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Molecular and Cellular Biology, April 1999, p. 3205-3215, Vol. 19, No. 4
Cancer Biology Program,
Received 29 May 1998/Returned for modification 16 July
1998/Accepted 14 January 1999
The nontransmembrane protein tyrosine phosphatase SHP-2 plays a
critical role in growth factor and cytokine signaling pathways. Previous studies revealed that a fraction of SHP-2 moves to focal contacts upon integrin engagement and that SHP-2 binds to SHP substrate
1 (SHPS-1)/SIRP-1 Complex processes such as cell
growth, differentiation, and migration require the integration of
multiple types of extracellular signals, including those delivered by
growth factors, cytokines, and hormones (soluble signals), and
solid-state signals, transmitted by cell-cell and cell-extracellular
matrix (ECM) interactions. Most of these signaling pathways involve
changes in cellular tyrosyl phosphorylation. Tyrosyl phosphorylation is
regulated by protein tyrosine kinases (PTKs) and protein tyrosine
phosphatases (PTPs). Although many PTKs are implicated in signaling
pathways for both soluble and solid-state signals, the roles of
specific PTPs are less well defined.
Multiple reverse-genetic studies suggest that the
nontransmembrane PTP SHP-2 is a required positive (i.e.,
signal-enhancing) component of growth factor and cytokine signal
transduction pathways (for reviews, see references 35,
56, and 63). Consistent with these
studies, fibroblasts from mice containing a deletion of SHP-2 exon 3 (hereafter referred to as SHP-2 mutant mice), which express low levels
of a defective SHP-2 protein that lacks its N-terminal SH2 domain
(48), exhibit impaired mitogen-activated protein kinase
(MAPK) activation in response to fibroblast growth factor (FGF),
epidermal growth factor, and insulin-like growth factor I (48,
55). SHP-2 functions similarly in lower organisms. Dominant-negative SHP-2 blocks FGF-induced mesoderm induction in
Xenopus ectodermal explants and completion of gastrulation in early embryos, leading to severe tail truncations (38,
57). Likewise, corkscrew (csw), the
Drosophila SHP-2 homolog (39, 40), is required
for multiple receptor tyrosine kinase (RTK) pathways involved in
early development (e.g., Torso, Sevenless, Breathless, and
Drosophila EGF receptor) (1, 39, 40), and the
recently described Caenorhabditis elegans homolog, Ptp-2, is
a component of the Let-23 pathway (13).
The precise mechanism by which SHP-2 orthologs function, as well as
their specific target(s), has remained unclear. SHP-2 binds directly to
and may dephosphorylate some growth factor and cytokine receptors (for
reviews, see references 35 and
63). In other pathways, however, SHP-2 binds to
distinct signaling intermediates. One class of SHP-2 binding proteins,
exemplified by the Drosophila daughter of sevenless
(dos) gene product, consists of an N-terminal pleckstrin
homology domain and multiple proline-rich stretches and potential
tyrosyl phosphorylation sites (42). Dos is essential for
Sevenless signaling (15, 42) and may be a direct substrate
for Csw (15). Mammalian cells express several groups of
molecules with overall topology similar to that of Dos (for a review,
see reference 63). including insulin receptor substrate family members (for a review, see reference
64), Gab-1 (16), P97/Gab-2 (11,
12), and FGF receptor substrate 2/SNT (26).
SHP substrate 1 (SHPS-1/SIRP- The major cellular receptors for most ECM ligands are members of the
integrin family (18). Integrin engagement activates several
downstream signaling pathways (5, 54), including multiple
PTKs, the best studied of which are members of the Src (hereafter
termed Src PTKs) and focal adhesion kinase (FAK) families (including
FAK and Pyk2/RAFTK). Recent work suggests that Abl also is activated
during integrin signaling (28), and integrin signaling in
hematopoietic cells involves activation of Syk (9). Although
the specific roles of each of these PTKs remain unclear, tyrosyl
phosphorylation is absolutely required for all integrin signaling
functions (for reviews, see reference 58).
Coordinating Src PTK and FAK function is critical for integrin
signaling. Activation of Src and FAK PTKs leads to the tyrosyl phosphorylation of several other proteins, including paxillin, tensin,
and p130Cas (58). These initial tyrosyl
phosphorylation events result in activation of downstream signaling
pathways, including phosphatidylinositol 3-kinase, protein kinase C,
and MAPK, which ultimately direct adhesion and cell shape changes, cell
motility, changes in gene expression, and the prevention of apoptosis
(24, 25, 29) (for reviews, see references
54 and 58).
The order of activation of and the relationship between Src and FAK
PTKs remain controversial. Some experiments suggest that FAK activation
precedes Src PTK activation, whereas others indicate that Src PTKs are
upstream of FAK (21, 49, 50, 59). It also is unclear how
initial Src or FAK activation is accomplished. Binding of Src PTKs (via
their SH2 domains) to tyrosyl-phosphorylated FAK could lead to Src PTK
activation, since Src family PTKs are negatively regulated by
interactions of their C-terminal phosphotyrosyl residues and their SH2
domains (for a review, see reference 58). In this
model, however, it remains unclear how FAK activation occurs, as well
as how and why Src PTKs are activated upon integrin engagement in
FAK-deficient cells (19). Alternatively, it is unclear how
integrin engagement alone could cause Src PTK activation. Some evidence
suggests that a fraction of c-Src is rapidly dephosphorylated at its
inhibitory C-terminal phosphotyrosyl residue upon integrin binding
(22), but the identity of the responsible phosphatase(s) is
unknown. Src PTK binding to FAK may also play a role in
integrin-induced activation of Src PTKs. Autophosphorylation of FAK (at
Tyr397) creates a binding site for the SH2 domains of Src PTKs
(50), whereas Src PTKs phosphorylate other residues on FAK,
including Tyr925, which directs binding of FAK to the SH2 domain of
Grb2 (52). Thus, full phosphorylation and activation of Src
and FAK may involve a FAK-Src positive-feedback loop.
The role(s) of specific PTPs in integrin signaling are even less well
defined. Several, including PTP-PEST (10), PTP-1B (31), and CD45 (44), have been suggested to
negatively regulate integrin signaling. The positive role of SHP-2 in
growth factor and cytokine signaling pathways, the finding that a
fraction of SHP-2 translocates to focal contacts upon integrin
engagement, the structure of SHPS-1, and the increased tyrosyl
phosphorylation of SHPS-1 upon integrin activation suggested to us that
SHPS-1-SHP-2 complexes might serve a positive role in integrin signal
transduction. Here we show that SHPS-1 is a target for Src PTKs upon
integrin engagement. Moreover, studies of SHP-2 mutant fibroblasts have established an essential role for SHP-2 in integrin signaling, leading
to cell spreading, and position SHP-2 action at the earliest times
following integrin engagement. We suggest a model in which SHP-2-SHPS-1 complexes promote a positive-feedback loop leading to Src
and FAK activation during integrin signaling.
Cells and cell culture.
Fibroblasts prepared from SHP-2 exon
3 Plasmids and plasmid constructions.
Clone pJ3/SHPS-1
consists of full-length wild-type rat SHPS-1 cDNA, generated by reverse
transcription-PCR (60), inserted into the expression vector
pJ3 (33), which directs transcription under the control of
the simian virus 40 promoter. Wild-type SHP-2 (6) and the
SHP-2 mutants C459S (3) and Plating on ECMs.
Tissue culture plates were coated with
different ECMs in accordance with the manufacturer's instructions
(Collaborative Biomedical Products). Briefly, FN, vitronectin (VN), and
laminin (LN) were diluted in SFM, added to tissue culture plates (final
coating concentration, 5 µg/cm2), and incubated at room
temperature for at least 1 h. Following washing with deionized
H2O, plates were blocked with 0.1% heat-inactivated bovine
serum albumin for 1 h and then washed with SFM. Collagen was
diluted in 0.05 N HCl instead of SFM, with coating accomplished as
described above. Poly(L-lysine) (10 µg/ml)-coated plates
were allowed to dry overnight in a fume hood, then washed one time with
water and once with SFM before using.
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Regulation of Early Events in Integrin Signaling by
Protein Tyrosine Phosphatase SHP-2
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
, a transmembrane glycoprotein with adhesion
molecule characteristics (Y. Fujioka et al., Mol. Cell. Biol.
16:6887-6899, 1996; M. Tsuda et al., J. Biol. Chem. 273:13223-13229). Therefore, we asked whether SHP2-SHPS-1 complexes participate in
integrin signaling. SHPS-1 tyrosyl phosphorylation increased upon
plating of murine fibroblasts onto specific extracellular matrices.
Both in vitro and in vivo studies indicate that SHPS-1 tyrosyl
phosphorylation is catalyzed by Src family protein tyrosine kinases
(PTKs). Overexpression of SHPS-1 in 293 cells potentiated integrin-induced mitogen-activated protein kinase (MAPK) activation, and potentiation required functional SHP-2. To further explore the role
of SHP-2 in integrin signaling, we analyzed the responses of SHP-2 exon
3
/ and wild-type cell lines to being plated on
fibronectin. Integrin-induced activation of Src family PTKs, tyrosyl
phosphorylation of several focal adhesion proteins, MAPK activation,
and the ability to spread on fibronectin were defective in SHP-2 mutant
fibroblasts but were restored upon SHP-2 expression. Our data suggest a
positive-feedback model in which, upon integrin engagement, basal
levels of c-Src activity catalyze the tyrosyl phosphorylation of
SHPS-1, thereby recruiting SHP-2 to the plasma membrane, where, perhaps
by further activating Src PTKs, SHP-2 transduces positive signals for
downstream events such as MAPK activation and cell shape changes.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1; hereafter referred to as SHPS-1) is
the prototype of a second class of SHP-2 binding proteins which
includes the more distantly related PECAM (46, 47) and PIR-B/p91A (60). Initially observed by several groups to be an SHP-2 binding protein whose tyrosyl phosphorylation increases in
cells expressing catalytically impaired SHP-2 mutants (for reviews, see
references 35 and 63). SHPS-1
contains three immunoglobulin domains and four cytoplasmic tyrosyl
residues (8). Several highly related cDNAs have been
identified, suggesting that a family of SHPS-1-like molecules (termed
SIRPs) may exist (23, 36). The precise function(s) of SHPS-1
like glycoproteins and their relatives remains unclear. Growth factor
stimulation of quiescent cells leads to enhanced SHPS-1 tyrosyl
phosphorylation and increased SHP-2-SHPS-1 association, suggesting a
role for SHPS-1-SHPS-2 complexes in RTK signaling (8, 23).
Overexpression of SHPS-1 was found to impair EGF-induced mitogenesis
and v-Fms-mediated transformation, which led to the conclusion that
SHPS-1-like proteins were negative signaling molecules (23).
Conceivably, however, SHP-2 must bind both Dos-like and SHPS-1-like
molecules to exert positive signaling functions. Thus, overexpressed
SHPS-1 might inhibit signaling gratuitously by sequestering SHP-2 from
other signaling complexes. The structure of SHPS-1-like molecules,
which is reminiscent of cell adhesion molecules, suggests that they may
be involved in other signaling pathways, for example, pathways transducing cell-cell or cell-ECM interactions. Consistent with this
notion, SHPS-1 tyrosyl phosphorylation increases upon plating of
fibroblasts onto some ECMs (8), and PECAM tyrosyl
phosphorylation increases upon plating of rat basophilic leukemia cells
onto fibronectin (FN) (47).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ (Ex3/) mutant mice (B1) and
wild-type (J3) littermates (48) were immortalized by
following a 3T3 protocol and maintained in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS),
penicillin, and streptomycin. The human embryonic kidney epithelial
cell line 293 was obtained from the American Type Tissue Collection.
Cell lines derived from mice bearing targeted mutations in Src and/or Fyn, Csk alone or in combination with Src and/or Fyn mutations (59), and Csk mutant cells reconstituted with wild-type or
kinase-inactive Csk (17) have been described previously.
These cells and 293 cells were maintained in DMEM plus 10% FBS and
antibiotics as described above.
P (57) have been described previously. These were excised from pBluescript vectors (Stratagene) as EcoRI fragments, filled in by using the
Klenow fragment of DNA polymerase, and ligated into
BamHI-digested and blunted pEBB (a generous gift of Bruce
Mayer, Children's Hospital, Boston, Mass.), which directs expression
under the control of the elongation factor 1- promoter.
Transient transfections.
Transient transfections of 293 cells were performed essentially as described previously
(3). Briefly, cells (2 × 106) were plated
on 6-cm-diameter dishes, incubated at 37°C for 24 h, and then
transfected with hemagglutinin-tagged MAPK, alone or together with
SHP-2 (4 µg for the wild type or the C
S mutant; 8 µg for the
P mutant) or SHP-2 and 2 µg of pJ3/SHPS1, in the presence of 25 µM chloroquine. After 10 h at 37°C, the medium was replaced
with 4 ml of fresh DMEM containing 10% FBS, and after 12 to 14 h,
the cells were starved in DMEM containing 0.2% FBS for an additional
24 h prior to stimulation.
Generation of and infections with recombinant SHP-2-containing
adenoviruses.
Wild-type SHP-2 cDNA was cloned into the plasmid
pACCMV.pLpA (2) (provided by C. Newgard, University of Texas
Southwestern Medical Center, Dallas), generating the plasmid
pACCMV.pLpA-SHP-2. Recombinant adenoviruses were generated as
previously described (2, 7). Briefly, pACCMV.pLpA-SHP-2 was
cotransfected with the plasmid pJM-17 into 80% confluent 293 cells by
using a Transfection MBS kit (Stratagene, La Jolla, Calif.) with 0.7 µg of pACCMV.pLpA-SHP-2 and 1 µg of pJM-17 (2) per
35-mm-diameter dish. Cell lysis, indicating a recombination event,
occurred 1 to 2 weeks following cotransfection. Several clones of
recombinant virus were tested for the successful integration of the
SHP-2 coding region by Western blotting with lysates of transduced 293 cells. One clone was amplified further in 293 cells and purified by
cesium chloride centrifugation, generating high-titer stocks of a
recombinant virus (1 × 1012 to 2 × 1012 PFU/ml, as determined by limiting dilution). A
recombinant adenovirus encoding 
-galactosidase was provided by C. Newgard and amplified and purified as described above.
-galactosidase-encoding recombinant adenoviruses were performed overnight in DMEM plus 15% FBS with constant agitation on a rocking platform. The media were then exchanged for 10-ml volumes of fresh DMEM
plus 15% FBS, and following an additional 12-h incubation, the cells
were serum starved by incubating them with DMEM containing 0.2% FBS
overnight and then used for experiments.
Antibodies. Rabbit polyclonal antibodies against a glutathione S-transferase (GST)-SHPS-1 cytoplasmic domain fusion protein (GST-cytoSHPS-1) (60) and against GST-SHP-2 (27) have been described previously. Antipeptide antibodies against the SHP-2 C terminus were purchased from Santa Cruz Biotechnology, Inc. Polyclonal anti-phospho-specific p44/42 MAPK antibodies were purchased from New England Biolabs. Monoclonal antiphosphotyrosine (anti-pTyr) antibodies (4G10) were obtained from Upstate Biotechnology, Inc. Monoclonal antipaxillin antibodies were from Transduction Laboratories. Polyclonal anti-MAPK antibodies (C1; anti-Erk1/Erk2) were gifts of John Blenis (Harvard Medical School, Boston, Mass.). Monoclonal (2A7) and polyclonal (BC3) anti-FAK antibodies were provided by J. Tom Parsons (University of Virginia Medical School), and monoclonal anti-Src antibodies (327) were from Joan Brugge (Harvard Medical School). Polyclonal anti-p130Cas antibodies were provided by Amy Bouton (University of Virginia Medical School).
Immunoprecipitation and Immunoblotting. After the plate contents were washed twice with phosphate-buffered saline, the cells were lysed (500 µl/10-cm-diameter plate) in NP-40 buffer (50 mM Tris, pH 8.0; 150 mM NaCl; 1% Nonidet P-40 [NP-40]; 10 mM NaF; 2 mM Na3VO4) containing a protease inhibitor cocktail (1 µg of aprotinin/ml, 1 µg of antipain/ml, 10 µg of leupeptin/ml, 1 µg of pepstatin A/ml, and 20 µg of phenylmethylsulfonyl fluoride/ml). Cell lysates were clarified by centrifugation at 10,000 × g for 15 min at 4°C. For immunoprecipitations, each sample (containing 200 to 1,000 µg of total protein) was incubated with the relevant antibody for 2 h at 4°C; this was followed by the addition of protein A-Sepharose beads (Sigma) and a 1-h incubation. The antibodies were used as follows: antipaxillin, anti-FAK, and anti-Cas, 2 µg; anti-Src, 1 µg; anti-SHPS-1 serum, 15 µl; and anti-GST-SHP-2 antibodies, 30 µg. Immune complexes were collected by centrifugation, washed three times with NP-40 buffer, resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled, and analyzed by SDS-PAGE (8% gel). Proteins were electroblotted onto Immobilon P membranes (Millipore) and probed with appropriate primary and peroxidase-conjugated secondary antibodies (Amersham). Signals were detected by enhanced chemiluminescence (ECL; Amersham). The concentrations of primary antibodies used in immunoblotting were as follows: 1:500 for anti-SHPS-1 serum; 1:1,000 for antipaxillin, anti-FAK, anti-p130Cas, anti-Src, anti-SHP-2 C-terminal peptide, and anti-phospho-MAPK; 1:5,000 for anti-MAPK; and 1:7,500 for anti-pTyr. In some experiments, total-cell lysates (30 µg) were used for anti-phospho-MAPK. Immune complex kinase assays for monitoring Src activity were performed as described previously (9).
In vitro phosphorylation and tryptic phosphopeptide
analysis.
GST-cytoSHPS1 was prepared and purified on
glutathione-agarose beads as described previously (60).
GST-FAK C-terminal fusion protein (GST-FAK-CT) was kindly provided by
David Schlaepfer (Scripps Institute). Purified fusion proteins (3 µg)
were phosphorylated by incubating them with 1.5 µg of purified
recombinant c-Src (a generous gift of Michael J. Eck, Dana Farber
Cancer Institute) in kinase buffer {30 mM HEPES [pH 7.4], 5 mM
MgCl2, 1 mM dithiothreitol, 20 µM ATP (5 µCi of
[32P-
]ATP)} at 37°C for various periods of time.
After being washed twice with kinase buffer, GSTicytoSHPS-1 and bound
proteins were recovered by brief centrifugation, boiled in SDS-PAGE
sample buffer, and analyzed by SDS-PAGE (10% gel).
Cell spreading assays. For cell spreading experiments, cells were plated on FN-coated 60-mm-diameter tissue culture dishes as described above. At various time points, three different fields were photographed. Only cells that were flat and dark were considered to be spread. All bright cells were counted as nonspread cells. Cells were visualized with a Nikon microscope, and images were processed by using a Photometrics digital camera and Phase 3 Imaging software. Quantification was carried out by a blinded observer (i.e., one with no knowledge of the cells used in a given experiment).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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SHPS-1 tyrosyl phosphorylation is mediated by Src PTKs. A previous report revealed that SHPS-1 tyrosyl phosphorylation increased when Chinese hamster ovary (CHO) cells were plated onto FN-coated plates (8). We confirmed that SHPS-1 becomes tyrosyl phosphorylated upon cell-ECM interaction in our mouse embryonic fibroblasts (J3). Plating on FN induced a rapid increase in tyrosyl phosphorylation of SHPS-1 (Fig. 1A). In addition, adhesion to VN, LN, or collagen induced tyrosine phosphorylation of SHPS-1 to various extents (Fig. 1B). The increased tyrosyl phosphorylation of SHPS-1 upon plating on FN was accompanied by an increase in both total and tyrosyl-phosphorylated SHPS-1 associated with SHP-2. SHP-2 itself was also tyrosyl phosphorylated, although weakly, upon plating on FN (data not shown).
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/ mice. Since Csk negatively regulates
Src PTKs by catalyzing phosphorylation of their C-terminal tyrosyl
residues, Csk/ fibroblasts exhibit elevated activity of
multiple Src PTKs (20, 34, 37). Consistent with a role for
Src PTKs in SHPS-1 phosphorylation, SHPS-1 was hyperphosphorylated on
tyrosyl residues in Csk/ fibroblasts (Fig. 2B) and in
Csk/ fibroblasts placed in suspension and then plated
onto FN (data not shown). Notably, SHPS-1 tyrosyl phosphorylation
was decreased by reconstituting expression of wild-type, but not
kinase-negative, Csk in Csk/ fibroblasts (Fig. 2B),
indicating that SHPS-1 hyperphosphorylation in the Csk/
cells was due to the absence of Csk per se rather than to clonal variation.
Previous studies suggested that some targets of Src family PTKs are
phosphorylated preferentially by individual family members (59). The concentration of PP1 required to inhibit SHPS-1
phosphorylation (Fig. 2A) suggested that Src itself or multiple Src
PTKs must be inhibited to block SHPS-1 tyrosyl phosphorylation. To
determine whether a particular Src PTK is required preferentially for
SHPS-1 tyrosyl phosphorylation, we examined SHPS-1 tyrosyl
phosphorylation in fibroblasts from mice bearing homozygotic mutations
in Csk alone, Csk and Src, Csk and Fyn, or Csk, Src, and Fyn. Loss of Src or Fyn in the Csk/ background partially suppressed
the SHPS-1- hyper-tyrosyl phosphorylation observed in fibroblasts
mutant for Csk alone, whereas loss of both Src and Fyn in the
Csk/ background almost completely suppressed SHPS-1
phosphorylation (Fig. 2B). These data suggest that multiple Src PTKs
regulate SHPS-1 tyrosyl phosphorylation, at least in fibroblasts. To
determine if integrin-induced tyrosyl phosphorylation of SHPS-1 also is regulated by Src PTKs, we analyzed SHPS-1 tyrosyl phosphorylation in wild-type, Src/, Fyn/, or
Src/ Fyn/ fibroblasts plated on
FN. Integrin-induced tyrosyl phosphorylation of SHPS-1 was partially
suppressed in the absence of either of the individual Src PTKs and was
more dramatically reduced in the absence of both Src and Fyn (Fig. 2C).
The above experiments strongly indicate that Src family PTKs are
required for proper SHPS-1 tyrosyl phosphorylation, but they do not
imply that Src PTKs directly phosphorylate SHPS-1. We asked whether
recombinant Src could phosphorylate SHPS-1 in vitro and, if so, how
this compared to the phosphorylation of other known Src substrates.
Recombinant Src efficiently phosphorylated GST-cytoSHPS-1, causing a quantitative mobility shift into at least two
more-slowly migrating forms. In addition, a fraction of Src associated
with phosphorylated SHPS-1 in vitro (Fig.
3A). Comparison of GST-cytoSHPS-1 with
two other known Src substrates, enolase and GST-FAK CT (Fig. 3B),
revealed much higher levels of phosphorylation of GST-cytoSHPS-1 (about 25-fold higher than for either of the other two substrates). Tryptic phosphopeptide analysis indicated that Src catalyzes
phosphorylation of SHPS-1 on two major and two minor sites (Fig.
3C), consistent with the four available sites present in
GST-cytoSHPS-1. Together with the quantitative mobility shift produced
by Src-catalyzed phosphorylation, these data argue that at least 2 mol
of phosphate is incorporated per mol of GST-cytoSHPS-1. Our results
indicate that SHPS-1 is a much better substrate for Src than
either enolase or the FAK C-terminal domain and suggest
that Src PTKs directly catalyze the tyrosyl phosphorylation
of SHPS-1 at multiple sites.
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/ Fyn/ cells (data not shown).
Therefore, we treated cells with cytochalasin D, which prevents
integrin-induced FAK phosphorylation (30) but does not
affect integrin-induced Src activation (32a). Pretreatment of cells with cytochalasin D (3 µM) substantially diminished (>80%) integrin-mediated FAK phosphorylation, but SHPS-1 tyrosyl
phosphorylation was largely unaffected (Fig.
4). Although it remains possible that FAK
phosphorylates SHPS-1 on some sites, both the in vitro and in vivo data
strongly argue that SHPS-1 is a direct substrate of Src PTKs.
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) or were
treated with cytochalasin D (+) as described in Materials and Methods,
detached, and plated on FN in the presence or absence of cytochalasin
D. Cell lysates were subjected to immunoprecipitation (IP) with
anti-SHPS-1 (SHP-2 regulates integrin-evoked tyrosyl phosphorylation and MAPK activation. The above-described studies indicated that upon plating on FN, Src PTKs directly phosphorylate SHPS-1 and cause the assembly of an SHPS-1-SHP-2 complex at the plasma membrane (where SHPS-1 resides). These data suggested that SHP-2 might play a functionally important role in integrin signaling. To test this possibility, we analyzed integrin-evoked signaling events in fibroblast cell lines derived from an SHP-2 mutant mouse or its wild-type littermate.
A number of focal adhesion proteins, including FAK, paxillin, and p130Cas, are tyrosyl phosphorylated upon plating on FN (for a review, see reference 58). We assessed FN-induced tyrosyl phosphorylation of these focal adhesion proteins in wild-type and SHP-2 mutant fibroblasts (Fig. 5). Total cellular tyrosyl phosphorylation was reduced in SHP-2 mutant cells plated on FN (data not shown). More specifically, tyrosyl phosphorylation of FAK, paxillin, and p130Cas were diminished in the SHP-2 mutant cells (Fig. 5).
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P; see Materials and Methods)
were cotransfected with epitope-tagged MAPK into 293 cells. Following
transfection, the cells were detached and replated on FN, and MAPK
activation was assessed with phosphospecific antibodies. Both
catalytically inactive SHP-2 mutants impaired MAPK activation in this
assay, consistent with a requirement for SHP-2 in integrin-stimulated
MAPK activation (Fig. 7C).
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P blocked SHPS-1-mediated enhancement of
MAPK activation. These results suggest that the SHPS-1-SHP-2 complex
transmits a positive signal to activate MAPK.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Integrin engagement activates a signal transduction cascade that leads to changes in cellular tyrosyl phosphorylation, activation of MAPKs, alterations in the cytoskeletal architecture, changes in gene expression, and prevention of apoptosis. Relatively little is known about how specific PTPs help regulate these processes. The results presented here implicate SHP-2, acting via one of its specific binding proteins, SHPS-1, as a critical positive component in integrin signaling. We have shown that (i) Src PTKs catalyze tyrosyl phosphorylation of SHPS-1 upon plating on FN; (ii) SHP-2 is required for normal integrin-induced activation of Src (and probably other Src PTKs), tyrosyl phosphorylation of FAK, paxillin, and p130Cas, and MAPK activation; (iii) increasing SHPS-1 expression can potentiate integrin-induced MAPK activation and this requires SHP2 function; and (iv) biological responses, such as spreading on FN, require normal SHP-2 function.
These results and those of previous studies suggest a possible model
for the role of SHP-2 and SHPS-1 in integrin signal transduction (Fig.
10). We propose that upon integrin
engagement by appropriate ECM ligands, Src PTKs catalyze the
phosphorylation of SHPS-1 on multiple tyrosyl residues. A recent report
indicates that Fyn can be recruited to 1 integrins by
the adapter protein caveolin and that a fraction of the
caveolin-associated Fyn is activated. While the mechanism of Fyn
activation is unclear, this population could catalyze the
phosphorylation of SHPS-1. At least two of the SHPS-1 phosphorylation
sites presumably function to recruit SHP-2. SHP-2 would then act in a
positive-feedback loop to sustain high levels of Src PTK activity
and/or to activate additional Src PTK molecules, potentially by
catalyzing dephosphorylation of their inhibitory C-terminal
phosphotyrosyl residues (e.g., Y527 in Src). The high level of Src PTK
activation is necessary to promote effective phosphorylation of FAK,
binding of Src PTKs to the FAK autophosphorylation site, and further
phosphorylation of FAK (50, 52) and downstream targets. This
model may also apply to complexes formed by SHP-2 with other
transmembrane glycoproteins (e.g., PECAM and PIR-B/P91A; see the
introduction). In addition to tyrosine dephosphorylation and
activation of Src upon integrin engagement, activation of Src by
tyrosyl dephosphorylation following treatment of platelets with
thrombin (4) or of Swiss 3T3 cells with bombesin
(45) has also been reported. It will be interesting to
determine whether ligand-inducible complexes of SHP-2- and SHPS-1-like molecules (eg., PECAM in platelets) play a role in c-Src
activation by these G protein-coupled receptors as well.
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Previous work showed that SHPS-1 tyrosyl phosphorylation increases upon plating of CHO cells on FN. Our results, along with recent work of others, confirm and extend these findings to other fibroblast cell lines and other ECM ligands (62). Not all ECMs evoke tyrosyl phosphorylation of SHPS-1 to equal extents, with FN and VN causing strong tyrosyl phosphorylation of SHPS-1 and Col and LN evoking only weak induction (Fig. 1B). These differences in ECM effects could reflect intrinsic differences in signal transduction from different integrins. However, we cannot exclude the trivial possibility that such differences reflect differential levels of integrin expression in the cell lines we have studied. Further work is required to distinguish among these possibilities.
Integrin engagement is known to activate FAK and Src family PTKs (see
the introduction). The following multiple lines of evidence strongly
implicate Src family rather than FAK family PTKs in SHPS-1 tyrosyl
phosphorylation: (i) SHPS-1 tyrosyl phosphorylation is dramatically increased in Csk/ fibroblasts, in which
Src PTK activity is elevated, whereas it is decreased in
Csk/ cells in which Src PTK deficiency is superimposed;
(ii) integrin-induced SHPS-1 tyrosyl phosphorylation is decreased in
cells lacking one or more Src PTKs; (iii) treatment of cells with the
Src PTK inhibitor PP1 dramatically decreases SHPS-1 phosphorylation
(Fig. 2); (iv) recombinant Src phosphorylates the cytoplasmic domain of
SHPS-1 in vitro (better than FAK or enolase), and transient
cotransfection of Src and SHPS-1 expression constructs leads to
increased tyrosyl phosphorylation of SHPS-1 (Fig. 3 and data not
shown); and (v) blocking phosphorylation of FAK by pretreating cells
with cytochalasin D has little effect on Src activation
(32a) or SHPS-1 tyrosyl phosphorylation (Fig. 4). While our
manuscript was under review, Tsuda and coworkers also reported
decreased SHPS-1 phosphorylation in Src- and Fyn-deficient cells.
However, they observed a decrease in SHPS-1 tyrosyl
phosphorylation in FAK-deficient cells and in cells treated with
cytochalasin D (62). It is unclear to us why our results
with cytochalasin D differ from those of Tsuda and coworkers;
nevertheless, their in vitro studies suggest that Src, but not FAK, can
phosphorylate SHPS-1. Thus, our data, along with these recent results,
argue that while FAK may contribute to the regulation of SHPS-1 tyrosyl
phosphorylation, Src PTKs directly phosphorylate SHPS-1 early upon
integrin engagement.
The precise order in which Src family PTKs and FAK are activated during
integrin signaling has been a subject of controversy. One school of
thought holds that FAK is activated directly upon integrin engagement
and that autophosphorylated FAK then recruits and activates Src and
other Src family PTKs by engaging their SH2 domains (50). In
this model, activated Src family PTKs bound to FAK then further
phosphorylate FAK on additional sites and phosphorylate other
substrates. Consistent with this model, Src PTKs can be
coimmunoprecipitated with FAK, and Src-FAK association is dependent on
the FAK autophosphorylation site. However, other evidence suggests that
Src PTK activation precedes FAK activation. For example, FAK is
hyperactivated in Csk/ cells (59) in which
Src PTK activity is high (20, 34), as well as in
v-Src-transformed cells (21, 50), and kinase-negative FAK
can be phosphorylated upon integrin engagement (43, 53). In
addition, FAK tyrosyl phosphorylation is absent in cells deficient for
both Src and Fyn (data not shown).
Although we favor the latter activation order, our proposed role for SHPS-1-SHP-2 complexes can be accommodated within either model. If FAK is upstream of Src, then initial FAK activation upon integrin engagement could lead to the recruitment and activation of a small amount of Src (and other Src family PTKs), which in turn could phosphorylate SHPS-1. Tyrosyl-phosphorylated SHPS-1 would then recruit SHP-2, which could then either activate additional molecules of Src or promote sustained Src activation. Interestingly, previous studies have shown that Src activation persists far longer than Src-FAK complexes are detectable by coimmunoprecipitation (51), suggesting that sustained Src activation results from a mechanism other than SH2 domain engagement. Alternatively, basal levels of Src activity could phosphorylate SHPS-1, leading to a positive-feedback loop consisting of further Src activation, FAK activation, and downstream signals. Either model also can accommodate previous suggestions that SHPS-1 is a substrate of SHP-2 (8, 23): by promoting SHPS-1 dephosphorylation, SHP-2 would turn off the very process that it initiates, thereby limiting PTK activation following integrin engagement.
At first glance, the positive role for SHPS-1-SHP-2 complexes in
integrin-mediated signaling suggested by our results seems to conflict
with the previous suggestion that SHPS-1 negatively regulates growth
factor receptor-induced signaling and mitogenesis (23). It
is conceivable that SHPS-1-SHP-2 complexes have distinct effects on
different signaling pathways
for example, inhibiting RTK signaling
while activating ECM signaling. However, if distinct SHP-2 complexes
are required in multiple signaling pathways, then the previous
experiments, in which the role of SHPS-1 was inferred on the basis of
the results of overexpression experiments, may have been misleading:
overexpression of SHPS-1 could titrate SHP-2 needed for other
positive signaling functions (e.g., direct binding to RTKs or binding
to downstream scaffolding molecules such as IRS family members, Gab1,
or FRS-2; see the introduction). Since SHPS-1 is implicated in both RTK
and integrin signaling, a particularly attractive possibility, and one
that we strongly favor, is that SHPS-1-SHP-2 complexes help explain
one or more well-known modulatory effects of ECM on growth factor and
cytokine receptor signaling. In this way, SHPS-1-SHP-2 complexes could
serve as integrators of these two types of signaling pathways. The
ability of SHPS-1 to potentiate MAPK activation in an SHP-2-dependent
manner is consistent with this hypothesis (Fig. 8).
Although our data implicate SHP-2 in integrin-induced Src family kinase
regulation, other PTPs also play roles in Src PTK regulation. Two
receptor PTPs, PTP and CD45, have been proposed to regulate Src
family PTKs. Overexpression of PTP results in dephosphorylation of
the negative regulatory tyrosine of Src PTKs (66), whereas
CD45 can have either negative or positive effects on Src PTK
activation, depending on the cellular context. In lymphocytes, CD45 has
been proposed to play a positive role in activation of Lck and, to a
lesser extent, Fyn (for a review, see reference 61).
In contrast, recent results indicate that in macrophages, CD45 is
targeted to focal contacts and the absence of CD45 results in elevated
levels of Src PTK activity, suggesting that CD45 is a negative
regulator of integrin signaling (44). Accordingly, we cannot
exclude the possibility that SHP-2's effects on Src PTK activity are
indirect, e.g., via effects on HPTP or other PTPs.
As a consequence of the essential role of SHP-2 in integrin signaling,
cytoskeletal changes (e.g., spreading) (Fig. 9) and apoptosis
prevention (unpublished observations) are impaired in SHP-2 mutant
fibroblasts. Since the exon 3/ SHP-2 mutant protein has
elevated PTP activity (41), it remains possible that defects
we have discussed are due to increased SHP-2 activity. However, the
rescue of these defects upon reconstitution of SHP-2 expression
suggests that the defects in integrin signaling most likely arise as a
consequence of the diminished SHP-2 function. Consistent with our
findings, while this article was under review, Yu and coworkers
also reported that SHP-2 mutant cells have defects in migration as
well as spreading and have an increased number of focal adhesions
(65). Our results confirm and extend their study by
showing that SHP-2's effects on MAPK activation are mediated by its association with SHPS-1 and that SHP-2 either directly or
indirectly regulates Src kinase activity and downstream tyrosyl phosphorylation events.
Defects in cell adhesion and migration may account for the developmental effects of defective SHP-2 function. Dominant-negative SHP-2 blocks FGF-induced elongation of Xenopus animal caps and prevents completion of gastrulation (57), a process which is nonmitogenic and involves the reorganization of existing cells. SHP-2 mutant mice also fail to gastrulate properly, and many of the defects in mutant embryos have been postulated to be a result of improper organization or migration of mesodermal cells (48). The results presented here support this hypothesis by demonstrating a role for SHP-2 in integrin biology, including cell spreading and survival.
Src PTKs have been implicated in the regulation of both of these
processes; thus, it is interesting to postulate that the defects
observed both in vivo and in cultured cells could be due primarily to
effects of SHP-2 on Src PTKs. For example, Src/ cells,
like SHP-2 mutant cells, show a reduced rate of spreading (22). Thus, the suppression of Src PTK activation observed
in SHP-2 mutant cells could be the primary mechanism contributing to
the phenotypic defects in vivo and in culture. Additional genetic and
biochemical studies will be performed to test this hypothesis.
Finally, given that attachment to an appropriate ECM is critical to allow growth factors to promote mitogenesis, it is conceivable that some or even all of the previously observed effects on loss of SHP-2 function on growth factor-induced signaling instead reflect interference with what would otherwise be a constitutive signal from the ECM. Further study is required to delineate just how defective SHP-2 function affects growth factor and ECM signaling pathways and their integration and the role of SHP-2 in the regulation of complex biological responses.
| |
ACKNOWLEDGMENTS |
|---|
E.-S.O., H.G., T.M.S., and J.F.T. contributed equally to this work.
This work was supported by NIH grants R01 CA49152 (to B.G.N.), R01 CA75621 (to S.M.T.), and R01 DK43051 (to B.B.K.) and by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada (to T.P.). T.P. is an International Scholar of the Howard Hughes Medical Institute. H.G. was supported by NIH NRSA CA72144, J.F.T. was supported by a postdoctoral fellowship from the Leukemia Society, and S.H. was the recipient of a postdoctoral fellowship from The Medical Foundation. E.U.F. was supported by a Physician Scientist Award (NIA no. AG 00294).
We are grateful to C. Newgard for providing adenovirus plasmids, Mike Eck for the kind gift of recombinant c-Src, Karen Zoller and Cindy Miranti for helpful discussions, and Joan S. Brugge and Alana M. O'Reilly for insightful comments and critical reading of the manuscript.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Cancer Biology Program, Division of Hematology-Oncology, Beth Israel Deaconess Hospital and Harvard Medical School, Boston, MA 02215. Phone: (617) 667-4174. Fax: (617) 667-0610. E-mail for B.G.N.: bneel{at}bidmc.harvard.edu. E-mail for S.M.T.: sthomas{at}bidmc.harvard.edu.
Present address: Ariad Pharmaceuticals, Cambridge, MA 02139.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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