From the Department of Immunology, The Scripps
Research Institute and ¶ The Salk Institute,
La Jolla, California 92037
Received for publication, October 12, 2000, and in revised form, January 23, 2001
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ABSTRACT |
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Integrins facilitate cell attachment
to the extracellular matrix, and these interactions generate cell
survival, proliferation, and motility signals. Integrin signals are
relayed in part by focal adhesion kinase (FAK) activation and the
formation of a transient signaling complex initiated by Src homology 2 (SH2)-dependent binding of Src family protein-tyrosine
kinases to the FAK Tyr-397 autophosphorylation site. Here we show that
in viral Src (v-Src)-transformed NIH3T3 fibroblasts, an
adhesion-independent FAK-Src signaling complex occurs. Co-expression
studies in human 293T cells showed that v-Src could associate with and
phosphorylate a Phe-397 FAK mutant at Tyr-925 promoting Grb2 binding to
FAK in suspended cells. In vitro, glutathione
S-transferase fusion proteins of the v-Src SH3 but not
c-Src SH3 domain bound to FAK in lysates of NIH3T3 fibroblasts. The
v-Src SH3-binding sites were mapped to known proline-X-X-proline (PXXP)
SH3-binding motifs in the FAK N- (residues 371-377) and C-terminal
domains (residues 712-718 and 871-882) by in vitro
pull-down assays, and these sites are composed of a PXXPXX Viral Src (v-Src),1 the
transforming gene product of Rous sarcoma virus, encodes a mutated and
constitutively activated version of its normal cellular homologue,
c-Src (1). c-Src is a non-receptor protein-tyrosine kinase (PTK) that
is a component of various intracellular signaling pathways (reviewed in
Refs. 2 and 3). Both v-Src and c-Src share the same overall domain
structure and interact with target proteins through Src homology 2 (SH2) or SH3 domain-mediated interactions (reviewed in Ref. 4).
Investigations over the past 15 years have demonstrated that many of
the proteins interacting with v-Src are also phosphorylated after c-Src
activation in cells (5-9). Whereas in normal cells, the activation and
interaction of Src family PTKs with target proteins is transient and
regulated by external stimuli, v-Src-mediated cell transformation
occurs in part through the engagement of SH2 and SH3 domain target
proteins in a stimulus-independent fashion.
The activation of v-Src is due in part to a truncation in the
C-terminal region resulting in the loss of a regulatory tyrosine residue. In c-Src, this tyrosine at position 527 (Tyr-527 in chicken c-Src or Tyr-529 in murine c-Src) is phosphorylated by the
p50csk PTK. Upon phosphorylation, Tyr-527 is recognized by
the c-Src SH2 domain, and this intramolecular binding acts to maintain
the c-Src kinase in an inactive or closed conformation (10, 11). The
inactive conformation of c-Src is further stabilized by an intramolecular interaction of the c-Src SH3 domain with a proline-rich sequence in the linker region between the c-Src SH2 and kinase domains
(11, 12). Stimulated activation of c-Src can be initiated by the
de-phosphorylation of Tyr-527 (13, 14) or the binding of high affinity
ligands to the c-Src SH2 or SH3 domains (15).
Activators of c-Src include cell surface proteins such as integrins and
growth factor receptors (reviewed in Refs. 2 and 16). The combined
overexpression of the epidermal growth factor receptor with c-Src is
sufficient to promote increased c-Src PTK activity and the
transformation of normal fibroblasts (17, 18). In the case of
integrins, which do not possess catalytic activity, overexpression of
integrin-associated signaling proteins such as the focal adhesion
kinase (FAK) can interact with and enhance c-Src PTK activity (19). In
normal cells, FAK and c-Src association and activation occur in a
transient fashion, and both events are tightly regulated. Upon integrin
stimulation, the c-Src SH2 domain binds to the motif surrounding a
major autophosphorylation site (Tyr-397) on FAK (20, 21). The formation
of this FAK-Src complex enhances FAK PTK activity (22), and
Src-mediated phosphorylation of FAK at Tyr-925 promotes Grb2 adaptor
protein binding to FAK (21, 23). The combined Src-FAK signaling complex
also promotes the enhanced phosphorylation of adaptor proteins such as
Shc and p130Cas (24, 25). Downstream targets that are
activated by this Src-FAK complex include the ERK and c-Jun
N-terminal kinase cascades as well as phosphatidylinositol 3'-kinase
and Akt (reviewed in Ref. 16).
In Src-transformed cells, a stable complex between FAK and activated
Src has been described; however, the molecular mechanism(s) stabilizing
this interaction remain undetermined (21, 26). Recent studies have
shown that v-Src can promote increased FAK tyrosine phosphorylation
independent of v-Src SH2-mediated binding to the motif surrounding FAK
Tyr-397 (27). Previous studies have shown that a motif upstream of FAK
Tyr-397 (residues 368-376 encoding RALPSIPKL) resembles an Src
SH3-binding consensus motif (28), and the integrity of this site
contributes to Src-FAK interactions (29). However, the c-Src SH3 domain
alone is not able to associate stably with FAK in pull-down assays (21,
29, 30). In contrast, we find that the v-Src SH3 domain strongly binds
FAK in vitro at three proline-X-X-proline (PXXP)-containing sites in FAK. Two amino acids in the v-Src SH3 domain RT loop region
(Trp-97 and Ile-98) are responsible for this enhanced binding to FAK.
Substitution of Trp-97 and Ile-98 into the c-Src SH3 domain is
sufficient to facilitate and sustain a c-Src complex with Phe-397 FAK
in vivo. Our results showing that the v-Src SH3 domain in addition to the v-Src SH2 domain can bind FAK provide a molecular explanation for the stable and adhesion-independent signaling complex
formed between the two PTKs in v-Src-transformed cells.
Cells and Cell Culture--
NIH3T3 fibroblasts (21),
v-Src-transformed NIH3T3 (pSrc11) (31), and 293T cells were grown in
Dulbecco's modified Eagle's medium containing 10% calf serum and
supplemented with pyruvate, penicillin, and streptomycin. Lysates were
made from either adherent serum-starved (0.5% calf serum overnight)
cells; cells detached by limited trypsin treatment (0.06% trypsin/1
mM EDTA for 2 min), collected in the presence of soybean
trypsin inhibitor (0.25 mg/ml), resuspended in Dulbecco's modified
Eagle's medium with 0.1% bovine serum albumin, and held in suspension
for 1 h at 37 °C; or cells held in suspension for 1 h at
37 °C and then replated onto fibronectin-coated dishes (10 µg/ml
in phosphate-buffered saline) for 30 min at 37 °C as described previously.
Metabolic Labeling--
Proliferating NIH3T3 cells were
incubated in the presence of 5% dialyzed calf serum containing 100 µCi/ml [35S]methionine [35S]cysteine
Express label (PerkinElmer Life Sciences) for 16 h before cell
lysis and extensive pre-clearing with glutathione-agarose beads as
described below.
Antibodies--
A monoclonal antibody (mAb) to phosphotyrosine
(P.Tyr) (4G10) and a avian-specific mAb to v-Src (EC10) were from
Upstate Biotechnology, Inc. (Lake Placid, NY). Hemagglutinin (HA)
epitope tag mAb (16B12) was from Covance Research (Berkeley, CA). A Myc
epitope tag mAb (9E10) and a c-Src-specific mAb (2-17) were used as
described (32, 33). A mAb to p130Cas (clone 21) was from BD
Phar- Mingen/Transduction Laboratories (San Diego, CA).
Affinity-purified polyclonal antibodies to c-Src (Src-2) were from
Santa Cruz Biotechnology (Santa Cruz, CA). Affinity-purified polyclonal
antibodies to FAK and Grb2 were used as described (33). Peptides
corresponding to murine FAK residues 865-884 (GKPDPAAPPKKPPRPGAPGH) or
a control peptide (GKPDPAAAAKKAARPGAPGH) were
kindly synthesized and purified by Jill Meisenhelder (The Salk Institute).
GST Fusion Constructs--
The murine c-Src SH3 and SH2 domains
cloned into pGEXKT were expressed and purified as described (21). The
SRA v-Src SH3, v-Src SH3 (Leu-133), and v-Src SH3 (Arg-118) domains
were generously provided by Tony Pawson (Mount Sinai Hospital, Canada)
and were used as described (34). QuikChange (Stratagene, La Jolla, CA) oligonucleotide-directed mutagenesis protocol was used to change codons
for murine c-Src SH3 domain residues Arg-97 to Trp and Thr-98 to Ile
singly or in combination within the pGEXKT vector using the
sense primers 5-CTATGAGTCATGGACAGAGAC-3', 5-GAGTCACGGATAGAGACTGAC-3', and 5'-TATGAGTCATGGATAGAGACTGAC-3', respectively. Mutations were verified by DNA sequencing. All fusion proteins were expressed in BL21
Escherichia coli and affinity-purified in batch by
glutathione-agarose chromatography. Proteins were dialyzed (20 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM
EDTA, and 10% glycerol), concentrated, and stored frozen at
DNA Constructs--
HA-tagged constructs for wild type FAK,
Phe-397, Phe-407, Arg-454, Ala-712/713, Phe-861, and Phe-925 FAK were
used as described (35). The QuikChange mutagenesis protocol was used to
change codons for FAK Pro-872 and Pro-873 to Ala using the sense
primer 5'-CCTGCAGCTGCAGCAAAGAAACCTCC-3', to change codons for FAK
Pro-876 and Pro-877 using the sense primer
5'-CCACCAAAGAAAGCTGCTCGCCCTGGA-3', and to change codons for FAK
Pro-872, Pro-873, Pro-876, and Pro-877 to Ala was performed using the
sense primer 5'-GATCCTGCAGCTGCAGCAAAGAAAGCTGCTCGCCCTGGAGC-3' within a
SmaI/XbaI FAK fragment cloned into
pBlue-script. The mutant FAK SmaI/XbaI
fragment was cloned into pcDNA3-HA-FAK (19) cut with the same
enzymes. FAK Pro
Mutagenesis of the c-Src SH3 domain to Trp-97/Ile-98 was performed (as
described above for the GST fusion proteins) in a murine c-Src cDNA
cloned into pBS. A SacI fragment was cloned into the same
sites of a murine c-Src cDNA containing a Phe-529 mutation (37) to
generate c-Src (Trp-97/Ile-98/Phe-529). The Arg-177 to Ser mutation was
introduced into the different c-Src constructs by site-directed
mutagenesis using the sense primer
5'-CCGAGAGGTACCTTCCTTGTGAGTGAGAGTGAGACCAC-3'. All mutations were
verified by DNA sequencing. Kinase-defective mouse c-Src
(Trp-97/Ile-98/Met-297) was constructed by cutting pcDNA3.1 c-Src
K297M (37) with MscI and XbaI and inserting the fragment into MscI- and XbaI-restricted pcDNA
c-Src (Trp-97 + Ile-98). All Src cDNA constructs were subcloned as
full-length BamHI restriction fragments into pcDNA3.1
(Invitrogen). A mammalian expression vector for v-Src (Prague C) in
pRCMV was generously provided by Walter Eckhart (The Salk Institute).
Cell Transfection--
For co-IP studies, human 293T cells were
transfected with the indicated constructs by a standard calcium
phosphate precipitation protocol as described previously (19).
Immunoprecipitation, in Vitro Binding, and
Immunoblotting--
Cells were solubilized in a modified RIPA lysis
buffer containing 1% Triton X-100, 1% sodium deoxycholate, and 0.1%
SDS (25), preincubated with agarose beads, and lysates cleared by
centrifugation. Antibodies (2.5 µg) or GST fusion proteins (5 µg)
pre-bound to glutathione-agarose beads (Sigma) were incubated with
lysates for 2.5 h at 4 °C. Antibody complexes were recovered by
addition of either Protein G-plus (Oncogene Research Products,
Calbiochem)- or Protein A (Repligen, Cambridge, MA)-agarose beads for
1 h at 4 °C. Antibody or GST-complexed proteins were washed
once with RIPA lysis buffer, twice with 1% Triton-only lysis buffer
(33), twice with HNTG buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol), and then
analyzed by SDS-PAGE. Proteins were transferred to polyvinylidene
difluoride membranes (Millipore, Bedford, MA), and the membranes were
blocked (2% bovine serum albumin in Tris-buffered saline containing
0.05% Tween 20) for 2 h at room temperature, incubated in either
1 µg/ml monoclonal or a 1:1000 dilution of polyclonal antibodies for
2 h at room temperature or at 4 °C overnight, and visualized by
enhanced chemiluminescent detection methods. Sequential re-probing of
membranes was performed as described (25).
Adhesion-independent Association of v-Src with FAK--
Several
groups have shown that FAK tyrosine phosphorylation and activity are
regulated by integrin interactions with extracellular matrix proteins
(16, 38). Importantly, when NIH3T3 fibroblasts are deprived of these
contacts by placing them in suspension, FAK is rapidly dephosphorylated
and inactivated (Fig. 1, lanes 1 and 3). In v-Src transformed fibroblasts
(v-Src3T3), FAK is associated with v-Src (21, 26), and this
interaction leads to increased FAK tyrosine phosphorylation in adherent
cells (Fig. 1, lane 2). Interestingly, in
suspended v-Src3T3s, FAK remains phosphorylated and associated with
v-Src (Fig. 1, lanes 4 and 6). Activated v-Src
can phosphorylate FAK at Tyr-925 in vivo which creates a
Grb2 adaptor protein SH2-binding site (23). Significantly, previous
studies have shown that an active signaling complex between v-Src and
FAK occurs in both adherent and suspended v-Src3T3 cells as measured by
Grb2 co-immunoprecipitation with FAK (21).
Signaling in Suspended v-Src Transfected Cells--
In normal
cells, c-Src association with FAK is mediated by c-Src SH2 domain
binding to the motif surrounding the FAK Tyr-397 autophosphorylation
site (20, 21). To test whether this was the molecular mechanism that
stabilized the v-Src/FAK interaction in suspended cells, either HA
epitope-tagged wild type FAK (FAK-WT), kinase-inactive FAK (Arg-454),
or various FAK tyrosine phosphorylation site mutants (Phe-397, Phe-407,
Phe-861, and Phe-925) were transfected into 293T cells (Fig.
2). When the cells were held in
suspension prior to cell lysis, all of the HA-FAK constructs were
expressed (Fig. 2A, top panel), but none were detectably
tyrosine-phosphorylated nor associated in signaling complexes with
endogenous c-Src or Grb2 (Fig. 2A). Upon fibronectin (FN)
replating of FAK-transfected and suspended 293T cells, all of the
HA-FAK constructs were tyrosine-phosphorylated, including the Phe-397
autophosphorylation site and Arg-454 kinase-inactive FAK mutants,
respectively, albeit at lower levels than WT FAK (Fig. 2B).
FN stimulation promoted c-Src association with all FAK constructs
except for Phe-397 FAK which is mutated at the c-Src SH2-binding site
(Fig. 2B, lane 3). Significantly, a stimulated signaling
complex with Grb2 was formed with WT, Phe-407, and Phe-861 FAK but not
with Phe-397, Arg-454 kinase-inactive, and Phe-925 FAK which is mutated
at the Grb2 SH2-binding site (Fig. 2B). These results are
consistent with published studies showing the requirement of both
enhanced FAK kinase activity and the integrity of the autophosphorylation/Src SH2-binding site at Tyr-397 for full FAK function in promoting integrin-stimulated signaling events leading to
the activation of targets such as the ERK2/mitogen-activated protein(MAP) kinase (19, 39).
In contrast to the suspended 293T results (Fig. 2A),
co-transfection of 293T cells with v-Src in addition to FAK resulted in
the enhanced tyrosine phosphorylation of all the different FAK
constructs in an anchorage-independent manner (Fig. 2C).
These elevated FAK tyrosine phosphorylation events were maintained in the absence of integrin stimulation and were higher than FN-stimulated FAK phosphotyrosine (P.Tyr) levels. Notably, all transfected FAK constructs including Phe-397 and Arg-454 FAK co-immunoprecipitated with
v-Src (Fig. 2C). Although v-Src binding to Phe-397 and
Arg-454 FAK is reduced compared with WT FAK, these results show that
v-Src can associate with FAK independently of both the Src SH2-binding site at Tyr-397 and FAK kinase activity.
Moreover, FAK signaling complexes with Grb2 were stabilized in the
suspended and v-Src-transfected 293T cells with the exception of
Phe-925 FAK, which is mutated at the Grb2 SH2-binding site (Fig.
2C). Surprisingly, both Phe-397 and Arg-454 FAK were
associated in a signaling complex with Grb2 albeit at lower levels
compared with WT FAK (Fig. 2C, lanes 3 and 5). An
in vivo signaling complex between endogenous Grb2 and FAK
has been detected in suspended v-Src3T3s (21), and our 293T results
with Phe-397 FAK suggest that this signaling complex may be enhanced
through novel v-Src binding contacts with FAK in addition to v-Src SH2
domain binding to the FAK Tyr-397 site.
v-Src SH3 but Not c-Src SH3 Domain Binding to FAK--
To address
the possibility that SH3 domain-mediated contacts may stabilize v-Src
interactions with FAK, GST fusion proteins encompassing either the
c-Src or v-Src SH3 domains were employed in pull-down assays using
lysates from suspended NIH3T3 cells where FAK was not
tyrosine-phosphorylated (Fig. 3). As
documented previously (21, 30), the isolated c-Src SH3 domain does not stably bind FAK in vitro (Fig. 3, lane 1),
whereas this GST c-Src SH3 construct is capable of binding known
targets such as p130Cas (see Fig. 6B). In
contrast, the GST-v-Src SH3 domain readily associated with FAK in a
P.Tyr-independent manner (Fig. 3, lane 2). These results
show that there are significant binding differences between the v-Src
and c-Src SH3 domains. Additionally, these results support the
hypothesis that v-Src may directly bind to Phe-397 FAK in
vivo (Fig. 2C) through novel SH3 domain-mediated
interactions.
Identification of v-Src SH3 Domain Binding Sites on FAK--
Since
the FAK C-terminal (CT) domain contains two known SH3 domain binding
sites for proteins such as p130Cas and Graf (40, 41), a
peptide encompassing the second FAK-CT domain PXXP SH3-binding site
(FAK residues 865-884) was synthesized and used as competitive
inhibitor in GST v-Src SH3 domain pull-down assays (Fig.
4A). When added to lysates of
NIH3T3 fibroblasts, the FAK 865-884 peptide inhibited v-Src SH3 domain
binding to FAK in a concentration-dependent manner (Fig.
4A). When a control FAK 865-884 peptide was
synthesized with alanine substitutions to disrupt the central PXXP
motifs, addition of this control peptide did not inhibit GST v-Src SH3
domain binding to FAK present in NIH3T3 lysates (Fig. 4A).
These results support the hypothesis that the v-SrcSH3 domain binds to
specific PXXP-containing sites in FAK.
To evaluate directly the contribution of the first or second FAK-CT
PXXP-rich motifs for v-Src SH3 binding, proline to alanine substitutions were introduced into full-length FAK as indicated (Fig.
4B). HA-tagged FAK constructs were transiently transfected into 293T cells, and expression of the various FAK mutants was verified
by HA tag blotting of whole cell lysates (WCLs) (Fig. 4C).
Results from pull-down assays with the GST v-Src SH3 domain showed
reduced binding to Ala-712/713 FAK compared with WT FAK (Fig. 4C,
lane 2). This result suggests that the first FAK-CT PXXP-rich
motif serves as a v-Src SH3 domain binding site. Interestingly, alanine
substitutions at FAK residues 872 and 873 only weakly affected v-Src
SH3 binding compared with WT FAK (Fig. 4C, lane 3), whereas
an Ala-876/877 FAK mutant exhibited reduced v-Src SH3 association (Fig.
4C, lane 4). This result is consistent with the inhibition
of binding after the addition of the FAK 865-884 peptide (Fig.
4A) and supports the conclusion that the second FAK-CT
PXXP-rich motif encompassing FAK Pro-866 and Pro-867 also serves as a
v-Src SH3-binding site. Notably, six combined alanine mutations in the
first and second FAK-CT domain PXXP-rich motifs yielded a protein (FAK
Pro
However, the binding of FAK Pro
To define further the v-Src SH3 domain binding site in the FAK-NT
domain, a truncated FAK-NT Pro c-Src and v-Src Differ in RT Loop SH3 Domain Residues--
Since
we found that the v-Src SH3 but not c-Src SH3 domain could stably bind
targets such as FAK in pull-down assays using lysates of NIH3T3
fibroblasts (Fig. 3), sequence comparisons were performed to determine
a potential molecular basis for this differential binding activity
(Fig. 6A). Interestingly, the
Schmidt-Ruppin (SRA), Prague C (PR-C), and Bryan high titer (BH)
strains of v-Src all contain amino acid substitutions in one or two
positions in the RT loop region of the SH3 domain (42-44).
Structural analyses have shown that this RT loop region of the Src SH3
domain lies in close proximity to the surface groove that
constitutes the PXXP ligand-binding site (28).
v-Src RT Loop SH3 Domain Residues Trp-97 and Ile-98 Confer FAK
Binding Activity--
To determine the effect of v-Src RT loop residue
differences on SH3 domain binding activity, mouse c-Src SH3 domain
residues were changed from Arg-97 to Trp or Thr-98 to Ile singly or in combination and expressed as GST fusion proteins (Fig. 6B).
By using metabolically 35S-labeled lysates from NIH3T3
fibroblasts in pull-down assays, the GST c-Src SH3 domain bound to a
number of different cellular proteins compared with the GST control
(Fig. 6B, lane 2). Specificity of this was verified by
re-probing the membrane containing the 35S-labeled proteins
by anti-P.Tyr blotting. The GST c-Src SH3 domain but not the GST
control bound an ~130-kDa tyrosine-phosphorylated protein, the
identity of which was verified by anti-p130Cas blotting
(Fig. 6B, lane 2). The p130Cas adaptor protein
is a highly tyrosine-phosphorylated protein and a known target for
c-Src SH3 domain binding (45).
Compared with the 35S-labeled protein binding profile of
the normal c-Src SH3 domain, the GST c-Src (Trp-97) SH3 domain
associated with additional cellular proteins ranging in size from ~60
to 200 kDa (Fig. 6B, lane 3, indicated by
arrows). By anti-P.Tyr blotting, proteins of ~130, ~116,
and ~85 kDa were detected in association with the c-Src (Trp-97) SH3
domain. In addition to p130Cas association with the c-Src
(Trp-97) SH3 domain, the ~116-kDa P.Tyr-containing protein was
positively identified as FAK by anti-FAK blotting (Fig. 6B, lane
3). The amount of FAK associated with the c-Src (Trp-97) SH3
domain was less than that bound to the GST Src SH2 domain fusion
protein (Fig. 6B, lane 9) which binds with high affinity to
a phosphorylated Tyr-Ala-Glu-Ile motif at FAK Tyr-397. Nevertheless,
these results show that the singular residue change to Trp-97 found
within the SH3 domain of the Prague C v-Src isoform is sufficient to
confer FAK binding activity without disrupting the binding of other
known c-Src SH3 domain targets.
Interestingly, the single substitution of Thr-98 to Ile found within
the Bryan high titer v-Src isoform did not dramatically alter the
35S-labeled cellular protein binding profile to the c-Src
(Ile-98) SH3 domain compared with the normal c-Src SH3 domain (Fig.
6B, lane 4). However, this singular mutation was sufficient
to confer weak FAK binding activity as detected by FAK blotting.
Notably, the combined substitutions (Trp-97 + Ile-98) in the GST c-Src SH3 potentiated both 35S-labeled cellular protein and FAK
binding (Fig. 6B, lane 5) equivalent to that observed for
the GST SRA v-Src SH3 domain (Fig. 6B, lane 6). Specificity
of these in vitro GST v-Src SH3 pull-down assays was
assessed by the lack of FAK and 35S-labeled cellular
protein binding to the inactivated (Leu-133 and Arg-118) GST v-Src SH3
domains (Fig. 6B, lanes 7 and 8). Significantly, the amount of FAK associated with either the c-Src (Trp-97 + Ile-98) or
v-Src SRA SH3 domains was equivalent to that bound to the c-Src SH2
domain (Fig. 6B). These results provide a molecular
explanation for the stabilization of an adhesion-independent v-Src
signaling complex with FAK (Figs. 1 and 2) and also show that FAK
differs from other SH3 targets such as p130Cas that
interact equally with the c-Src and v-Src SH3 domains.
Trp-97 + Ile-98 Substitutions in the c-Src SH3 Domain Are
Sufficient to Promote Adhesion-independent c-Src Association with FAK
in Vivo--
To determine if the addition of v-Src-specific residues
into the mouse c-Src SH3 domain could promote FAK association with c-Src in an SH2-independent manner in vivo, mutagenesis was
used to introduce Trp-97 + Ile-98 substitutions into the c-Src SH3 domain and an activating mutation (Phe-529) at the C-terminal regulatory region within murine c-Src (Fig.
7A). These substitutions were
combined with inactivating mutations introduced into the c-Src SH2
domain (Ser-177) and within the kinase domain (Met-297) as indicated
(Fig. 7A). Previous studies have shown that mutation of the
conserved FLVRES Arg to Ser at position 177 in the Src SH2 domain
inactivates this domain and that mutation of Lys to Met within the
kinase domain ATP-binding site at position 297 inhibits intrinsic c-Src
kinase activity (37).
Co-transfection and co-immunoprecipitation assays were performed with
c-Src constructs containing the Ser-177 mutation in combination with
Phe-397 FAK in order to analyze SH2-independent interactions of c-Src
with FAK (Fig. 7B). Equal expression levels of Phe-397 FAK
were observed in lysates of suspended 293T cells (Fig. 7B,
lanes 1-6). Importantly, Phe-397 FAK did not
detectably associate with either endogenous c-Src, c-Src (Ser-177), or
c-Src (Ser-177/Phe-529) (Fig. 7B, lanes 1, 2, and
5), confirming that no Src-FAK complex was formed after
overexpression of an activated c-Src construct that also contained an
inactive SH2 domain (Ser-177/Phe-529). In contrast, overexpression of
either c-Src (Trp-97 + Ile-98/Ser-177) or c-Src (Trp-97 + Ile-98/Ser-177/Phe-529) readily associated with Phe-397 FAK in
vivo (Fig. 7B, lanes 3 and 4). In addition, Phe-397 FAK was tyrosine-phosphorylated when complexed with these (Trp-97 + Ile-98) SH3 domain-containing and SH2 domain-inactivated c-Src constructs when isolated from suspended 293T cells (Fig. 7B, lanes 3 and 4).
Interestingly, kinase-inactive c-Src (Trp-97 + Ile-98/Ser-177/Met-297)
associated with Phe-397 FAK in a P.Tyr-independent manner (Fig.
7B, lane 6) albeit at lower levels than c-Src (Trp-97 + Ile-98/Ser-177) (Fig. 7B, lane 3). Whereas these in
vivo results support the in vitro binding analyses
showing that FAK tyrosine phosphorylation is not required for v-Src SH3
domain binding (Fig. 3), the integrity of c-Src kinase domain activity
positively enhanced SH3 domain-mediated FAK association in an
undetermined manner. Taken together, these results show that
gain-of-function Trp-97 + Ile-98 substitutions in the c-Src SH3 domain
can promote the formation of an Src-FAK complex in vivo that
is independent of both FAK Tyr-397 phosphorylation and c-Src SH2 domain function.
Both integrin and growth factor receptor stimulation of fibroblast
and smooth muscle cells promote the recruitment of Src family PTKs into
a signaling complex with FAK (35, 36, 46, 47). This interaction is
mediated by c-Src SH2 domain binding to the FAK autophosphorylation
site at Tyr-397 (20). Within this complex, Src-mediated phosphorylation
of FAK at Tyr-925 promoting Grb2 binding is one pathway contributing to
ERK/MAP kinase activation after integrin stimulation of cells (25). In
normal cells, both c-Src and Grb2 binding to FAK after cell
adhesion-triggered integrin stimulation is transient and tightly
regulated. Whereas FAK is dephosphorylated and inactivated when normal
cells are placed in suspension, FAK remains highly
tyrosine-phosphorylated and associated with Grb2 in a signaling complex
in v-Src-transformed cells (21).
In this study, we show that v-Src and Grb2 can form a signaling complex
with wild type, kinase-inactive, or Phe-397 FAK in an
anchorage-independent manner. As a potential explanation for the
enhanced stability of this adhesion-independent v-Src-FAK signaling
complex, we found that the v-Src SH3 domain specifically bound to
PXXP-containing sites within the FAK-NT and FAK-CT domains. This
enhanced binding activity was not shared by the c-Src SH3 domain and
was promoted by specific v-Src amino acid substitutions in the RT loop
region of the v-Src SH3 domain found in the Prague C, Byran high titer,
and SRA v-Src isoforms. Substitution of c-Src SH3 domain residues
Arg-97 and Thr-98 singly or doubly with Trp-97 and Ile-98 promoted the
specific SH3 domain-mediated binding to FAK and other proteins in
lysates of NIH3T3 fibroblasts. Notably, the c-Src (Trp-97 + Ile-98) SH3
and v-Src SH3 domains bound to FAK in a phosphorylation-independent
manner equally as well as the c-Src SH2 domain that binds to FAK with
high affinity to a phosphorylated Tyr-Ala-Glu-Ile motif at Tyr-397.
This c-Src (Trp-97 + Ile-98) SH3 domain-mediated binding was strong
enough to mediate an in vivo association with Phe-397 FAK in
a c-Src SH2 domain-independent manner. This novel SH3 domain-mediated
binding interaction between v-Src and FAK provides a molecular
explanation for the observed switch from a transiently stimulated
integrin- and adhesion-dependent c-Src-FAK signaling
complex in normal cells to an anchorage-independent and stable
association between v-Src and FAK in v-Src-transformed cells.
By a combination of peptide competition binding assays and proline to
alanine mutagenesis, the v-Src SH3-binding sites in the FAK-CT domain
were mapped to known binding sites (FAK residues 712-718 and 871-882)
for other SH3 domain-containing proteins such as p130Cas
and Graf (40, 41). A weaker binding interaction of the v-Src SH3 domain
was mapped to a putative c-Src SH3-binding site (residues 367-377) in
the FAK-NT domain (29). SH3 domains bind to left-handed polyproline
helices, and studies have identified a PXXP c-Src SH3 class I consensus
motif as RXLPPLP and a class II motif as XPPLPXR, where X is not a selected amino acid
residue and the position of a basic arginine residue determines SH3
domain binding orientation (28, 48-50). Known ligands for c-Src SH3
domains are shown in Table I (34, 48,
51), including p130Cas and FAK consensus c-Src class I
SH3-binding motifs (29, 45). Interestingly, p130Cas readily
associates with the c-Src SH3 domain in vitro, whereas we
and other previously published studies (21, 30, 52, 53) have not
detected FAK as an isolated c-Src SH3-binding partner.
(where
is a hydrophobic residue) v-Src SH3 binding consensus.
Sequence comparisons show that residues in the RT loop region of the
c-Src and v-Src SH3 domains differ. Substitution of c-Src RT loop
residues (Arg-97 and Thr-98) for those found in the v-Src SH3 domain
(Trp-97 and Ile-98) enhanced the binding of distinct NIH3T3 cellular
proteins to a glutathione S-transferase fusion protein of
the c-Src (Trp-97 + Ile-98) SH3 domain. FAK was identified as a c-Src
(Trp-97 + Ile-98) SH3 domain target in fibroblasts, and co-expression
studies in 293T cells showed that full-length c-Src (Trp-97 + Ile-98)
could associate in vivo with Phe-397 FAK in an
SH2-independent manner. These studies establish a functional role for
the v-Src SH3 domain in stabilizing an adhesion-independent signaling
complex with FAK.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. Protein purity was analyzed by SDS-PAGE, and protein
concentrations were determined by bicinchoninic acid assays (Pierce).
(Ala-712/713/872/873/876/877) was
created by subcloning the FAK Ala-872/872/876/877
SmaI/XbaI fragment into the same sites of pcDNA3-FAK Ala-712/713. Expression of the FAK C-terminal domain as
HA-FRNK was used as described (35). FRNK Pro
(Ala-712/713/872/873/876/877 using the same number as FAK) was created
by subcloning an AflII/XbaI fragment into
pcDNA3.1 (Invitrogen, Carlsbad, CA). The translational FRNK start
site occurs at FAK Met-691, and FRNK Pro
contains a
triple HA epitope tag at the C-terminal end. Expression of the FAK
N-terminal domain (FAK-NT) residues 1-402 containing a 6×-repeated
Myc epitope tag was used as described (36). Polymerase chain reaction
was used to amplify Myc-FAK-NT using an antisense primer
5'-TTTATCGATTTTCACCGTTCACCTTCTTTCTGGGT-3' that contains a
ClaI site following a stop codon to create Myc-FAK-NT
Pro
(FAK residues 1-368). All mutations were verified by
DNA sequencing.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Stable association of v-Src and FAK in
suspended cells. IPs with FAK (lanes 1-4) or Src
(lanes 5 and 6) antibodies were made from lysates
of NIH3T3 fibroblasts or v-Src-transformed NIH3T3 fibroblasts
(v-Src3T3) as indicated. Lysates were prepared from adherent
cells (Attached) or from cells held in suspension for 60 min
(Suspended), and the proteins associated with the IPs were
resolved by SDS-PAGE and analyzed by anti-phosphotyrosine
(P.Tyr) or anti-FAK blotting.
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Fig. 2.
Formation of a v-Src-FAK signaling complex in
suspended cells independent of v-Src SH2 binding to FAK Tyr-397.
Human 293T cells were transfected (2.5 µg) with either control vector
(pCDNA3) or the indicated HA-tagged FAK constructs or
co-transfected with the indicated FAK constructs and v-Src (2.5 µg
each) as indicated. A, cells were serum-starved and held in
suspension for 1 h prior to lysis and IPs. B, cells
were serum-starved, placed in suspension for 1 h, and then
replated onto FN-coated dishes (10 µg/ml) for 30 min prior to lysis
and IPs. C, cells co-transfected with v-Src were
serum-starved and held in suspension for 1 h prior to lysis and
IPs. A-C, IPs were made with antibodies to the HA tag to
directly isolate FAK constructs and were analyzed by either HA tag or
P.Tyr blotting. Association of transfected FAK mutants with endogenous
c-Src or co-transfected v-Src was analyzed by mAb Src IPs followed by
HA tag blotting. Visualization of FAK-associated signaling complexes
containing Grb2 was performed by polyclonal Grb2 IPs followed by HA tag
blotting.
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Fig. 3.
The v-Src but not the c-Src SH3 domain binds
to FAK in a phosphorylation-independent manner. In
vitro binding assays with GST fusion proteins containing either
the chicken c-Src SH3 domain (lane 1), Schmidt-Ruppin strain
A v-Src SH3 domain (lane 2), GST alone (lane 3),
or polyclonal FAK-IPs (lanes 4 and 5) were
performed with cell lysates of suspended (S, lanes
1-4) or adherent (A, lane 5) NIH3T3 fibroblasts. The
proteins associated with the IPs or GST fusion proteins were resolved
by SDS-PAGE and analyzed by either FAK (upper panel) or
P.Tyr (middle panel) blotting. Comparable amounts of GST
fusion proteins were employed as shown by Coomassie Blue staining of
the membrane (lower panel).
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Fig. 4.
v-Src SH3 domain binding to sites in the
FAK-CT domain. A, in vitro pull-down assays
using the GST v-Src SH3 domain and lysates from NIH3T3 cells were
performed in the presence of increasing concentrations of synthetic
peptides (0-100 µM) corresponding to FAK residues
865-884 (FAK 865-884, upper panel) or a control 865-884
peptide with alanine substitutions (control, lower panel).
FAK association was detected by FAK blotting. B, diagram of
paired proline (P) to alanine (A) substitutions
introduced either into the first (residues 711-721) or second
(residues 869-879) SH3 domain binding regions located in the CT domain
of HA-tagged FAK. C, wild type (WT) or the
indicated FAK mutants (2.5 µg each) were transfected into human 293T
cells, and lysates were prepared. In vitro pull-down assays
using the GST v-Src SH3 domain were performed, and FAK association was
visualized by HA tag blotting (upper panel). The panel on
the right compares GST v-Src SH3 binding to FAK and
background binding of FAK to GST-agarose beads. Expression levels of
the FAK mutants was verified in WCLs of transfected cells (50 µg
each) by HA tag blotting (lower panel).
) that was strongly expressed but only weakly bound by
the v-Src SH3 domain (Fig. 4C, lane 6).
to the v-Src SH3 domain
was reproducibly stronger than the level of nonspecific FAK WT
association with the GST control (Fig. 4C, right panel),
indicating that other binding sites for the v-Src SH3 domain may exist
on FAK. Since a c-Src SH3-binding motif has been identified in the FAK
N-terminal (NT) domain (residues 368-377) (29), expression vectors for the FAK-NT or FAK-CT (herein referred to as FRNK) domains were constructed with the addition of either Myc or HA epitope tags, respectively (Fig. 5A). After
exogenous expression of FAK-NT domain, FRNK or a FRNK Pro
mutant (Ala-712/713/872/876/877 using the FAK numbering) in human 293T
cells, in vitro pull-down assays were performed with either GST, the GST v-Src SH3 domain, or with GST v-Src SH3 domain containing an inactivating point mutation (Pro to Leu-133) (Fig. 5B).
FRNK strongly bound to the v-Src SH3 domain, and mutations of both PXXP-rich motifs in FRNK Pro
disrupted this binding
interaction. Neither FRNK nor FRNK Pro
detectably
associated with the GST or GST v-Src SH3 Leu-133 controls (Fig.
5B). Interestingly, the FAK-NT domain also associated with the GST v-Src SH3 domain but not with the GST or GST v-Src SH3 Leu-133
controls (Fig. 5B).
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Fig. 5.
The v-Src SH3 domain binds to specific sites
in the FAK-CT and FAK-NT domains. A, diagram of the
Myc-tagged FAK-NT domain and the HA-tagged FAK-CT domain (termed
FRNK). FRNK Pro contains alanine
(A) substitutions in both the first (residues 711-721) and
second (residues 869-879) SH3 domain binding regions. Myc-tagged
FAK-NT Pro
contains a stop codon following Arg-368.
B, FAK-NT, FRNK, or FRNK Pro
(2.5 µg each)
were transfected into human 293T cells, and lysates were prepared.
In vitro pull-down assays using either GST, the
GST v-Src SH3, or the GST v-Src L-133 SH3 domain
(2 µg each) were performed, and FRNK or FRNK Pro
association (upper panel) or FAK-NT association
(middle panel) was visualized by either anti-HA or Myc tag
blotting, respectively. Equal amounts of GST fusion proteins were
employed as shown by Coomassie Blue staining of the membrane
(lower panel). C, WCLs of either non-transfected
(Control), FAK-NT, or FAK-NT Pro
-transfected
(2.5 µg) human 293T cells were prepared (50 µg each), and FAK-NT
protein expression was visualized by anti-Myc tag blotting. In
vitro pull-down assays using the GST v-Src SH3 domain (Coomassie
stain, lower panel) were performed, and FAK-NT association
was visualized by anti-Myc tag blotting (upper panel).
mutant (residues 1-368)
was created and expressed in 293T cells (Fig. 5C). Whereas
the full-length FAK-NT domain (residues 1-402) associated with the GST
v-Src SH3 domain, FAK-NT Pro
was strongly expressed but
only weakly bound by the v-Src SH3 domain (Fig. 5C).
Although residual binding of both FRNK Pro
(Fig.
5B) and FAK-NT Pro
(Fig. 5C) to the
GST v-Src SH3 domain was greater than binding to the GST control,
specific mutations or truncations to remove existing PXXP motifs
resulted in the loss of strong in vitro v-Src SH3 binding to
FAK. These results support the conclusion that v-Src SH3
domain-mediated binding to FAK occurs in a site-specific manner
in vitro. Our results show that a single PXXP-rich motif in
the FAK-NT domain and either of two PXXP-rich motifs in the FAK-CT
domain constitute the major v-Src SH3 domain binding sites on FAK.
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Fig. 6.
Substitution of c-Src SH3 domain RT
Loop residues for those in v-Src promote the binding of
novel target proteins such as FAK. A, comparison of Src
SH3 domain amino acid sequences. The one-letter amino acid code is used
to compare the normal chicken c-Src sequence with that of normal mouse
c-Src, Schmidt-Ruppin A v-Src (SRA), Prague C v-Src
(PR-C), and Bryan high titer v-Src (BH). The
common v-Src-specific changes in the RT loop region are
boxed. Secondary structure assignments according to Ref. 48
are indicated. B, the mouse GST c-Src SH3 domain (Src
SH3, lane 2) expression vector was altered by
site-directed mutagenesis to encode the indicated RT loop substitutions
as follows: c-Src SH3 Arg-97 to Trp (W-97, lane
3), c-Src SH3 Thr-98 to Ile (I-98, lane 4),
and c-Src SH3 Arg-97 and Thr-98 to Trp and Ile, respectively
(W-97+I-98, lane 5). Lysates of metabolically
35S-labeled NIH3T3 fibroblasts were incubated with 5 µg
of the indicated GST fusion proteins, GST alone (lane 1),
the GST SRA v-Src SH3 domain (lane 6), the GST v-Src SH3
with a Pro-133 to Leu mutation (L-133, lane 7),
the GST v-Src SH3 with a Trp-118 to Arg mutation (R-118,
lane 8), or the GST c-Src SH2 domain (lane 9). In
addition, FAK was immunoprecipitated (IP) from
35S-labeled NIH3T3 cell lysates (lane 10). The
proteins associated with the various GST fusion proteins in the
pull-down assays were resolved by SDS-PAGE, transferred to a membrane,
and visualized by autoradiography (upper panel). Proteins
associated with the GST v-Src SH3, Src SH3 (W-97), and Src
SH3 (W-97+I-98), but not the c-Src SH3 domain, are indicated
on the left by arrows. Following autoradiography,
the membrane was sequentially analyzed by anti-P.Tyr,
p130Cas, and FAK blotting. Comparable amounts of GST fusion
proteins were employed as shown by Coomassie Blue staining (lower
panel).
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Fig. 7.
The Trp-97 + Ile-98 mutations in the c-Src
SH3 domain facilitate an SH2-independent binding interaction with FAK
in vivo. A, schematic overview of
either combined Trp-97 + Ile-98 substitutions, an activating Phe-529
mutation, or inactivating (Ser-177 and Met-297) mutations introduced
into murine c-Src constructs. B, human 293T cells were
co-transfected with HA-tagged Phe-397 FAK (2.5 µg) and empty vector
(pCDNA) or the indicated c-Src mutants (2.0 µg)
containing an inactivated SH2 domain (Ser-177). Transfected cells were
serum-starved, held in suspension for 45 min, and lysed in RIPA buffer.
Src was immunoprecipitated, and the Src-associated proteins were
visualized by sequential anti-HA-tag, P.Tyr, or c-Src blotting. FAK
Phe-397 expression was verified by HA tag blotting of whole cell
lysates (WCLs).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Sequence alignments of c-Src SH3-binding motifs
represents hydrophobic
residues; and p represents residues that tend to be proline. Amino
acids that are important for binding are boxed.
Interestingly, proteins such as the p85 subunit of phosphatidylinositol
3'-kinase (PI3K) have been shown to bind both the c-Src and v-Src SH3
domains (34, 54). Residues 88-101 from the p85 PI3K subunit conform to
the c-Src SH3 class I consensus (Table I). However, no structural
studies have been performed to our knowledge to establish whether the
v-Src SH3 domain binds to PXXP target motifs in a similar or distinct
manner as the c-Src SH3 domain. Another protein that has been shown to
associate preferentially with the v-Src SH3 but not c-Src SH3 domain is
connexin-43 (55). Sequence comparisons between the v-Src SH3-binding
site in connexin-43 (residues 274-284) and in the p85 PI3K subunit
(residues 88-99) reveal an overlapping potential consensus motif of
PXXPXX (where
is a hydrophobic residue)
(Table II). Significantly, this motif is
also found in the three identified v-Src SH3-binding sites in FAK.
Residues 871-882 of FAK contain an overlapping PXXPXXP motif, and a
synthetic peptide encompassing this motif potently inhibited v-Src SH3
binding to FAK in a dose-dependent manner. Notably,
mutation of FAK residues Pro-871 and Pro-872 that did not detectably
disrupt v-Src SH3 binding (Fig. 4C) also does not completely
disrupt the overlapping and remaining PXXPXX
consensus at FAK
residues 876-882.
|
Since the RT loop region of the c-Src SH3 domain lies in close
proximity to the ligand-binding groove on the surface of the SH3 domain
(28), we speculate that the Trp-97 + Ile-98 substitutions found in the
v-Src SH3 domain may provide an extended hydrophobic binding interface
for a PXXPXX motif in addition to the normally selected class I
c-Src SH3 binding consensus. This hypothesis is supported by our
findings that 1) targets such as p130Cas bind equally well
to the c-Src and v-Src SH3 domains and 2) that the (Trp-97 + Ile-98)
substitutions within the c-Src SH3 domain promoted the selective gain
(without noticeable loss) of bound target proteins in pull-down assays
using 35S-labeled NIH3T3 cell lysates. Whereas targets such
as the p85 PI3K subunit contain both c-Src and v-Src SH3 domain
consensus binding motifs (Tables I and II), proteins such as
connexin-43 and the FAK-CT domain that contain PXXPXXP motifs exhibit
stronger in vitro v-Src SH3 binding activity than targets
with a PXXPXX
consensus.2
What may be the biological significance of additional v-Src SH3-mediated binding interactions with target proteins such as FAK? Recent studies using a temperature-sensitive v-Src isoform have found that v-Src induces increased FAK tyrosine phosphorylation at sites other than Tyr-397 based on the reactivity of a phospho-specific antibody (27). Our previous studies showed that FAK Tyr-397 is phosphorylated in v-Src3T3 cells by in vivo 32P labeling and phosphopeptide mapping (23). We have also reported that v-Src forms a stable complex with FAK in both adherent and suspended v-Src3T3s (21), and we now show that this complex is formed with a Phe-397 FAK mutant and can be mediated by c-Src SH3 (Trp-97 + Ile-98) binding to FAK in the absence of a functional SH2 domain. It is likely that wild type v-Src employs both its SH2 and SH3 domains in binding to FAK and that this contributes to the stability of the adhesion-independent signaling complex formed by v-Src and FAK. Although it is intriguing that v-Src may promote FAK tyrosine phosphorylation at sites distinct from that initiated after integrin stimulation of normal cells (27), in vivo FAK phosphopeptide mapping yielded only minor differences between tyrosine-phosphorylated FAK from v-Src3T3 and fibronectin-stimulated NIH3T3 fibroblasts (23).
Other potential biological connections come from studies showing that
changes in chicken c-Src SH3 domain RT loop residues Arg-95 and Thr-96
to Trp-95 and Ile-96, respectively (note that in chicken c-Src Arg-95
corresponds to mouse c-Src Arg-97), were sufficient to convert c-Src
into a transforming protein promoting anchorage-independent cell growth
and tumor formation in newborn chickens (56). Variants of chicken c-Src
encoding a Trp-95 substitution also produced alterations in cell
morphology as well as changes in the in vivo pattern of
tyrosine-phosphorylated proteins (57). Although recent studies have
shown that overexpression of Phe-397 FAK in temperature-sensitive
v-Src-expressing cells did not block a morphological conversion
phenotype (27), our studies show that Phe-397 FAK forms a
Grb2-containing signaling complex in cells co-transfected with v-Src.
Notably, studies have also shown that FAK positively contributes to
ERK2/MAP kinase activation and the anchorage-independent growth of
v-Src-transformed cells in soft agar (58). Future studies using the
overexpression of the FAK-CT domain as a competitive inhibitor of v-Src
SH3-targeted interactions and as a dominant-negative inhibitor of
endogenous FAK function will be aimed at elucidating the specific role
of v-Src SH3-FAK interactions in cell transformation events.
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ACKNOWLEDGEMENTS |
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We thank Amanda Moore and Datsun Hsia for administrative and technical assistance, respectively.
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FOOTNOTES |
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* This work was supported in part by American Cancer Society Grant RPG-98-109-01-TBE and in part by NCI Grants R29-CA75240 (to D. D. S.) and CA39780 (to T. H.) from the National Institutes of Health. This is manuscript number 13622-IMM from The Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported in part by the Deutsche Forschungsgemeinschaft Grant HA-2856/1-1.
Frank and Else Schilling American Cancer Society Research Professor.
** To whom correspondence and requests for materials should be addressed: The Scripps Research Institute, Dept. of Immunology, IMM26, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8207; Fax: 858-784-8227; E-mail: dschlaep@scripps.edu.
Published, JBC Papers in Press, March 1, 2001, DOI 10.1074/jbc.M009329200
2 D. Schlaepfer, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: v-Src, viral Src; PTK, protein-tyrosine kinase; CT, C-terminal; GST, glutathione S-transferase; HA, hemagglutinin; FAK, focal adhesion kinase; FN, fibronectin; IP, immunoprecipitation; MAP, mitogen-activated protein; NT, N-terminal; PXXP, proline-X-X-proline, where X is not a selected amino acid; P.Tyr, phosphotyrosine; PAGE, polyacrylamide gel electrophoresis; SH, Src homology; WCL, whole cell lysate; mAb, monoclonal antibody; WT, wild type; PI3K, phosphatidylinositol 3'-kinase; ERK, extracellular signal-regulated kinase.
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