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Mol Cell Biol, May 1998, p. 2571-2585, Vol. 18, No. 5
Department of Immunology,
Received 30 October 1997/Returned for modification 8 December
1997/Accepted 5 February 1998
Fibronectin receptor integrin-mediated cell adhesion triggers
intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we
show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and
focal adhesion kinase (FAK) can be independently activated after
fibronectin (FN) stimulation and that their combined activity promotes
signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase
through multiple pathways upstream of Ras. FN stimulation of NIH 3T3
fibroblasts promotes c-Src and FAK association in the Triton-insoluble
cell fraction, and the time course of FN-stimulated ERK2 activation
paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to
Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK
in vitro kinase activity and signaling to ERK2, but it only partially
inhibited c-Src activation. Treatment of fibroblasts with protein
kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1
resulted in reduced Src PTK activity, no Grb2 binding to FAK, and
lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was
not significantly affected by herbimycin A treatment and, under these
conditions, FAK autophosphorylation promoted Shc binding to FAK. In
vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and
Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN
stimulation of human 293T cells and its expression did not inhibit
signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317
Shc also did not block signaling to ERK2, whereas FN-stimulated
signaling to ERK2 was inhibited by coexpression of an SH3
domain-inactivated mutant of Grb2. Our studies show that FN receptor
integrin signaling upstream of Ras and ERK2 does not follow a linear
pathway but that, instead, multiple Grb2-mediated interactions with
Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets
represent parallel signaling pathways that cooperate to promote maximal
ERK2 activation.
Cell adhesion to extracellular
matrix (ECM) proteins can generate transmembrane signals important for
cell survival and can promote directed cell migration events. In a
variety of cell types, integrin stimulation by ECM proteins such as
fibronectin (FN) leads to changes in intracellular protein tyrosine
phosphorylation events. In fibroblasts, the focal adhesion kinase
(FAK), a nonreceptor protein-tyrosine kinase (PTK), colocalizes with
integrin receptors at sites of cell attachment to ECM proteins. FAK may
associate directly with In addition to integrin stimulation of FAK, ECM protein binding to
cells can lead to changes in the tyrosine phosphorylation of a number
of different signaling proteins, including p130Cas, Shc,
and Cbl, as well as structural proteins such as paxillin and tensin.
Integrin stimulation can also promote increases in intracellular
calcium levels (51), protein kinase C activity (32,
56), and phosphatidylinositol (PI) 3-kinase activity (7,
28). One downstream target for integrin-initiated signaling events is the activation of the extracellular signal-regulated kinase
2/mitogen-activated protein (ERK2/MAP) kinase pathway (9, 38, 39,
47, 59). Although integrin-initiated signaling to ERK2 is
dependent on the integrity of the actin cytoskeleton and involves the
activation of both the Rho and the Ras families of small GTPase
proteins (12, 40), the integrin signaling pathways upstream
of Ras have not been clearly defined.
Attempts to delineate the molecular mechanisms of integrin-stimulated
signaling to ERK2 have yielded potentially conflicting results. In NIH
3T3 fibroblasts, Grb2 transiently binds to a motif surrounding FAK
Tyr-925 after FN stimulation (47), with the binding of
Src-family PTKs to the motif surrounding the FAK autophosphorylation site (Tyr-397) being important for Src-mediated phosphorylation of FAK
Tyr-925 in vivo (48). Direct Grb2 binding to FAK and association with the Sos GDP-GTP exchange protein for the small G
protein Ras is one pathway through which integrins may activate ERK2.
However, partial ERK activation in NIH 3T3 fibroblasts can be achieved
by antibody-mediated clustering of a chicken The PTK responsible for integrin antibody-mediated Shc tyrosine
phosphorylation in suspended cells has not been defined, but overexpression of Shc mutated at the Tyr-317 Grb2 binding site can
block integrin-stimulated ERK2 activation (57), a finding that underscores the importance of Grb2-Shc interactions in integrin signaling events. Interestingly, subsequent studies have shown that
full integrin and Shc-mediated activation of Ras requires physical
attachment and/or cell spreading on ECM ligands (37). Cell
attachment and spreading on ECM ligands are conditions that stimulate
FAK activation, suggesting that FAK also could play a role in Shc
phosphorylation. Overexpression of FAK in 293T cells enhances
FN-stimulated ERK2 activation, and this signaling event is dependent
upon FAK autophosphorylation at Tyr-397 (49). However, FAK-enhanced FN-stimulated signaling to ERK2 is not dependent on Grb2
binding to FAK Tyr-925. Significantly, an alternate FAK-mediated signaling route is through enhanced c-Src PTK activation, Shc tyrosine
phosphorylation, and Grb2 binding to Shc after FN stimulation of cells
(49). Overexpression of the c-Src binding site mutant of FAK
(Phe-397) inhibits FN-stimulated signaling to ERK2, implying that
Src-family PTK binding may be essential for FAK-mediated signaling
events (49).
Studies performed with Src-deficient fibroblasts show that
FN-stimulated signaling to ERK2 is significantly reduced compared to
the same cells reexpressing normal mouse c-Src (46).
Although FAK is activated normally in Src-deficient cells, the
FN-stimulated tyrosine phosphorylation of targets such as
p130Cas and Shc are reduced compared to cells reexpressing
c-Src. Surprisingly, expression of a dominant-negative fragment of
c-Src in the Src-deficient cells enhanced FAK and p130Cas
phosphotyrosine (P.Tyr) levels and did not block FN-stimulated signaling to ERK2 (46). This result supports the existence
of an integrin-activated and Src-independent signaling pathway. In this
current study, we extend these findings by showing that FAK and c-Src
can be regulated independently after FN stimulation of NIH 3T3
fibroblasts and that in the absence of Src PTK activity, FAK can
associate and directly phosphorylate Shc at Tyr-317 to promote Grb2
binding and low-level signaling to ERK2. Moreover, through the combined
use of pharmacological inhibitors with NIH 3T3 fibroblasts and protein
overexpression studies in 293T epithelial cells, we find that
FN-stimulated signaling to ERK2 is dependent upon PTK activity,
involves signaling inputs from protein kinase C, and is not blocked by
Phe-317 Shc overexpression but instead may be mediated by multiple
Grb2-mediated interactions with Shc, FAK, or other yet-to-be-determined
phosphorylated targets. Our findings support the conclusion that
multiple and potentially parallel signaling pathways can regulate the
extent and duration of FN receptor integrin-stimulated ERK2 activation.
Reagents.
Calphostin C, cytochalasin D, enolase, bovine
plasma-purified FN, herbimycin A, PP1, and tyrphostin A47 (AG213) were
purchased from Calbiochem (San Diego, Calif.). With calphostin C, cell
treatments were performed in the presence of a fluorescent light source
to photoactivate the drug. All other reagents or chemicals were
purchased from Sigma Chemical Co. (St. Louis, Mo.) unless stated
otherwise. Purified recombinant mouse c-Src was a generous gift from M. Broome (The Salk Institute).
Cells, fusion proteins, plasmids, and antibodies.
Mouse NIH
3T3 fibroblasts and human kidney epithelial 293T cells were maintained
in Dulbecco's modified Eagle medium (DMEM) supplemented with 10%
bovine calf serum (CS), penicillin (50 U/ml), and streptomycin (50 µg/ml). Glutathione S-transferase (GST) fusion proteins
encompassing either the c-Src or the Grb2 SH2 domains were produced and
purified as described previously (47). Epitope-tagged wild-type (WT), Phe-397, Arg-454, and Phe-925 mouse FAK cloned by using
pCDNA3 (Invitrogen, La Jolla, Calif.) and epitope-tagged ERK2 cloned by
using pLNC were used as described previously (49). Epitope-tagged WT and Phe-317 human Shc (54) cloned by using pCDNA3 were kindly provided by P. van der Geer (University of California, San Diego, Calif.). WT and an SH3 domain-inactivated mutant
of human Grb2 (Leu-49 and Leu-206) cloned by using pSLX were used as
described (14). 293T cells attached to cell culture dishes
precoated with 25 µg of poly-L-lysine (PLL) per ml were transfected by standard calcium phosphate methods with either 5 µg
(FAK, Shc, and Grb2) or 1 µg of ERK2 plasmid constructs in growth
medium containing chloroquine (25 µM). Control pCDNA3 vector was
added as necessary to equalize the total amount of DNA transfected. The
cells were incubated at 37°C (5% CO2) for 8 h, the
precipitate was removed by washing with phosphate-buffered saline
(PBS), and the cells were incubated with DMEM containing 10% CS for
24 h. Cells were serum starved in DMEM containing 0.5% CS for
24 h prior to cell lysis or FN-replating experiments.
Cell stimulation with FN or adherence to PLL.
Cells were
serum-starved in DMEM containing 0.5% CS for 24 h and harvested
by limited trypsin-EDTA treatment (0.05% trypsin and 2 mM EDTA in
PBS). The trypsin was inactivated by soybean trypsin inhibitor (0.5 mg/ml) with 0.25% bovine serum albumin (BSA; Fraction V; ICN
Biomedicals, Aurora, Ohio) in DMEM, and cells were collected by
centrifugation, resuspended in DMEM containing no serum with 0.1% BSA,
and held in suspension for 1 h at 37°C (2 × 105 cells/ml). Cell culture dishes (10 cm diameter) were
precoated with FN purified from bovine plasma (10 to 20 µg/ml) or PLL
(100 µg/ml) in PBS overnight at 4°C, rinsed with PBS, and warmed to 37°C for 1 h prior to replating. Suspended cells were
distributed onto ligand-coated dishes (106 cells per dish)
and incubated at 37°C; at various times following plating, the
attached cells were rinsed in PBS (4°C) and lysed in 0.75 ml of
modified radioimmunoprecipitation assay (RIPA) lysis buffer (see
below). Total cell protein in lysates from serum-starved, suspended, or
replated cells was determined by a colorimetric assay (Bio-Rad
Laboratories, Hercules, Calif.) and standardized prior to further
analyses.
Cell lysis, immunoprecipitation, and immunoblotting.
Unless
otherwise described, cells in suspension or on 10-cm-diameter culture
dishes were lysed at 4°C with 0.75 ml of modified RIPA lysis buffer
(1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl
sulfate [SDS], 50 mM HEPES [pH 7.4], 150 mM NaCl, 10% glycerol,
1.5 mM MgCl2, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM
sodium pyrophosphate, 100 mM NaF, leupeptin [10 µg/ml], aprotinin
[100 U/ml], and 1 mM phenylmethylsulfonyl fluoride) and insoluble
material was removed by centrifugation (16,000 × g, 10 min at 4°C). Antibodies or purified GST fusion proteins (5 µg) were
added to the cell lysates and incubated for 2 h at 4°C, and the
antibodies were collected on protein A (Repligen, Cambridge, Mass.) or
protein G-plus (Calbiochem, La Jolla, Calif.) agarose beads, whereas
the GST fusion proteins were collected by binding to
glutathione-agarose beads. The precipitated protein complexes were
washed at 4°C in Triton-only lysis buffer (1% Triton X-100, 50 mM
HEPES [pH 7.4], 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2,
1 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM NaF, leupeptin [10 µg/ml], aprotinin [100 U/ml], and 1 mM
phenylmethylsulfonyl fluoride) followed by washing in HNTG buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 0.1% Triton X-100, 10% glycerol)
prior to direct analysis by SDS-polyacrylamide gel electrophoresis
(PAGE) or in vitro 32P labeling.
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Multiple Grb2-Mediated Integrin-Stimulated
Signaling Pathways to ERK2/Mitogen-Activated Protein Kinase: Summation
of Both c-Src- and Focal Adhesion Kinase-Initiated Tyrosine
Phosphorylation Events
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
integrin cytoplasmic domains
(44) or may cocluster with integrin receptors through
interactions with other integrin-associated proteins (4, 8,
22). FAK tyrosine phosphorylation is stimulated by cell binding
to ECM proteins (for a review, see reference 50), by
overexpression of the integrin cytoplasmic domains (52) and also by other growth factor or serum mitogens (for a review, see
reference 24). Since integrin receptors lack
catalytic activity, FAK association and activation may be important for
integrin-mediated signal transduction events (for a review, see
reference 20). This hypothesis is supported by gene
knockout results in which both the FN- and FAK-null mice die as a
result of similar developmental gastrulation defects (15,
25).
1 integrin subunit lacking the cytoplasmic domain without concomitant activation of FAK (35). Although the components of this pathway remain to be defined, other studies also find that antibody-mediated clustering of 
11,
51, and v3
integrins in suspended cells can stimulate Shc tyrosine
phosphorylation, Grb2 binding to Shc, and ERK2 activation without
detectable tyrosine phosphorylation of FAK (57).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-mercaptoethanol at 75°C
for 30 min. Prior to reprobing with different primary antibodies,
stripped membranes were washed extensively in TBST and placed in
blocking buffer overnight.
Immune complex 32P-labeled kinase reactions.
FAK
or c-Src immunoprecipitates (IPs) were washed in Triton-only lysis
buffer, followed by HNTG buffer, and then kinase buffer (20 mM HEPES
[pH 7.4], 10% glycerol, 10 mM MgCl2, 10 mM
MnCl2, 150 mM NaCl). To initiate kinase reactions, excess
buffer was removed from the IPs, 2.5 µl of [
-32P]ATP
(3,000 Ci/mmol, 10 µCi/µl) was added, and the IPs were incubated for 15 min at 37°C (~30 µl, total volume). For direct analyses, reactions were stopped by the addition of 2× SDS-polyacrylamide gel
electrophoresis (PAGE) sample buffer. To dissociate protein complexes
for subsequent immunoprecipitation steps, kinase reactions were stopped
by the addition of 100 µl of kinase-SDS stop solution (1% SDS, 1 mM
EDTA, 5 mM dithiothreitol, 20 mM NaPO4 [pH 7.4]) and
boiled for 2 min; the agarose beads were then removed by
centrifugation. The supernatant was divided in half and analyzed
directly by SDS-PAGE or diluted 15-fold with Triton-only lysis buffer
and subjected to antibody reimmunoprecipitation for 2 h at 4°C.
The secondary IPs were washed in HNTG buffer and analyzed directly by
SDS-PAGE.
-32P]ATP
per nmol), incubated at 37°C for 15 min, and stopped by the addition
of a 2× SDS-PAGE sample buffer. The phosphorylated MBP was resolved on
a 17.5% acrylamide gel and visualized by autoradiography, and the
amount of 32P incorporated into MBP was determined by
Cerenkov counting.
To measure quantitatively c-Src kinase activity, IPs (monoclonal
antibody 2-17 covalently coupled to protein G agarose
[47]) from ~500 µg of total NIH 3T3 cell protein
in RIPA lysis buffer were washed in Triton lysis buffer, HNTG buffer,
and then enolase kinase buffer (20 mM PIPES [pH 7.0], 10 mM
MnCl2, 1 mM dithiothreitol). Acid-denatured enolase (2.5 µg) was added to each IP. Kinase reactions (~35 µl, total volume)
were initiated by the addition of an ATP mix (5 µl; final
concentration of 20 µM ATP and 10 µCi [-32P]ATP
per nmol), incubated at 30°C for 10 min, and stopped by the addition
of 2× SDS-PAGE sample buffer, and the products were resolved on a 10%
acrylamide gel. The enolase band was visualized by autoradiography and
cut from the gel, and the amount of 32P incorporated was
determined by Cerenkov counting. FAK kinase activity in Triton-only
cell lysates was measured by autophosphorylation as described above in
the presence of 20 µM ATP (10 µCi of [-32P]ATP per
nmol) incubated at 30°C for 10 min.
Phosphopeptide mapping. 32P-labeled FAK was digested with trypsin and subjected to two-dimensional phosphopeptide mapping as described previously (3). Electrophoresis in pH 1.9 buffer (2.2% formic acid, 7.8% acetic acid) was performed for 1 h at 1,000 V, and ascending chromatographic separation was performed overnight in phosphochromo buffer (37.5% n-butanol, 25% pyridine, and 7.5% acetic acid in water).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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FAK, p130Cas, paxillin, and Shc are tyrosine phosphorylated in quiescent as well as FN-stimulated NIH 3T3 fibroblasts. Several studies have shown that integrin receptor activation, mediated either by antibody clustering or through cell interactions with ECM proteins, can lead to the activation of the Ras/ERK2 MAP kinase pathway. However, the critical initial signaling events involved and the regulatory factors that modulate these pathways still remain relatively undefined. To investigate the role of tyrosine phosphorylation in FN-stimulated signaling events to ERK2, whole-cell lysates (WCLs) (Fig. 1A) or specific IPs of p130Cas (Fig. 1B), FAK (Fig. 1C), paxillin (Fig. 1D), or Shc (Fig. 1E) were made from either serum-starved NIH 3T3 fibroblasts, starved NIH 3T3 fibroblasts detached by limited trypsin-EDTA treatment and held in suspension for 30 min, or suspended NIH 3T3 fibroblasts that were replated onto either FN (10 µg/ml)- or PLL (100 µg/ml)-coated cell culture dishes for the times indicated (Fig. 1 and 2). This assay activates integrin receptors by presenting cells to an immobilized ECM substrate.
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51
integrin receptor, P.Tyr levels in p130Cas, FAK, and Shc
were rapidly and maximally elevated within 20 min (Fig. 1 and 2) as the
cells started to spread (see Fig. 3A to D). Maximal ERK2
phosphorylation (ERK2-P) and activation also occurred within 20 min
after FN plating as detected by anti-P.Tyr blotting of WCLs (Fig. 1A,
lanes 3 to 8) or monitored by the appearance of a second protein band
with decreased SDS-PAGE mobility by ERK2 blotting (Fig. 2D). Control
experiments were performed by plating suspended cells onto PLL-coated
dishes, where the cells "stick" independently of integrin
activation. On PLL-coated plates, the NIH 3T3 fibroblasts remained
rounded for the first 60 min (Fig. 3E to
H) and began to spread slowly after 60 min, at which time p130Cas and FAK become weakly tyrosine phosphorylated (Fig.
2E). Significantly, the cell spreading and tyrosine phosphorylation
events on PLL-coated plates after 60 min did not stimulate signaling to
ERK2 (Fig. 2H).
|
FAK and Shc "activation" as measured by Grb2 SH2-dependent binding. Anti-P.Tyr blotting analyses of p130Cas, FAK, and Shc IPs showed that these proteins were rapidly rephosphorylated after FN plating and that their P.Tyr levels remained elevated beyond the period of maximal ERK2 activation (Fig. 1B, C, and E). In contrast, paxillin tyrosine phosphorylation was low during the period of maximal signaling to ERK2 (Fig. 1D, lanes 3 to 6) and became elevated during the formation of actin stress fibers and focal contacts 1 to 2 h after FN stimulation (data not shown). Although the tyrosine phosphorylation of FAK, p130Cas, and Shc is important for these proteins to interact with Src homology 2 (SH2) domain-containing signaling proteins, measures other than P.Tyr blotting analyses are required to determine the "activation" states of these proteins. With respect to FAK, it has been shown that Tyr-397 is phosphorylated in vivo in serum-starved as well as in FN-plated cells (48). In a finding consistent with this observation, in vitro binding of the c-Src SH2 domain to FAK was observed in quiescent serum-starved cells (Fig. 2A, lane 1) within 20 min of FN stimulation (Fig. 2A, lane 3) and within 60 to 120 min of PLL plating (Fig. 2F) and was therefore directly correlated with the extent of Tyr-397 phosphorylation. However, in vivo SH2-dependent c-Src PTK binding to this site is tightly regulated and occurs transiently only after FN stimulation of NIH 3T3 cells (47) (see Fig. 4).
Even though in vitro c-Src SH2 domain binding to FAK does not necessarily correlate with FN-stimulated ERK2 activation events, FAK phosphorylation at Tyr-397 is necessary for the recruitment of Src family PTKs in the formation of an integrin-stimulated signaling complex (49). The formation of an FAK-c-Src complex permits c-Src to phosphorylate FAK within the catalytic domain (5) and at Tyr-925 (47), thus enhancing FAK kinase activity and promoting Grb2 binding, respectively. The Grb2 SH2 domain, which specifically binds to the FAK peptide containing phosphorylated Tyr-925 (48), only bound to FAK in vitro during the initial period (20 to 80 min) of FN-stimulated signaling to ERK2 (Fig. 2B). Control plating of NIH 3T3 cells onto PLL did not promote Grb2 SH2 binding to FAK (Fig. 2G), indicating that this binding assay can be used as an indirect measure of FAK "activation." As with FAK, Grb2 binding to Shc has been shown to be an integrin-stimulated signaling event upstream of ERK2 (37, 46, 57). In vitro, the GST-Grb2 SH2 domain bound to Shc from lysates made after serum starvation and FN replating but not from lysates of suspended or PLL-plated NIH 3T3 cells (data not shown). Although Shc is tyrosine phosphorylated under serum-starved conditions (Fig. 1E), ERK2 is not activated (Fig. 2D) and in vitro Grb2 binding to Shc under these conditions may not reflect in vivo signaling complexes. To determine conditions in which Grb2 binding to Shc parallels signaling events in NIH 3T3 fibroblasts, Shc IPs were made from serum-starved and FN-plated NIH 3T3 fibroblasts and blotted for the presence of associated Grb2 (Fig. 2C). Grb2 did not coimmunoprecipitate with Shc in lysates from serum-starved cells or suspended cells (Fig. 2C, lanes 1 and 2), whereas Grb2 was associated with Shc within 20 min of FN plating (Fig. 2C, lane 3). Interestingly, this Grb2-Shc association paralleled the time course of FN-stimulated ERK2 activation (20 min to 2 h) and persisted beyond the period of Grb2 binding to FAK (20 to 80 min). The Grb2-Shc association declined after 2 h (Fig. 2C, lanes 9 and 10) even though Shc remained tyrosine phosphorylated at these later time points (Fig. 1E).Activated c-Src associates with FAK during the period of
FN-stimulated signaling to ERK2.
Activation of Src family PTKs is
required for the phosphorylation of FAK at Tyr-925 to promote Grb2
binding, but the kinase(s) responsible for integrin-stimulated Shc
tyrosine phosphorylation has not been clearly defined. Since FAK
overexpression enhances both Shc tyrosine phosphorylation and c-Src PTK
activity after FN stimulation (49), experiments were
performed to measure the kinetics c-Src PTK activation after FN
stimulation of NIH 3T3 fibroblasts. In vitro kinase assays in the
presence of [-32P]ATP were performed from RIPA
cell lysates containing equal amounts of immunoprecipitated c-Src
(data not shown) from either serum-starved, suspended, or FN-plated
cells for the times indicated (Fig. 4). Assays were performed in either the presence (see Fig. 6A; data not
shown) or absence (Fig. 4A) of added acid-denatured enolase protein
substrate, and the labeled proteins were resolved by SDS-PAGE. Enhanced
32P incorporation into c-Src (Fig. 4A) or into enolase
(data not shown) was observed 20 to 40 min following FN stimulation.
Significantly, a labeled ~120-kDa protein coimmunoprecipitated with
c-Src at the early time points after FN stimulation (Fig. 4A). This
band was subsequently identified as FAK by denaturation of duplicate 32P-labeled c-Src IPs prior to analyzing secondary IPs with
antibody specific for FAK (Fig. 4B). This transient FAK association
with c-Src correlated with the period of GST-Grb2 binding to FAK (Fig. 2B) and supports the hypothesis that FAK autophosphorylation after FN
stimulation is a signal to recruit Src family PTKs to sites of integrin
clustering (43, 47).
|
-32P]ATP (Fig. 4C). Whereas FAK kinase activity as
measured by autophosphorylation was low in serum-starved and suspended
cells, FAK in vitro phosphorylation and association with a strongly
labeled protein at ~60 kDa, as well as with a weakly labeled band at
~52 kDa, were significantly enhanced 20 to 60 min after FN
stimulation (Fig. 4C, lanes 3 to 6).
The FAK-associated ~60-kDa band was identified as c-Src by
denaturation of duplicate 32P-labeled FAK IPs prior to
performing secondary immunoprecipitations with a monoclonal antibody
specific for c-Src (Fig. 4D). Phosphopeptide mapping of the
FAK-associated 52-kDa protein were similar to those of in
vitro-phosphorylated Shc (data not shown, see Fig. 8). No significant
c-Src association with FAK could be detected by immunoblotting in FAK
IPs from serum-starved cell lysates (data not shown), even though the
c-Src binding site at FAK Tyr-397 is phosphorylated under these
conditions (Fig. 2A and F) (48). These results confirm that
the association between c-Src and FAK after FN stimulation of NIH 3T3
cells is tightly regulated. Although these in vitro kinase assays (Fig.
4) cannot discriminate FN-stimulated FAK autophosphorylation from c-Src
transphosphorylation of FAK, the time course of enhanced FAK kinase
activity between 20 min and 2 h in the FN-stimulated NIH 3T3 cells
was similar to the extended time course of FN-stimulated FAK in vitro
kinase activity from Src-deficient fibroblasts (46). In
addition, our NIH 3T3 fibroblast results show that the time course of
enhanced PTK activity of both c-Src and FAK parallels the period of
maximal FN-stimulated signaling to ERK2.
FAK and c-Src associate with the Triton-insoluble cell fraction after FN-stimulation of NIH 3T3 fibroblasts. The fact that the dissociation of the FAK-c-Src complex (~80 min after FN stimulation) occurred prior to the complete down-regulation of FAK or c-Src in vitro kinase activities (both occur at ~2 h after FN stimulation) (Fig. 4A and C) suggests that the regulation of both FAK and c-Src association and PTK activity is complex and partly independent. Significantly, if NIH 3T3 fibroblasts were extracted with 1% Triton cell lysis buffer instead of RIPA lysis buffer, both FAK and c-Src IPs exhibited lower levels of FN-stimulated in vitro kinase activity and the amount of coimmunoprecipitation of the other PTK was significantly reduced (data not shown), suggesting that this PTK complex may be predominantly localized to the Triton-insoluble cell fraction.
Previous studies have shown that activated c-Src partitions to Triton-insoluble cytoskeletal complexes after platelet stimulation (11) and fibroblast stimulation (27). To determine if FAK partitions to a Triton-insoluble fraction after FN stimulation (30 min) of NIH 3T3 fibroblasts, cells were extracted with 1% Triton lysis buffer, and the remaining insoluble cell material was reextracted with RIPA lysis buffer prior to FAK immunoprecipitation analyses (Fig. 5). Although greater amounts of FAK protein were recovered from the Triton-soluble fraction, FAK extracted from the insoluble pellet fraction was highly tyrosine phosphorylated and exhibited a two- to threefold-higher specific in vitro kinase activity than Triton-soluble FAK (Fig. 5A). In serum-starved or suspended cells, FAK was not associated with the Triton-insoluble fraction (data not shown). Significantly, phosphopeptide mapping and immunoblotting (data not shown) confirmed that the labeled (Fig. 5A, lane 2) and the tyrosine-phosphorylated (Fig. 5A, lane 4) 60-kDa protein in the Triton-insoluble FAK IP was c-Src.
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FAK and c-Src can be activated independently after FN stimulation. Although our results show that FAK and c-Src transiently associate (Fig. 4) and that these signaling complexes are localized to the Triton-insoluble cell fraction after FN stimulation, Triton-soluble FAK also exhibited increased FN-stimulated in vitro kinase activity (Fig. 5, lane 1) compared to the low levels of FAK PTK activity in serum-starved cells (Fig. 4C, lane 1). Since only very low levels of c-Src are associated with Triton-soluble FAK and since FN stimulation of Src-deficient fibroblasts activates FAK (46), it is possible that integrin signals may be able to promote FAK in vitro kinase activation without significant input from c-Src. Similarly, studies in platelets have shown that integrin-stimulated increases in c-Src PTK activity occur prior to detectable FAK activation (23).
To explore the regulation of FAK and c-Src PTK activities, in vitro kinase measurements were performed on either FAK or c-Src IPs made from either serum-starved, suspended, or FN-plated NIH 3T3 fibroblasts over a time course of 20 min to 6 h (Fig. 6; data not shown). FAK PTK activity was measured by autophosphorylation in IPs after 1% Triton cell lysis. Measurements of c-Src PTK activity after RIPA cell lysis were performed with added acid-denatured enolase as a substrate. The input of associated FAK PTK activity in the c-Src IPs is minimal, since FAK does not efficiently transphosphorylate enolase (49). Both FAK and c-Src exhibited low levels of in vitro kinase activity in lysates from serum-starved and suspended cells (Fig. 6A), and these results are consistent with the results of previous phosphorylation assays (Fig. 4). Maximal activities for both FAK and c-Src (four- to eightfold over basal levels) were observed 20 to 40 min after FN but not after PLL stimulation (Fig. 6A and data not shown).
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Inhibitors of protein kinase C decrease FN-stimulated c-Src activity and reduce but do not block signaling to ERK2. Previous studies have shown that down-regulation of phorbol ester-sensitive isoforms of protein kinase C (PKC) and pharmacological PKC inhibitors such as calphostin C result in reduced integrin-stimulated FAK phosphorylation (32, 56). To test the role of PKC in FN-stimulated FAK and c-Src activation, as well as in the activation of ERK2, NIH 3T3 cells were either treated with phorbol myristate acetate (PMA; 250 ng/ml) for 24 h to down-regulate PKC or calphostin C (250 nM) was added to suspended cells prior to FN plating. Surprisingly, these treatments did not significantly affect FN-stimulated FAK in vitro kinase activity as measured by autophosphorylation (Fig. 6A) or FAK Tyr-397 phosphorylation as measured by c-Src SH2 domain binding to FAK (Fig. 6B), but these treatments reduced both FAK tyrosine phosphorylation at Tyr-925 and FN-stimulated signaling to ERK2 (Fig. 6B and C). Interestingly, PMA or calphostin C treatments reduced FN-stimulated c-Src in vitro kinase activity toward enolase approximately threefold (Fig. 6A) and also resulted in a two- to threefold reduction of peak FN-stimulated ERK2 activity (Fig. 6C).
Both PMA and calphostin C treatments of FN-stimulated NIH 3T3 fibroblasts also resulted in the inhibition of p130Cas tyrosine phosphorylation (Fig. 6B). Since Src family PTK activity has been shown to be responsible for integrin-stimulated p130Cas tyrosine phosphorylation (46, 55), these results support the conclusion that a PKC-mediated pathway is important for integrin-stimulated c-Src PTK activation and subsequent p130Cas tyrosine phosphorylation. Although direct PKC phosphorylation of c-Src does not affect c-Src in vitro kinase activity (16), PMA stimulation of 293T cells can transiently activate both c-Src and FAK in vitro kinase activities (see Fig. 8), and PMA stimulation of fibroblasts can affect Src-family PTK activity in the absence of FAK tyrosine phosphorylation potentially through the activation of protein-tyrosine phosphatases (42). These results provide further evidence that FAK and c-Src can become activated independently after FN stimulation.Elucidation of a Src-independent signaling pathway involving FAK, Shc, and Grb2. In Src-deficient fibroblasts, FAK is activated by FN stimulation and signaling to ERK2 is reduced but not totally abolished (46). It is possible that Fyn or c-Yes partially compensates for the loss of c-Src in Src-deficient fibroblasts, or it is also possible that FAK may be able to promote low-level signaling to ERK2 in the absence of Src family PTK activity. To extend the results from the PKC inhibitor studies and explore the possibility of a direct FAK-mediated signaling pathway to ERK2, Src family PTKs were inhibited in NIH 3T3 fibroblasts by extended treatment with herbimycin A (875 nM, 24 h) or by the addition of the PP1 (19) Src PTK-specific inhibitor (10 µM) to suspended cells prior to FN stimulation. Herbimycin A treatment resulted in reduced levels of c-Src but not FAK protein expression (data not shown). FN-stimulated c-Src kinase activity (Fig. 6A) and tyrosine phosphorylation of FAK Tyr-925 and p130Cas (Fig. 6B) were inhibited in the presence of herbimycin A, but these treatments had only minor effects on FN-stimulated FAK autophosphorylation activity (Fig. 6A). Similar results were obtained with PP1 addition to suspended NIH 3T3 cells prior to FN stimulation (data not shown).
Significantly, extended herbimycin A treatment reduced but did not block FN-stimulated activation of ERK2 (Fig. 6B and C), a finding similar to the results obtained with PKC inhibitors or PP1 treatment of suspended cells. Combined IP-anti-P.Tyr blotting analyses identified FAK, Shc, and ERK2 as three of the five major P.Tyr-containing proteins in the FN-stimulated and herbimycin A-treated fibroblasts (Fig. 7A). To identify the PTK that phosphorylates Shc under these conditions, Shc IPs were made from herbimycin A-treated and FN-stimulated NIH 3T3 cells and incubated in the presence of [-32P]ATP to detect
associated kinase activity (Fig. 7B). At the early time points after FN
stimulation, a ~116-kDa phosphorylated protein coimmunoprecipitated
Shc (Fig. 7B), and immunoblotting of the same membrane identified this
protein as FAK (Fig. 7C). Interestingly, Grb2 was found to
coimmunoprecipitate with Shc during the same period as the FAK-Shc
association in the herbimycin A-treated and FN-stimulated NIH 3T3 cells
(Fig. 7D). Therefore, FAK may be able to promote signaling to ERK2
through Shc tyrosine phosphorylation in NIH 3T3 fibroblasts. Although
this FAK-Shc association was best visualized under conditions of
reduced c-Src expression and PTK activity, phosphopeptide mapping
comparisons (data not shown) identified the labeled 52-kDa protein
associated with FAK after FN stimulation of NIH 3T3 fibroblasts as Shc
(Fig. 4C). These results show that FAK can promote the recruitment of
signaling proteins such as Shc, as well as Src family PTKs after FN
stimulation of cells.
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FAK can directly phosphorylate Shc to promote Grb2 binding. FAK autophosphorylation at Tyr-397 creates a binding site (P.Tyr-Ala-Glu-Ile) for SH2-containing proteins such as Src family PTKs. Since Shc coimmunoprecipitation with WT but not Phe-397 FAK after FN stimulation of 293T cells and a GST-Shc SH2 domain fusion protein binds to WT but not Phe-397 FAK in vitro (49), experiments were performed to determine whether FAK could directly phosphorylate Shc to promote Grb2 binding (Fig. 8). In vitro experiments were performed with epitope-tagged WT, Phe-397, and kinase-inactive (Arg-454) FAKs or WT and Phe-317 Shc constructs individually expressed in human 293T cells (Fig. 8A). Grb2 binding to Shc Tyr-317 has been characterized as an integrin-stimulated signaling pathway to ERK2 (37, 57).
|
FN-stimulated ERK2 activation is dependent upon Grb2-mediated signals. Previous studies have shown that Grb2 binding to Shc Tyr-317 is important for integrin activation of the MAP kinase pathway since Phe-317 Shc overexpression can block integrin-stimulated ERK2 activation (37, 57). Since FAK overexpression can potentiate FN-stimulated Shc tyrosine phosphorylation in 293T cells (49), experiments were performed to test the effects of FAK, Shc, and Grb2 overexpression on FN-stimulated signaling to ERK2 (Fig. 9A to F).
|
Multiple Grb2-mediated signaling pathways to ERK2. To determine whether FAK overexpression could enhance FN-stimulated signaling to ERK2 in the absence of direct Grb2 binding to FAK at Tyr-925 or to Shc at Tyr-317, coexpression experiments were performed with Phe-925 FAK and Phe-317 Shc in 293T cells. Expression of Phe-925 FAK with WT Shc enhanced signaling to ERK2 (Fig. 9, lane 13) to a level comparable to that stimulated by WT FAK alone (Fig. 9, lane 4) but less than that after combined expression of WT FAK and WT Shc (Fig. 9, lane 10). This result shows that FAK Tyr-925 contributes to maximal integrin-stimulated signaling to ERK2, but as previous results have documented (49), Grb2 binding to FAK Tyr-925 is not essential.
Coexpression of Phe-317 Shc with Phe-925 FAK resulted in reduced FN-stimulated activation of ERK2 compared to that obtained with Phe-925 FAK coexpressed with WT Shc (Fig. 9, lanes 13 and 14). FN-stimulated ERK2 activation in the presence of Phe-925 FAK and Phe-317 Shc was blocked by the coexpression of Leu-49/Leu-206 Grb2, but this Grb2 mutant did not block the FN-stimulated tyrosine phosphorylation of Shc Phe-317 (Fig. 9, lane 15). The level of FN-stimulated ERK2 activity with Phe-925 FAK and Phe-317 Shc expression was still elevated compared to the level of endogenous 293T ERK2 activation after FN stimulation (Fig. 9, lane 1). These results show that although FN-stimulated signaling to ERK2 in 293T cells is dependent upon Grb2 function, neither direct Grb2 binding to FAK Tyr-925 nor direct Grb2 binding to Shc Tyr-317 is essential for signaling. But since the combined expression of Phe-925 FAK with Phe-317 Shc led to reduced levels of FN-stimulated signaling to ERK2 compared to that of the WT FAK and Shc controls, our results support the hypothesis that Grb2 binding to these sites contributes to maximal FN-stimulated ERK2 activation. What targets other than FAK Tyr-925 and Shc Tyr-317 may mediate integrin signaling to ERK2? Grb2 was present in 12CA5 IPs in 293T cells expressing Phe-925 FAK and Phe-317 Shc (data not shown), and therefore it is possible that FAK-enhanced and Src-mediated phosphorylation of Shc at sites such as Tyr-239 may also promote Grb2 binding and signaling to ERK2. In addition, other Grb2 binding proteins such as Gab1 are tyrosine phosphorylated after FN stimulation and therefore could also contribute to the overall integrin-stimulated signal (45a). In summary, our studies support the hypothesis that multiple Grb2-mediated interactions with Shc, FAK, and other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.| |
DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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A number of different signaling pathways have been proposed to mediate integrin activation of ERK2. As summarized in a working model (Fig. 10), our kinetic and pharmacological studies with NIH 3T3 fibroblasts in combination with studies of protein function by coexpression analyses in human 293T cells show that FN-stimulated signaling to ERK2 is dependent on PTK activity and that both FAK and c-Src can promote ERK2 activation through more than one Grb2-mediated signaling pathway. Our studies also show that there are multiple and potentially parallel FN receptor integrin-stimulated signaling pathways upstream of Ras. Src-mediated phosphorylation of FAK Tyr-925 and both FAK and Src-mediated phosphorylation of Shc Tyr-317 promote Grb2 binding and signaling to ERK2. Importantly, although we find that these pathways contribute to maximal integrin-stimulated ERK2 activation, neither pathway is essential. Whereas conditions can be found such that one particular Grb2 pathway may be sufficient, our studies show that it is the summation of Grb2-mediated signals that regulate the extent and duration of ERK2 activation after integrin stimulation as performed by cell replating assays.
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Our study also provides evidence that FAK and c-Src may become independently activated after integrin stimulation of fibroblasts. This result may explain how integrin-generated signals in the absence of detectable FAK tyrosine phosphorylation can activate signaling pathways to ERK2 (35, 57) and are consistent with studies with FAK-deficient mouse embryonic fibroblasts whereby integrin-generated signals can activate Src-family PTKs (45b). How Src family PTKs are activated by integrin stimulation in the absence of FAK is not known, but it is possible that integrins could stimulate the activity of a protein-tyrosine phosphatase leading to the dephosphorylation of C-terminal Src family tyrosine residues and resulting in the conformational activation of Src family PTKs (41, 58). Another potential link between integrins and Src family PTK activation also involves PKC, which is also rapidly activated upon FN stimulation of cells (56).
Although previous studies have shown that integrin-stimulated PKC
activation precedes 51 FN-stimulated cell
spreading (56) and that inhibitors of PKC decrease
v5-dependent FAK tyrosine phosphorylation
(32), our results show that some of these effects may be
mediated through the prevention of full c-Src PTK activation after FN
stimulation. By evaluating known targets of c-Src PTK activity, such as
p130Cas and FAK Tyr-925 phosphorylation, and by
measurements of in vitro kinase activities, we show that these PKC
inhibitors prevented c-Src but not FAK activation after FN stimulation
and resulted in decreased but not abolished signaling to ERK2. Although
the use of pharmacological inhibitors is an indirect means for
assessing protein function, we also found that cytochalasin D treatment of cells prevented FN-stimulated FAK activation and signaling to ERK2.
These results with cytochalasin D are consistent with previous studies
(9, 36) and support the hypothesis that FN
receptor-stimulated FAK activation is indirect and mediated by actin
cytoskeletal associations or through binding interactions with other
integrin-associated proteins. The fact that cytochalasin D did not
completely inhibit increases in FN-stimulated c-Src activity further
supports our model that integrin signals can activate Src-family PTKs
in a FAK-independent manner.
Although FAK and Src-family PTKs respond differentially to specific integrin-generated signals downstream of the actin polymerization or PKC, we found that maximal c-Src and FAK activation after FN stimulation may require a physical association between these two PTKs. Previous studies have shown that c-Src preferentially partitions to the Triton-insoluble fraction of platelets (13) and localizes to cell substratum adhesion contact sites after integrin stimulation of fibroblasts (26). Our results here show that after FN stimulation of NIH 3T3 fibroblasts, activated FAK and c-Src are both associated with the Triton-insoluble fraction of cells. This is the first demonstration of FAK redistribution after integrin stimulation that reinforces the importance of using strong detergent cell lysis conditions such as RIPA buffer containing 1% Triton, 1% sodium deoxycholate, and 0.1% SDS to analyze FAK tyrosine phosphorylation events. Previous studies with such lysis conditions have demonstrated that FN-stimulated FAK tyrosine phosphorylation occurs early within 10 min (17) and that FAK tyrosine phosphorylation can occur prior to integrin-stimulated cell spreading events (30). Other studies with milder cell lysis conditions have failed to detect early FAK tyrosine phosphorylation events after integrin stimulation (35, 57).
Since we show that FAK and c-Src transiently associate after FN stimulation, this FAK-Src association may prolong the activation of both PTKs. Although recent structural determinations have confirmed that the non-ligand-bound SH2 and SH3 domains of c-Src can act to constrain c-Src kinase domain activity (58), much less is known about the regulation of FAK kinase activity. In this study, we show that although FAK is highly tyrosine phosphorylated in serum-starved NIH 3T3 fibroblasts, it exhibits only minimal in vitro kinase activity. FN but not PLL replating of NIH 3T3 cells promoted both FAK tyrosine phosphorylation and in vitro kinase activity compared to that measured in suspended cells. Interestingly, FN-stimulated FAK in vitro kinase activity decreased over time without significant alterations in total FAK P.Tyr content. This decrease in FAK activity began at the same time as that observed for actin stress fiber and focal contact formation in the spreading NIH 3T3 fibroblasts. We speculate that FAK incorporation into these mature focal contact structures may promote c-Src dissociation and a down-regulation of FAK kinase activity through conformational changes or serine phosphorylation events (6, 48) without a concomitant reduction in observable FAK P.Tyr content. Under these circumstances FAK may be held in an inactive but "ready" (autophosphorylated at Tyr-397) conformation.
This model accommodates the fact that FAK exhibits high levels of tyrosine phosphorylation in quiescent serum-starved NIH 3T3 fibroblasts and is consistent with observations that mechanical forces such as contractility (10), physical stretching (18), or fluid flow shear stress (34) can stimulate FAK kinase activity. Under these circumstances, FAK may become activated through a conformational release of inhibitory factors, and exposure of phosphorylated FAK Tyr-397 could promote the specific recruitment of Src family PTKs or other SH2 domain-containing proteins to promote signaling events. The integrin stimulated association of Src family PTKs could further enhance FAK PTK activity through Src-mediated phosphorylation of residues within the FAK catalytic domain activation loop (5).
In a finding consistent with our previous studies (46), we found that maximal FN-stimulated signaling to ERK2 required the coordinated activation of both FAK and c-Src PTK activities. We show that the transient association of FAK and c-Src after FN stimulation (from 20 to 80 min) was correlated with the kinetics of maximal in vitro kinase activities and the phosphorylation of FAK Tyr-925 to promote Grb2 binding. Interestingly, we found that FN-stimulated Grb2 binding to Shc in NIH 3T3 cells paralleled the extended time course of ERK2 activation for up to 2 h, but that the amount of Grb2 associated with Shc was constant during the period of maximal ERK2 activation (20 to 40 min) at early times after FN stimulation. Therefore, we speculate that it is the summation of signals generated by Grb2 binding to FAK and Grb2 binding to Shc at early times during FN-stimulated cell spreading that may be responsible for maximal ERK2 activation. Our direct studies in 293T cells support this hypothesis and also show that there may be additional integrin-stimulated Grb2-mediated signaling pathways in cells.
Significantly, we also provide evidence in this study of an Src-independent FN receptor-stimulated signaling pathway to ERK2. As shown previously with Src-deficient fibroblasts (46) and in the current study with NIH 3T3 fibroblasts treated with Src-specific PTK inhibitors, FN-stimulated FAK autophosphorylation can promote low-level signaling to ERK2. We show that in the absence of Src family PTK activity, FN stimulation can promote the transient association of Shc with FAK and that FAK PTK activity can directly phosphorylate Shc at Tyr-317 to promote Grb2 binding. In vitro, FAK exhibited phosphorylation site specificity on Shc since it did not phosphorylate other known sites such as Shc Tyr-239 and Tyr-240 (21, 54). Interestingly, the aspartate amino acid residues preceding Shc Tyr-317 (Asp-Asp-Pro-Ser-P.Tyr) match the positions of two acidic glutamate residues preceding the identified FAK phosphorylation sites on paxillin at Tyr-31 (Glu-Glu-Thr-Pro-P.Tyr) and on paxillin at Tyr-118 (Glu-Glu-His-Val-P.Tyr) (1, 45), and it is possible that these acidic residues may direct FAK kinase specificity. Although both WT and Phe-397 FAK were able to directly phosphorylate Shc in vitro, Shc associates with WT but not with Phe-397 FAK in vivo (49).
This result supports the hypothesis that FAK may interact with multiple signaling proteins through binding interactions at the motif surrounding the Tyr-397 autophosphorylation site. FAK Tyr-397 is the direct SH2 binding site for Src family PTKs (43), and mutation of this site indirectly regulates Grb2 binding to FAK Tyr-925 (48). Mutation of FAK Tyr-397 also disrupts the association of the p85 subunit of PI 3-kinase with FAK (7). Since the Shc SH2 domain binds directly to WT but not to Phe-397 FAK in vitro (49) and the Shc SH2 domain binding consensus (P.Tyr-Ile-X-Ile) matches the motif surrounding FAK Tyr-397 (P.Tyr-Ala-Glu-Ile), Tyr-397 is likely to be the direct Shc binding site on FAK. Interestingly, only low levels of FAK-Shc associations after FN stimulation can be detected without overexpression. This may be a result of competition between Shc and Src family PTKs for the same binding site on FAK. Nevertheless, although direct FAK tyrosine phosphorylation of Shc in normal cells occurs, it is also likely that Shc tyrosine phosphorylation after integrin stimulation is mediated by other PTKs such as c-Src.
It has been postulated that Shc tyrosine phosphorylation at Tyr-317 is critically important for integrin signaling to ERK2 (57). We show that c-Src can phosphorylate both WT and Phe-317 Shc to promote Grb2 binding in vitro. Since FAK and c-Src are activated after integrin stimulation and since FAK overexpression can also potentiate c-Src PTK activity after FN stimulation (49), it is not surprising that Phe-317 Shc is tyrosine phosphorylated after FN stimulation of 293T cells and that its expression did not block WT FAK-enhanced FN-stimulated ERK2 activation. Our results also are consistent with findings that Phe-317 Shc overexpression does not block middle T antigen-stimulated and potentially Src family PTK-mediated Grb2 binding to Shc and cell transformation events (2). Therefore, what may be the mechanism by which Phe-317 Shc could block integrin signaling in some cells? As shown for FAK, other integrin-activated PTKs such as c-Abl (31) may preferentially phosphorylate Shc Tyr-317 and thus limit the number of Grb2 binding sites on Shc. It is also possible that the documented association of integrin subunits with caveolin (57) may negatively regulate integrin-stimulated c-Src PTK activation and signaling events (33).
Our results do not discount the importance of Grb2 binding to Shc Tyr-317 in mediating integrin-stimulated signals to ERK2, but in the case with Grb2 binding to FAK Tyr-925 (49) our studies show that Grb2 binding to Shc Tyr-317 is not essential for FN-stimulated signaling to ERK2. Significantly, we find that the expression of a SH3 domain-inactivated Grb2 protein, which does not bind to downstream targets such as the Sos GDP-GTP exchange protein for Ras, blocked FN-stimulated signaling to ERK2. The fact that coexpression of FAK Phe-925 and Shc Phe-317 results in lower ERK2 activation shows that these sites on the WT proteins do indeed function to promote FN-stimulated signaling events and therefore we conclude that signaling upstream of Ras does not proceed through one linear pathway (Fig. 10). Moreover, our results support the hypothesis that FN-stimulated signals to ERK2 proceed through multiple Grb2-mediated binding interactions with FAK, Shc, or perhaps other unidentified tyrosine-phosphorylated proteins. This multiplicity of signaling events affecting ERK2 activation is consistent with other observations that PI 3-kinase activity can also modulate integrin signaling pathways downstream of Ras to regulate ERK2 activation (28).
Most studies on integrin-based signaling have used acute cell stimulation via antibodies or synthetic ligands to induce integrin receptor clustering or via plating detached cells on insoluble ECM proteins. Conditions of this sort will be found rarely in humans, except perhaps for cells leaving the circulation or at the leading edge of migrating cells. But with acute stimulation it has been possible to find assay conditions in which one signaling pathway predominates and may be sufficient for ERK2 activation. Whereas this information is important to the mechanistic aspects of integrin signaling, our results support the hypothesis that multiple inputs and pathways may act in combination to regulate the extent and duration of ERK2 activation in vivo. Future studies will be directed at better understanding the role of integrin-stimulated ERK2 activation as it may pertain to cell survival, gene transcription, or cell migration.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grant R29 CA75240 (D.D.S.) and in part by grants CA14195 (T.H.) and CA39780 (T.H.) from the National Cancer Institute. Tony Hunter is an American Cancer Society Research Professor.
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FOOTNOTES |
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* Corresponding author. Mailing address: The Scripps Research Institute, Department of Immunology, IMM26, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Phone: (619) 784-8207. Fax: (619) 784-8227. E-mail: dschlaep{at}scripps.edu.
Paper 10863-IMM of The Scripps Research Institute, La Jolla,
Calif.
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Materials & Methods
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Discussion
References
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