From the Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom
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ABSTRACT |
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We examined whether constitutively active mutants
of the G proteins G
12 and G
13,
which together comprise the G12 subfamily of G
proteins,
induce Rho-dependent tyrosine phosphorylation of the focal
adhesion proteins p125 focal adhesion kinase, paxillin, and p130
Crk-associated substrate. We report that transient expression of the
constitutively active mutants of G
12 or of
G
13 in human embryonic kidney 293 cells stimulates
tyrosine phosphorylation of a set of proteins of
Mr of 110,000-130,000, 97,000, and
60,000-70,000. We identified p125 focal adhesion kinase, paxillin, and
p130 Crk-associated substrate as prominent tyrosine-phosphorylated
proteins in human embryonic kidney 293 cells expressing constitutively
active G
12 and G
13. In common with the
increased tyrosine phosphorylation of these proteins mediated by
mitogens acting through heptahelical receptors, the G
12-
and G
13-mediated increase in tyrosine phosphorylation is
blocked by cytochalasin D, which specifically disrupts the actin
cytoskeleton, and by the Clostridium botulinum C3
exoenzyme, which ADP-ribosylates and inactivates Rho. Our results
support the hypothesis that G
12 and G
13
activate Rho and suggest that G
12 and G
13
may mediate the tyrosine phosphorylation of p125 focal adhesion kinase,
paxillin, and p130 Crk-associated substrate.
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INTRODUCTION |
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Many mitogens, including bioactive lipids such as lysophosphatidic acid (LPA)1 and neuropeptides such as bombesin, signal through distinct cell surface proteins that are members of the superfamily of G-protein-coupled receptors (GPCRs) with seven putative transmembrane domains. In Swiss 3T3 cells, a useful model system to analyze the mechanisms of growth factor signaling, an early event in the signal transduction pathways activated by these mitogens is the tyrosine phosphorylation of a common set of focal adhesion proteins (1). Treatment of Swiss 3T3 cells with GPCR agonists including bioactive lipids (2-4) and mitogenic neuropeptides such as bombesin (5-7) stimulates tyrosine phosphorylation of proteins with apparent Mr of 110,000-130,000, 97,000, and 60,000-70,000 on SDS-polyacrylamide gels. The nonreceptor tyrosine kinase p125fak (8, 9) and the adaptor proteins paxillin (10, 11) and p130cas (12), which localize to focal adhesion plaques, have been identified as prominent tyrosine-phosphorylated proteins in agonist-stimulated Swiss 3T3 cells (3, 4, 13-20). The increases in tyrosine phosphorylation of p125fak, paxillin, and p130cas are accompanied by profound alterations in the organization of the actin cytoskeleton in Swiss 3T3 cells, leading to the formation of actin stress fibers and the assembly of focal adhesions (3, 4, 21, 22). RhoA, a member of the Ras superfamily of low molecular weight GTPases, has been shown to direct stress fiber formation and focal adhesion assembly in Swiss 3T3 cells (23, 24). We proposed that the tyrosine phosphorylation of p125fak, paxillin, and p130cas mediated by heterotrimeric G-proteins is downstream of RhoA activation and focal adhesion assembly (1, 25-28). Signaling through p125fak, paxillin, and p130cas has been implicated in the regulation of cell migration, proliferation, and transformation (20, 29-32).
The immediate mechanism(s) coupling mitogen-induced activation of GPCRs
to tyrosine phosphorylation of focal adhesion proteins is not
understood. Although many GPCRs couple to the pertussis toxin-sensitive
Gi, Gi does not appear to
mediate increases in protein tyrosine phosphorylation (1, 33).
G12 and G
13, which together comprise the
ubiquitously expressed G12 subfamily of G
proteins, are
distantly related to other G-protein
subunits and are pertussis
toxin-insensitive (34, 35). There is increasing evidence indicating
that G
12 and G
13 are involved in cell
migration, proliferation, and transformation. Expression of
mutationally activated G
12 stimulates cellular entry
into DNA synthesis, proliferation, and malignant transformation in
NIH3T3 and Rat-1 cell lines (36-41) and promotes stress fiber
formation and focal adhesion assembly in Swiss 3T3 cells (42).
Furthermore, in astrocytoma cells, thrombin-induced stimulation of DNA
synthesis was prevented by the microinjection of
anti-G
12 antibody (43). Gene disruption experiments have
implicated G
13 in the regulation of cell migration (44). The downstream targets through which G
12 and
G
13 induce these effects have not been identified,
although Ras-, Rac-, Rho-, and Cdc42-dependent pathways
leading to cytoskeletal reorganization and to the activation of
mitogen-activated protein kinase, Jun N-terminal kinase, and the
Na+-H+ exchanger have been implicated (40,
45-48). However, it is not known whether activation of
G
12 and/or G
13 can also promote the
tyrosine phosphorylation of the nonreceptor protein tyrosine kinase
p125fak and the adaptor proteins paxillin and
p130cas.
To examine whether the G12 subfamily of heterotrimeric
G-proteins induce protein tyrosine phosphorylation, we have transiently transfected human embryonic kidney HEK 293 cells with expression vectors encoding constitutively active G12 and
G
13 proteins and determined the effect of the expression
of these activated G
subunits on the tyrosine phosphorylation of
p125fak, paxillin, and p130cas. Our results
demonstrate that expression of active G
12 and
G
13 in HEK 293 cells induces tyrosine phosphorylation of
these focal adhesion proteins through a pathway that requires the
integrity of the actin cytoskeleton and functional Rho.
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EXPERIMENTAL PROCEDURES |
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cDNA Constructs--
The murine G12 subunit
cDNAs in the eucaryotic expression vector pcDNA-1 (Invitrogen)
were gifts from Dr. H. R. Bourne (University of California at San
Francisco, CA) and included the constitutively active mutants
G
12-Q229L and G
13-Q226L
(G
12QL and G
13QL) (37). The
constitutively active mutant murine G
i-2-Q205L (G
i-2QL) and G
q-Q209L (G
qQL)
subunit cDNAs in pcDNA-1 (49, 50) were obtained from the
American Type Tissue Collection (Manassas, VA). The Clostridium
botulinum C3 exoenzyme (C3 toxin) cDNA in the eucaryotic
expression vector EFplink, EFC3, was a gift from Dr. R. Treisman
(Imperial Cancer Research Fund, London, UK). This construct encodes the
C3 toxin under the control of the human polypeptide chain elongation
factor 1
(EF-1
) promoter (51).
Cell Culture and Transfection--
Cultures of transformed HEK
293 cells were maintained in DMEM supplemented with 10% fetal bovine
serum in a humidified atmosphere containing 10% CO2 at
37 °C. HEK 293 cells were transfected with the various plasmids by
calcium phosphate precipitation (52) or with
LipofectAMINETM (Life Technologies, Inc.). The conditions
for transient transfection of HEK 293 cells using calcium phosphate
precipitation and LipofectAMINE were each optimized to yield peak
efficiency of transfection using an expression plasmid encoding
-galactosidase under the control of the human polypeptide chain
elongation factor 1
(EF-1
) promoter (pEFlacZ, a gift from R. Treisman). Both transfection methods yielded greater than a 50%
transfection efficiency optimally, and we were unable to match this
transfection efficiency with Lipofectin or DEAE-dextran-mediated
transfection methods. Briefly, HEK 293 cells were plated at a density
of 1.2 × 106 cells/60-mm Nunc dish and transfected
the following day. For calcium phosphate transfections, 12.5 µg of
plasmid DNA (precipitated in 0.25 ml of 140 mM NaCl, 0.75 mM Na2HPO4, 125 mM
CaCl2, 20 mM PIPES, pH 6.95) was added per dish
containing 2.25 ml of Optimem-1 medium, and the dishes were incubated
for 4 h at 5% CO2. For LipofectAMINE transfections,
HEK 293 cells were transfected with 3 µg of plasmid DNA and 25 µl
of LipofectAMINE in 3 ml of Optimem-1/dish for 18 h at 5%
CO2 according to the manufacturer's specifications. In the
cotransfection experiments, total amounts of DNA were kept constant,
and equal amounts of G
and of C3 expression constructs (or vector
without cDNA insert) were mixed. After transfection with calcium
phosphate or LipofectAMINE, the cells were washed and maintained in
DMEM supplemented with 10% fetal bovine serum. All cultures were used
for experimental purposes 4 days after plating.
Immunoprecipitation-- Cultures (one 60-mm dish/condition) were washed twice with DMEM and equilibrated in 5 ml of the same medium at 37 °C for 1-2 h. Some dishes were treated with inhibitors during this equilibration period or with growth factors for 10 min at the end of this period as indicated. Cultures were lysed in 1.4 ml of ice-cold lysis buffer A (10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 1% Triton X-100) supplemented with 2 mM Na3VO4, 1 mM 4-(aminoethyl)-benzenesulfonyl fluoride and 0.1% SDS. Lysates were clarified by centrifugation at 20,800 × g for 10 min at 4 °C. In some experiments, the cultures were washed and lysed as described above, with the exception that DMEM without phenol red was used, and the lysates were adjusted to 1 mg protein/ml after protein determination using the bicinchoninic acid protein assay (Pierce). 1 ml of the lysate was immunoprecipitated at 4 °C for 3-4 h with anti-mouse IgG-agarose-linked mAb directed against phosphotyrosine (4G10 or PY72), p125fak (2A7), paxillin, or p130cas or with Protein A-agarose-linked rabbit antisera directed against p125fak.
Western Blot Analysis-- Immunoprecipitates were washed three times by centrifugation with lysis buffer A supplemented with 1 mM Na3VO4 and extracted in 1× Laemmli sample buffer containing 1 mM EDTA for 10 min at 100 °C. The solubilized proteins were fractionated by discontinuous SDS-polyacrylamide gel electrophoresis (53) under reducing conditions on an 8% acrylamide resolving gel. Proteins were transferred onto polyvinylidene fluoride membranes (ImmobilonTM-P) at 4 °C for 2 h at 35 V and 2 h at 70 V in 48 mM Tris, 386 mM glycine, 0.1% SDS, and 20% methanol. The membranes were then blocked with 1% polyvinylpyrrolidone, 0.05% Tween 20, 0.02% sodium azide in phosphate-buffered saline, pH 7.4 (blocking buffer) (54) and incubated for 2 h with anti-phosphotyrosine (anti-Tyr(P)) mAb (4G10, 1 µg/ml) in blocking buffer. For some experiments, cultures were immunoprecipitated with anti-paxillin and with anti-p130cas mAb in duplicate, and parallel Western blots were probed with anti-Tyr(P) antibody and with the immunoprecipitating antibody to verify that equivalent amounts of protein were immunoprecipitated. Bound antibodies were visualized by the binding of 125I-labeled anti-mouse IgG (0.1 µCi/ml) followed by autoradiography. After autoradiographic detection of bound anti-Tyr(P), membranes from anti-p125fak immunoprecipitates were reprobed with rabbit antiserum directed against p125fak (1:500) and horseradish peroxidase-conjugated donkey antibodies to rabbit IgG (1:5,000). Immunoreactive p125fak was detected by enhanced chemiluminescence Western blotting ECLTM reagents (Amersham Pharmacia Biotech).
For analysis of GReproducibility--
Although both the calcium phosphate and the
LipofectAMINE transfection methods appeared to yield little variation
in levels of expression of G proteins between dishes in a
single transfection, both methods yielded variation in levels of
expression of G
proteins between experiments. Although this
variation in the levels of expression of the G
proteins was
reflected in the variation in the levels of increased protein tyrosine
phosphorylation, the reported experiments were repeated at least two
times with results equivalent to those illustrated.
Materials--
LipofectAMINETM and Optimem-1 were
obtained from Life Technologies, Inc., and the bicinchoninic acid
BCA*TM protein assay reagent was obtained from Pierce. The
anti-Tyr(P) mAb 4G10 and the anti-p125fak mAb 2A7 were
obtained from Upstate Biotechnology, Inc.; mAbs directed against
paxillin and p130cas were obtained from Transduction
Laboratories; the rabbit anti-p125fak antiserum C-20 and
rabbit anti-G 12 were obtained from Santa Cruz
Biotechnology, Inc.; and the rabbit
anti-G
i-1/G
i-2 was obtained from
Calbiochem. 125I-Labeled sheep anti-mouse IgG (100 µCi/ml), 125I-labeled protein A (100 µ Ci/ml),
horseradish peroxidase-conjugated donkey anti-rabbit Ig, and the
ECLTM Western blotting detection reagents were from
Amersham International. Polyvinylidene fluoride
ImmobilonTM-P Western blotting membranes were obtained from
Millipore. Anti-mouse IgG-linked agarose,
Na3VO4, thrombin, 1-oleoyl-LPA, phorbol
12,13-dibutyrate, epidermal growth factor, cytochalasin D, pertussis
toxin, polyvinylpyrrolidone 40, and thapsigargin were obtained from
Sigma. GF109203X and U73122 were obtained from LC Laboratories. The
synthetic G
13 peptide CLHDNLKQLMLQ was obtained from
Research Genetics. The anti-Tyr(P) mAb PY72 was obtained from the
Hybridoma Development Unit, Imperial Cancer Research Fund. All other
reagents were of the highest available grade from standard commercial
sources.
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RESULTS |
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Thrombin-mediated Tyrosine Phosphorylation of Focal Adhesion Proteins in HEK 293 Cells-- Elements of the signal transduction pathways mediating the tyrosine phosphorylation of focal adhesion proteins downstream of GPCRs have been well characterized in Swiss 3T3 cells. We were interested in using a transient transfection system to analyze the ability of the G12 subfamily of heterotrimeric G-proteins to induce tyrosine phosphorylation of focal adhesion proteins. We elected to use HEK 293 cells for these studies because these cells can be efficiently transfected. However, signal transduction pathways exhibit a degree of heterogeneity in different cells types. Consequently, we initially examined tyrosine phosphorylation induced by ligands of GPCRs in HEK 293 cells.
As thrombin causes GPCR-mediated activation of G
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Thrombin-induced Tyrosine Phosphorylation of p125fak, Paxillin, and p130cas Requires an Intact Cytoskeleton but Is Independent of PKC, Ca2+, and Pertussis Toxin-sensitive G-proteins-- Agonist-mediated tyrosine phosphorylation of focal adhesion proteins in Swiss 3T3 cells occurs through a PKC-, Ca2+-, and Gi-independent pathway that is critically dependent on the integrity of the actin cytoskeleton and of functional Rho (1, 3, 4, 14, 15, 25, 26). Direct PKC activation by phorbol esters can also lead to tyrosine phosphorylation of these proteins in Swiss 3T3 cells (14, 15, 20). As illustrated by Fig. 2 (upper left panel), the phorbol ester phorbol 12,13-dibutyrate also induced tyrosine phosphorylation of p125fak, paxillin, and p130cas in HEK 293 cells. The PKC inhibitor GF109203X (56, 57) prevented the tyrosine phosphorylation induced by phorbol 12,13-dibutyrate but did not inhibit the thrombin-induced tyrosine phosphorylation of p125fak, paxillin, or p130cas in HEK 293 cells, suggesting that PKC activation does not mediate agonist-induced tyrosine phosphorylation of focal adhesion proteins in HEK 293 cells. Similarly, treatment with pertussis toxin to inactivate Gi, thapsigargin to deplete the endoplasmic reticulum Ca2+ pool (58), or U73122 to inhibit phospholipase C-mediated phosphoinositide breakdown (59) did not affect thrombin-induced tyrosine phosphorylation of focal adhesion proteins in HEK 293 cells (Fig. 2).
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G12QL and G
13QL Induce Tyrosine
Phosphorylation of p125fak, Paxillin, and
p130cas in HEK 293 Cells--
Mutations in the catalytic
domain of G
subunits that inhibit their intrinsic GTPase activity
are known to convert these proteins into constitutively active
-subunits (60). To examine the effects of G
subunits on tyrosine
phosphorylation, HEK 293 cells were transiently transfected with
expression plasmids encoding the constitutively active G
mutants
G
12QL, G
13QL, G
i-2QL, and
G
qQL, which are deficient in GTPase activity (37, 49, 50). Given that pertussis toxin does not interfere with
agonist-stimulated tyrosine phosphorylation of focal adhesion proteins,
G
i was tested as a negative control. Conversely, because
PKC activation leads to tyrosine phosphorylation of
p125fak, paxillin, and p130cas,
G
q, which stimulates phospholipase C-mediated
phosphoinositide hydrolysis and thereby PKC (61-63), was included as a
positive control. The extracts of transfected cells were
immunoprecipitated with the anti-Tyr(P) mAb PY72, and the
immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis and Western blotting with the anti-Tyr(P) mAb 4G10. As
illustrated by Fig. 3 (upper
panel), HEK 293 cells overexpressing constitutively active mutant
G
12QL, G
13QL, or G
qQL
proteins exhibited increased tyrosine phosphorylation of a set of
proteins with apparent Mr of 110,000-130,000, 97,000, and 60,000-70,000. In contrast, transient transfection of HEK
293 cells with activated G
i-2QL expression plasmids did not increase tyrosine phosphorylation. Overexpression of wild-type G
12 and G
13 in HEK 293 cells also did not
induce tyrosine phosphorylation (data not shown), suggesting that the
effects of G
12QL, G
13QL, and
G
qQL were specific for the activated state of these G
subunits rather than due to the modulation of
subunit
availability. No stimulation of protein tyrosine phosphorylation was
observed after treatment of HEK 293 cells with medium conditioned by
cells transfected transiently with activated G
12QL or G
13QL
expression plasmids (data not shown), suggesting that the increased
tyrosine phosphorylation was not mediated by secreted factors.
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Tyrosine Phosphorylation in Response to G12QL and
G
13QL Requires an Intact Actin
Cytoskeleton--
Agonist-stimulated tyrosine phosphorylation of
p125fak, paxillin, and p130cas is inhibited by
treatment of Swiss 3T3 cells or HEK 293 cells with cytochalasin D,
which selectively disrupts the actin cytoskeleton (3, 14, 15, 64). To
determine whether the increase in the tyrosine phosphorylation of
p125fak and paxillin induced by activated
G
12 or G
13 is dependent upon an intact
actin cytoskeleton, we tested the effect of cytochalasin D on tyrosine
phosphorylation in HEK 293 cells transfected transiently with the
G
12QL or G
13QL expression vectors. As
shown in Fig. 4 (upper
panels), cytochalasin D inhibited the G
12QL- and
G
13QL-induced tyrosine phosphorylation of
p125fak and paxillin. Western blotting of total cell
lysates with antisera to the G
subunits demonstrated that
cytochalasin D treatment did not alter expression levels of the
transfected G
subunits (Fig. 4, lower panels).
Cytochalasin D also inhibited G
qQL- induced tyrosine
phosphorylation (data not shown).
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12QL and G
13QL Induce Tyrosine
Phosphorylation of p125fak, Paxillin, and
p130cas in a Rho-dependent Manner--
The
C. botulinum C3 toxin, which specifically ADP-ribosylates
RhoA, RhoB, and RhoC, resulting in the functional inhibition of these
GTP-binding proteins, can be used to determine whether a cellular
response requires functional Rho (65). This approach has been used to
demonstrate that in Swiss-3T3 cells, bombesin-, LPA- and
toxin-stimulated tyrosine phosphorylation of p125fak,
paxillin, and p130cas requires functional Rho (25-27). To
determine whether the G
12QL- or
G
13QL-induced increases in the tyrosine phosphorylation
of p125fak and paxillin are dependent upon Rho, we
cotransfected HEK 293 cells with either the G
12QL or the
G
13QL expression vector and with a C3 toxin expression
vector. We then immunoprecipitated lysates from the transfected cells
with the anti-Tyr(P) mAb PY 72, and the immunoprecipitates were
analyzed by Western blotting with the anti-Tyr(P) mAb 4G10. As
illustrated by Fig. 5 (upper panel), the increase in the tyrosine phosphorylation of proteins with apparent Mr of 110,000-130,000, 97,000, and 60,000-70,000 induced by either G
12QL or
G
13QL was inhibited by cotransfection with the C3 toxin
expression vector. The G
qQL-induced tyrosine phosphorylation of this set of proteins was also inhibited by cotransfection with the C3 toxin expression vector (data not shown). In
addition, we immunoprecipitated lysates of cotransfected cells with
mAbs directed against p125fak, paxillin, and
p130cas and subjected the immunoprecipitates to Western
blot analysis with the anti-Tyr(P) mAb 4G10. As shown in Fig. 5
(middle panels), the G
12QL- and
G
13QL-induced tyrosine phosphorylation of
p125fak, paxillin, and p130cas was inhibited by
cotransfection with the C3 toxin expression vector. Western blotting
with antisera to the G
subunits demonstrated that cotransfection
with C3 toxin plasmid did not alter the expression levels of the
transfected G
subunits (Fig. 5, lower panels).
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DISCUSSION |
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Activation of GPCRs for mitogenic neuropeptides and bioactive
lipids leads to a pertussis toxin-insensitive tyrosine phosphorylation of multiple protein substrates including the nonreceptor tyrosine kinase p125fak and the adaptor proteins paxillin and
p130cas. The mechanism(s) linking activation of GPCRs to
increases in protein tyrosine phosphorylation remains poorly
understood. In the present study, we have used transient transfection
of HEK 293 cells with expression plasmids encoding constitutively
active G12 and G
13 to investigate the
involvement of the G12 subfamily of G
proteins in
tyrosine phosphorylation of the nonreceptor tyrosine kinase
p125fak and of the adaptor proteins paxillin and
p130cas. We report for the first time that HEK 293 cells
expressing constitutively active G
12 and
G
13 mutants exhibit increased tyrosine phosphorylation of p125fak, paxillin, and p130cas. Identical
increases in tyrosine phosphorylation of these focal adhesion proteins
were observed in HEK 293 cells treated with thrombin. This is
noteworthy with reference to the previous reports that thrombin
receptors couple to G
12 and G
13 in
platelets and to G
12 in astrocytoma cells (43, 55) and,
in particular, that fibroblasts lacking G
13 exhibit a
greatly diminished migratory response to thrombin (44). Our
observations reveal a novel connection between activated
G
12 and G
13 and increases in tyrosine
phosphorylation of p125fak, paxillin, and
p130cas.
The signal transduction mechanism that mediates G12- and
G
13-induced increases in tyrosine phosphorylation of
p125fak, paxillin, and p130cas in HEK 293 cells
has several features in common with the mechanism extensively studied
in Swiss 3T3 cells mediating tyrosine phosphorylation of these proteins
in response to LPA and bombesin, which signal by activation of GPCRs.
The common features in the signal transduction pathway leading to
tyrosine phosphorylation of p125fak, paxillin, and
p130cas include a requirement for an intact actin
cytoskeleton, as indicated by the block of the tyrosine phosphorylation
response by treatment with cytochalasin D, which specifically disrupts
the actin cytoskeleton (3, 4, 14, 15, 20, 64). Another common feature
is the requirement for functional Rho, as indicated by inhibition with
the C. botulinum C3 toxin, which ADP-ribosylates
and functionally inactivates Rho (25, 26, 66). These observations
suggest that G
12 and G
13 may couple GPCRs
to increases in tyrosine phosphorylation of p125fak,
paxillin, and p130cas.
It has been proposed that the mitogen-induced increases in tyrosine
phosphorylation of p125fak, paxillin, and
p130cas are downstream of Rho activation, stress fiber
formation, and focal adhesion assembly (4, 25-28, 67, 68). Activated
G12 and G
13 have been shown to induce Rho
activation (69) and Rho-dependent biological responses,
including stress fiber formation and focal adhesion assembly (42, 70,
71). These results suggest that these G-proteins may couple GPCRs to
the activation of Rho. Our observation that activated
G
12 and G
13 mediate a
Rho-dependent increase in tyrosine phosphorylation of
p125fak, paxillin, and p130cas further supports
the hypothesis that G
12 and G
13, which
together comprise the G12 subfamily of G
proteins,
couple the activation of GPCRs to the activation of Rho. Taken
together, these findings suggest the existence of a signal transduction
pathway whereby ligand occupation of GPCRs activates G
12
and/or G
13, which subsequently activate Rho, presumably
by a mechanism involving recruitment and activation of a GDP/GTP
exchange factor. Activated Rho would induce the formation of actin
stress fibers and promote the assembly of focal adhesions, resulting in
recruitment of p125fak to focal adhesions and tyrosine
phosphorylation of p125fak, paxillin, and
p130cas. This hypothesis warrants further experimental
work.
Direct activation of PKC also leads to tyrosine phosphorylation of
focal adhesion proteins in either Swiss 3T3 cells (14, 15, 20) or in
HEK 293 cells (Fig. 2). Accordingly, we found that HEK 293 cells
expressing constitutive active Gq, which couples to
phospholipase C and PKC (61-63), exhibit increased tyrosine phosphorylation of focal adhesion proteins, which was dependent upon an
intact cytoskeleton and functional Rho. In light of the report that, in
contrast to G
12 and G
13,
G
q does not induce stress fiber formation and focal
adhesions in Swiss 3T3 cells (42), G
q has not been
thought to couple GPCRs to the activation of Rho. However, a recent
report has suggested that GPCRs can induce the phosphorylation of
Tiam1, a Rac-1-specific exchange factor, in a manner that is dependent
upon PKC and independent of pertussis toxin-sensitive G-proteins (72).
In Swiss 3T3 cells, Cdc42, Rac, and Rho have been placed in a
hierarchical cascade wherein Cdc42 activates Rac, which in turn
activates Rho (73). Therefore, G
q may induce the
Rho-dependent tyrosine phosphorylation of focal
adhesion proteins via an activation of Rac, which may subsequently
activate Rho. Because agonist-mediated tyrosine phosphorylation of
focal adhesion proteins occurs independently of PKC, the precise physiological significance of the PKC-dependent pathway is
unclear.
There is increasing evidence indicating that G12 and
G
13 are involved in cell migration, proliferation, and
transformation (36, 38-40, 43, 44, 48). Downstream targets through
which G
12 and G
13 act may include Ras-,
Rac-, Rho-, and Cdc42-dependent pathways leading to
cytoskeletal reorganization and to the activation of mitogen-activated
protein kinase, Jun N-terminal kinase, the Na+-H+ exchanger, and the c-fos
serum response element (40-42, 46-48). In this context, our results
revealing that activated G
12 and G
13
induce increases in the level of tyrosine phosphorylation of
p125fak, paxillin, and p130cas suggest novel
mechanisms of action of these G
subunits. These findings assume an
added importance in view of increasing evidence implicating
p125fak and p130cas in cell migration,
proliferation, and transformation. Gene disruption experiments have
demonstrated a critical role of p125fak in embryonic
development, cell migration, and turnover of focal adhesions (29, 74),
and microinjection of dominant negative fragments of
p125fak prevented serum stimulation of DNA synthesis (75).
The adaptor protein p130cas has also been implicated in
agonist-stimulated mitogenesis and in cell transformation (12, 20, 76)
and has recently been identified as a mediator of
p125fak-mediated cell migration (31). Interestingly,
embryonic fibroblasts lacking G
13 also display a greatly
impaired migratory response to thrombin receptor activation (44). Taken
together with the results presented here, these findings raise the
attractive possibility that p125fak and p130cas
are downstream targets of G
13 in a signal transduction
pathway that regulates cell migration in response to GPCR agonists.
Future studies should assess the contribution of increased tyrosine
phosphorylation of p125fak, paxillin, and
p130cas to the growth promoting and transforming activities
of G
12 and G
13.
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ACKNOWLEDGEMENTS |
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We thank Dr. Henry R. Bourne for the kind
gifts of the expression constructs for G12-Q229L and
G
13-Q226L and Dr. Richard Treisman for the kind gifts of
the expression constructs for C3 and
-galactosidase. We also thank
Dr. Thomas Seufferlein for advice on transfection protocols and Dr. Ray
Harris of Life Technologies, Inc. for sharing information on conditions
for transfecting HEK 293 cells with LipofectAMINETM.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Biochemistry, School of Medicine,
University of North Carolina, Chapel Hill, NC 27599-7260.
§ To whom correspondence should be addressed: 900 Veteran Ave., Warren Hall Room 11-124, Dept. of Medicine, School of Medicine, UCLA, Los Angeles, CA 90095-1786. Tel.: 310-794-6610; Fax: 310-267-2399.
1
The abbreviations used are: LPA,
lysophosphatidic acid; anti-Tyr(P), anti-phosphotyrosine; C3 toxin,
C. botulinum C3 exoenzyme; DMEM, Dulbecco's modified
Eagle's medium; G12QL, G
13QL,
G
i-2QL, and G
qQL, constitutively active
mutant G
12-Q229L, G
13-Q226L, G
i-2-Q205L, and G
q-Q209L subunits,
respectively; G-protein, guanine nucleotide regulatory protein; GPCR,
G-protein-coupled receptors; mAb, monoclonal antibody;
p125fak, p125 focal adhesion kinase; p130cas,
p130 Crk-associated substrate; PKC, protein kinase C; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid) ; EF,
elongation factor.
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REFERENCES |
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