY1086 (Fig. 6B).
from PC-12 cells stimulated with NGF or
EGF. GST-Crk-SH2 or GST-Grb2-SH2 fusion proteins were preincubated in
the absence or in the presence of 100 µM of the following
phosphopeptides: DADEpYLIPQQG (pY992), VPEpYINQSVPK (pY1068), and
NPVpYHNQPLN (pY1086) for 30 min. The lysates from PC-12 cells
unstimulated (C) or stimulated with NGF (100 nM) or
EGF (100 ng/ml) for 1 min at 37 °C, were then added, and the
incubation continued for 90 min. The SH2 bound proteins were separated
by SDS-PAGE and identified by immunoblotting with anti-phosphotyrosine
antibodies (anti-pY) 4G10 and PY20 (panel A). The
blot from panel A was stripped and reprobed with
anti-p130
antibodies (panel
B).
(39) . Both phosphotyrosine peptides, pY1068
and pY1086, which contain potential Grb2-SH2 recognition sites,
completely inhibited GST-Grb2-SH2 binding to the
tyrosine-phosphorylated EGF receptor from PC-12 cells treated with EGF.
However, the pY992 peptide that does not contain this motif had no
effect on GST-Grb2-SH2 binding (Fig. 6A).
tyrosine phosphorylation and enhanced its
association with endogenous Crk-II. Because p130
contains
15 potential Crk-SH2 recognition sites, we suspected that p130
might associate with the SH2 domain of the later protein. This
interaction was directly demonstrated using a GST-fusion protein
containing the SH2 domain of Crk. These data suggest that upon
stimulation of PC-12 cells with NGF or EGF, p130
undergoes tyrosine phosphorylation, further inducing its binding
to Crk. Although the significance of this association is not known, Crk
can be constitutively associated with the nucleotide exchange factor
C3G. Thus, one possible physiological role for the
p130
-Crk-C3G complex in tyrosine kinase-induced signaling
may involve targeting the activation of p21
or a member
of the Ras family of GTPases.
that is already associated
with Crk could be detected in nonactivated PC-12 cells (Fig. 2A and Fig. 4A). While this work
was under review, several reports have demonstrated adhesion-induced
tyrosine phosphorylation of p130
, suggesting a role for
p130
in integrin-mediated signal
transduction(44, 45) . It is therefore possible that
the low level of p130
tyrosine phosphorylation seen in
unstimulated PC-12 cells is due to the adhesion of the cells to the
dishes. However, it is possible that even a 3-4-fold increase in
the tyrosine phosphorylation of p130
in response to NGF
or EGF and its interaction with Crk further amplify and propagate NGF
and EGF signaling.
(37) . The exact
role of paxillin tyrosine phosphorylation in cytoskeleton organization
is not completely understood. It has been shown that the tyrosine
phosphorylation of paxillin by p125
creates binding sites
for the Crk-SH2 domain(37) . Interestingly, we detected
p125
with Crk-SH2-associated proteins. However, the
presence of p125
in this complex may be mediated through
its interaction with paxillin. Moreover, p125
may also
phosphorylate other proteins present in this complex, including
p130
or Crk itself.
in
anti-Crk immunoprecipitates. Similarly, we did not observe the
coimmunoprecipitation of the EGF receptor with Crk in PC-12 cells. This
may reflect the relative instability of such interactions or may
indicate that the association is below the limit of detection.
Consistent with this later possibility, both the EGF receptor and
paxillin are coimmunoprecipitated with v-Crk in PC-12 cells expressing
v-Crk(26) . On the other hand, the association of Crk with
tyrosine-phosphorylated p130
is easily detected in PC-12
cells in a growth factor-dependent manner.
and C3G via SH2 and
SH3 domains suggest that Crk may play a key role as an adapter
targeting nucleotide exchange factors for Ras and its homologs.
However, the growth factor-dependent interaction of Crk with paxillin
and therefore the potential interaction with p125
suggests that it may play a separate role in regulating
cytoskeletal interactions that result from tyrosine phosphorylation.
Thus, Crk proteins belong to a family of bifunctional adapter molecules
that link tyrosine phosphorylation to a multitude of downstream
cellular processes.
), 125-kDa focal
adhesion kinase; PAGE, polyacrylamide gel electrophoresis.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
