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(Received for publication, March 1, 1996, and in revised form, May 10, 1996)
From the ABSTRACT Several G protein-coupled receptors that interact
with pertussis toxin-sensitive heterotrimeric G proteins mediate
Ras-dependent activation of mitogen-activated protein (MAP)
kinases. The mechanism involves G INTRODUCTION Many receptors that couple to heterotrimeric G proteins have been
shown to mediate the rapid activation of
MAP1 kinases. Among these are receptors for
several substances either present in the general circulation, released
as neurotransmitters, or produced locally by vascular endothelium or
activated platelets. These include catecholamines, acetylcholine,
pituitary glycopeptide hormones, adenosine, angiotensins, bombesin,
endothelins, LPA, and Heterogeneity exists in the mechanisms whereby G protein-coupled
receptors activate MAP kinases. Depending upon receptor and cell type,
MAP kinase activation may be mediated by pertussis toxin-sensitive or
-insensitive G proteins and be either PKC- or
Ras-dependent. In COS-7 cells, for example, activation of
MAP kinase via the pertussis toxin-insensitive, Gq-coupled, We have shown previously that Ras-dependent MAP kinase
activation via LPA and The identity of the tyrosine kinase(s) and their mechanism of
activation by G protein-coupled receptors remains unclear. Several cell
surface receptors that lack intrinsic tyrosine kinase activity,
including the antigen receptors on T and B cells as well as the
receptors for growth hormone, erythropoietin and several cytokines,
stimulate tyrosine phosphorylation through association with Src family
tyrosine kinases such as Lck, Lyn, and Fyn (18). Similar recruitment of
nonreceptor tyrosine kinases might play a role in G protein-coupled
receptor signaling. To test this possibility, we have investigated the
role of Src kinases in LPA receptor and G EXPERIMENTAL PROCEDURES The cDNAs encoding G COS-7 cells were maintained
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and 100 µg/ml gentamicin at 37 °C in a humidified 5%
CO2 atmosphere. Transfections were performed on 80-90%
confluent monolayers in 100-mm dishes for immunoprecipitation and c-Src
kinase assays or in 6-well tissue culture plates for MAP kinase assays.
For transient transfection, cells were incubated at 37 °C in
serum-free Dulbecco's modified Eagle's medium (4 ml containing 6-10
µg of DNA/100-mm dish or 1 ml containing 1-2 µg of DNA/well) plus
6 µl of LipofectAMINE reagent (Life Technologies, Inc.)/µg of DNA.
Empty pRK5 vector was added to transfections as needed to keep the
total mass of DNA added per dish constant within an experiment. After
3-5 h of exposure to the transfection medium, monolayers were refed
with growth medium and incubated overnight. Assays were performed
48 h after transfection. LipofectAMINE transfection of COS-7 cells
consistently resulted in transfection efficiencies of greater than 80%
(data not shown). Transient expression of G Stimulations were
carried out at 37 °C in serum-free medium as described in the figure
legends. After stimulation, monolayers were washed once with ice-cold
phosphate-buffered saline, lysed in RIPA buffer (150 mM
NaCl, 50 mM Tris-HCl, pH 7.5, 0.25% sodium deoxycholate,
0.1% Nonidet P-40, 1 mM NaVO4, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotonin, and 10 µg/ml leupeptin), sonicated briefly,
clarified by centrifugation, and diluted with RIPA buffer to a protein
concentration of 2 mg/ml. Endogenous Shc was immunoprecipitated from 1 ml of lysate using 4 µg/sample of polyclonal anti-Shc antibody
(Transduction Laboratories) plus 50 µl of a 50% slurry of Protein G
plus/Protein A agarose (Oncogene Science) agitated for 1 h at
4 °C. Immune complexes were washed twice with ice-cold RIPA buffer
and once with phosphate-buffered saline and denatured in Laemmeli
sample buffer. Following resolution by SDS-polyacrylamide gel
electrophoresis (PAGE) and transfer to nitrocellulose membranes,
immunoblots were performed to detect Shc phosphotyrosine or the
presence of coprecipitated proteins. Shc phosphotyrosine was detected
using a 1:1000 dilution of horseradish peroxidase-conjugated
antiphosphotyrosine monoclonal antibody (Transduction Laboratories).
Shc protein was detected using a 1:1000 dilution of rabbit polyclonal
anti-Shc IgG (Transduction Laboratories), and Grb2 was detected using a
1:1000 dilution of rabbit polyclonal anti-Grb2 IgG (Santa Cruz
Biotechnology), each with horseradish peroxidase-conjugated donkey
anti-rabbit IgG (Amersham) as secondary antibody. Wild-type and mutant
c-Src were detected using a 1:100 dilution of mAb 327 anti-Src
monoclonal antibody (31) with horseradish peroxidase-conjugated donkey
anti-mouse IgG (Jackson Laboratories) as secondary antibody. Fyn and
c-Yes immunoblots were performed using rabbit polyclonal anti-Fyn and
anti-Yes antibodies (Santa Cruz Biotechnology). Immune complexes on
nitrocellulose were visualized by enzyme-linked chemiluminescence
(Amersham) and quantified by scanning laser densitometry.
Shc
immune complexes on agarose beads were prepared from RIPA lysates of
appropriately stimulated cells as described. To detect coprecipitated
tyrosine kinase activity, washed pellets were incubated for 15 min at
20 °C in 30 µl of reaction mix (10 mM PIPES, pH 7.0, 10 mM MnCl2, 5 mM
Val5-angiotensin II (Sigma), 10 mM ATP, and 10 µCi [ Rabbit antiserum
specific for Y416-phosphorylated Src was the generous gift
of M. Weber. To detect endogenously autophosphorylated c-Src, clarified
RIPA whole-cell lysates of appropriately stimulated or transfected
cells (50 µg of whole-cell protein/lane) were resolved by SDS-PAGE
and transferred to nitrocellulose. Y416-phosphorylated
c-Src was detected using a 1:500 dilution of anti-pY416-Src
antibody, with horseradish peroxidase-conjugated donkey anti-rabbit IgG
(Amersham) as secondary antibody. Identical samples were immunoblotted
with mAb 327 as controls. Immune complexes on nitrocellulose were
visualized by enzyme-linked chemiluminescence (Amersham) and
quantified by scanning laser densitometry.
Activation of
epitope-tagged p44HA-mapk was determined using
myelin basic protein (MBP) as substrate (30). Appropriately
transfected, serum-starved cells in 6-well plates were stimulated as
described in the figure legends, lysed in 200 µl of ice-cold RIPA/SDS
lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH
8, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 10 mM NaF, 10 mM sodium pyrophosphate, and 0.1 mM phenylmethylsulfonyl fluoride) and clarified by
centrifugation. Immunoprecipitation of p44HA-mapk
from the clarified supernatants was performed using 6.5 µg of anti-HA
12CA5 antibody (Boehringer Mannheim) plus 25 µl of a 50% slurry of
protein A-agarose (Oncogene Science) for 1 h at 4 °C. Immune
complexes were washed twice with lysis buffer and twice with kinase
buffer (20 mM HEPES, pH 7.4, 10 mM
MgCl2, and 1 mM dithiothreitol). MBP
phosphorylation was performed at 20 °C for 30 min in 40 µl of
kinase buffer containing 250 µg/ml MBP, 20 µM ATP, and
4 µCi [ RESULTS Fig. 1A
depicts the effects of endogenous LPA or epidermal growth factor (EGF)
receptor stimulation, or transient coexpression of G
To determine whether stimulation of endogenous LPA receptors or
transient expression of G
The Src family kinases Src, Fyn, and Yes are expressed in COS-7 cells
(data not shown). To determine whether recruitment of Src family
kinases could account for the increase in Shc-associated tyrosine
kinase activity, Shc immunoprecipitates were immunoblotted with
antibodies specific for Src, Fyn, or Yes. Only c-Src was detected in
Shc immunoprecipitates from cells following LPA or EGF stimulation, as
shown in Fig. 3A. Coprecipitation of a c-Src
with Shc was also observed in cells transiently expressing a
constitutively activated human c-Src mutant (Y530F) (23, 24, 25). As shown
in Fig. 3B, the LPA-stimulated association between c-Src and
Shc was rapid and transient, reaching a maximum within 1-2 min of
stimulation. Thus, the time course of c-Src·Shc protein complex
formation paralleled the time course of Shc tyrosine phosphorylation
and recruitment of tyrosine kinase activity into Shc
immunoprecipitates.
Two- to 3-fold increases in c-Src autophosphorylation and kinase
activity have been reported following stimulation of LPA (32),
The transforming viral oncogene
product v-Src is known to mediate tyrosine phosphorylation of Shc (36),
suggesting that the cellular homologue might play a similar role. As
shown in Fig. 5A, transient overexpression of
wild-type c-Src or the Y530F and K298M mutants resulted in increased
c-Src·Shc complex formation, detected in c-Src immunoblots performed
on Shc immunoprecipitates. As shown in Fig. 5B, Shc
immunoprecipitates from wild-type c-Src and Y530F-expressing cells
contained increased tyrosine kinase activity, whereas cells expressing
the kinase inactive K298M mutant exhibited less than basal levels of
Shc-associated tyrosine kinase activity, suggesting that the
overexpressed kinase inactive mutant competed with endogenous kinase
for Shc binding. Fig. 5C depicts the effects of c-Src
overexpression on Shc tyrosine phosphorylation and Shc·Grb2 complex
formation. Transient expression of wild-type c-Src or the activated
Y530F mutant increased Shc phosphorylation and Shc·Grb2 association
to a level comparable to that observed following EGF stimulation. The
ability of wild-type c-Src to induce Shc phosphorylation comparable to
the constitutively active Y530F mutant probably results from the high
levels of expression achieved in the transient transfection system.
To determine whether c-Src expression could mimick the effects of LPA
stimulation and G
To directly determine whether Src family kinase
activity is necessary for LPA receptor- and G Fig. 7A depicts the effects of Csk
overexpression on LPA- and EGF-stimulated Shc tyrosine phosphorylation
and Shc·Grb2 association in transfected COS-7 cells. EGF-induced Shc
phosphorylation was reduced by approximately 40%, whereas the
LPA-mediated signal was abolished. The inhibition of Shc·Grb2 complex
formation paralleled the effects on Shc phosphorylation. As shown in
Fig. 7B, LPA-stimulated Shc tyrosine phosphorylation and
Shc·Grb2 complex formation were reduced to levels not significantly
different from basal in cells overexpressing Csk.
The effects of Csk expression on MAP kinase activation are depicted in
Fig. 8. In Csk-transfected cells, LPA-stimulated MAP
kinase activation was reduced by 60% and G
DISCUSSION Gi-coupled receptors transduce intracellular signals via the
stimulation or inhibition of several effectors, including phospholipase
C and adenylyl cyclase isoforms and some ion channels. Recently,
pertussis toxin-sensitive activation of the Src family kinases Src,
Fyn, Yes, and Lyn in various cell types has been reported (14, 33, 34),
suggesting that these kinases may also function in Gi-coupled receptor
signaling. Here, we demonstrate that recruitment and activation of
c-Src is involved in Gi-coupled receptor-mediated activation of the
Ras/MAP kinase pathway. In COS-7 cells, LPA receptor stimulation leads
to the rapid and transient formation of protein complexes containing
Shc and c-Src, which parallels the time course of LPA-stimulated Shc
tyrosine phosphorylation and Shc·Grb2 association. These events are
mimicked both by cellular expression of G Our data directly implicate c-Src in G The detection of endogenous or transiently expressed c-Src in Shc
immunoprecipitates may reflect either a direct interaction between the
two molecules or an association of both with an unknown intermediate.
However, in stimulated neutrophils (14), the Lyn kinase can be
precipitated by a Shc-SH2 domain containing fusion protein, supporting
the hypothesis that the interaction between Shc and this Src family
kinase is direct and SH2 domain-mediated.
Activation of c-Src via G protein-coupled receptors may also provide a
direct link between this class of receptor and other receptor pathways
involved in the regulation of cell growth and differentiation. Src
associates with activated platelet-derived growth factor receptor, EGF
receptor, and ErbB2 (42) and phosphorylates EGF receptor and ErbB2 on
nonautophosphorylation sites required for the binding of Src and
possibly other signaling molecules (43). Genistein-sensitive tyrosine
phosphorylation of insulin-like growth factor-1 receptor and IRS-1
following thrombin stimulation of rat aortic smooth muscle cells has
been reported (44). Thus, Src activation might provide a mechanism for
G protein-coupled, receptor-mediated assembly of a mitogenic signaling
complex directly on a tyrosine kinase growth factor receptor scaffold.
Recent data have suggested such a role for platelet-derived growth
factor receptors in vascular smooth muscle cells (45) and for EGF
receptor and p185neu in Rat-1 fibroblasts (46). Src is also
known to redistribute into a cytoskeletal compartment upon activation,
where it associates with integrin-dependent cytoskeletal
complexes. Bombesin, vasopressin, endothelin, thrombin, and LPA
receptors stimulate tyrosine phosphorylation of focal adhesion kinase
through both PKC-dependent and -independent pathways. In
addition to focal adhesion kinase and Src, integrin signaling complexes
contain Csk, the protein tyrosine phosphatase PTP1B, PI3K, and
Grb2/mSos, suggesting that these complexes may regulate intracellular
signal transduction pathways as well as integrin-mediated cell adhesive
interactions (47).
The focal adhesion kinase-related protein-tyrosine kinase PYK-2, which
is highly expressed in brain, has been implicated in Shc·Grb2·Sos
complex formation. Activation of PYK-2 is Ca2+ and
PKC-dependent and occurs following stimulation of the
Gq-coupled bradykinin receptor in PC12 cells (48). Thus, in appropriate
tissues, G protein-coupled, receptor-mediated phospholipase C
activation and Ca2+ influx might mediate
Ras-dependent MAP kinase activation via PYK-2-induced
tyrosine phosphorylation. The mechanism whereby G The Src family tyrosine kinases Fyn, Lyn, and Hck have been reported to
interact with the Bruton's tyrosine kinase (Btk) in hematopoietic
cells via an SH3 domain-mediated interaction (53). Src/Btk interaction
is associated with Btk autoactivation (54). Btk, and the related
tyrosine kinases Itk, Tsk, and TecA, like the serine/threonine kinases
FOOTNOTES Acknowledgments We thank D. Addison and M. Holben for
excellent secretarial assistance.
REFERENCES
Volume 271, Number 32,
Issue of August 9, 1996
pp. 19443-19450
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit-mediated Activation of Mitogen-activated
Protein Kinases*
§,,,
The Howard Hughes Medical Institute and the
Departments of Medicine and Biochemistry, Duke University Medical
Center, Durham, North Carolina 27710 and the ¶ Department of
Molecular Cell Biology, Glaxo Wellcome Inc., Research Triangle Park,
North Carolina 27709
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
subunit-mediated increases in
tyrosine phosphorylation of the Shc adapter protein, Shc·Grb2 complex
formation, and recruitment of Ras guanine nucleotide exchange factor
activity. We have investigated the role of the ubiquitous nonreceptor
tyrosine kinase c-Src in activation of the MAP kinase pathway via
endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or
by transient expression of G subunits in COS-7 cells. In
vitro kinase assays of Shc immunoprecipitates following LPA
stimulation demonstrated rapid, transient recruitment of tyrosine
kinase activity into Shc immune complexes. Recruitment of tyrosine
kinase activity was pertussis toxin-sensitive and mimicked by cellular
expression of G subunits. Immunoblots for coprecipitated proteins
in Shc immunoprecipitates revealed a transient association of Shc and
c-Src following LPA stimulation, which coincided with increases in
Shc-associated tyrosine kinase activity and Shc tyrosine
phosphorylation. LPA stimulation or expression of G subunits
resulted in c-Src activation, as assessed by increased c-Src
autophosphorylation. Overexpression of wild-type or constitutively
active mutant c-Src, but not kinase inactive mutant c-Src, lead to
increased tyrosine kinase activity in Shc immunoprecipitates, increased
Shc tyrosine phosphorylation, and Shc·Grb2 complex formation. MAP
kinase activation resulting from LPA receptor stimulation, expression
of G subunits, or expression of c-Src was sensitive to dominant
negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates
Src family kinases by phosphorylating the regulatory C-terminal
tyrosine residue, inhibited LPA stimulation of Shc tyrosine
phosphorylation, Shc·Grb2 complex formation, and MAP kinase
activation. These data suggest that G subunit-mediated formation
of Shc·c-Src complexes and c-Src kinase activation are early events
in Ras-dependent activation of MAP kinase via pertussis
toxin-sensitive G protein-coupled receptors.
-thrombin (1). Receptors for these substances,
activated in response to systemic or locally generated ligands, may in
turn play significant roles in the endocrine or paracrine regulation of
cell proliferation.
1B
adrenergic and M1 muscarinic acetylcholine receptors is significantly
inhibited by PKC depletion but insensitive to expression of a
dominant-negative mutant of Ras. In contrast, activation of MAP kinase
via the pertussis toxin-sensitive Gi-coupled 2A adrenergic and M2
muscarinic acetylcholine receptors is PKC-independent but requires Ras
activation and is sensitive to inhibitors of tyrosine protein kinases
(2). Similarly, LPA, a potent stimulator of mitogenesis in quiescent
fibroblasts that signals via a G protein-coupled receptor coupling to
both pertussis toxin-sensitive and -insensitive G proteins (3, 4, 5),
activates MAP kinase via a pertussis toxin-sensitive pathway involving
Ras and Raf activation (6, 7). LPA-mediated MAP kinase activation is
sensitive to tyrosine kinase inhibitors (7, 8) but independent of its
effects on phosphatidylinositol hydrolysis and its ability to inhibit
adenylyl cyclase (4, 8). In COS-7 cells, Ras-dependent MAP
kinase activation via 2A adrenergic (9), M2 muscarinic
acetylcholine, D2 dopamine, and A1 adenosine receptors (10) is mediated
largely by G subunits derived from pertussis toxin-sensitive G
proteins. Indeed, overexpression of G subunits, but not
constitutively activated Gi1 or Gi2 mutants, is sufficient to
activate MAP kinase (9, 10, 11) in these cells.
2A adrenergic receptors in COS-7 cells is
associated with increased tyrosine phosphorylation of the Shc adapter
protein and is dependent upon Grb2-mediated recruitment of the Ras
guanine nucleotide exchange factor mSos-1 (9). Stimulation of LPA,
2A adrenergic (9), thyrotropin-releasing hormone (12), endothelin 1 (13), and formyl methionyl peptide receptors (14) has been reported to
cause rapid and transient increases in Shc tyrosine phosphorylation and
Shc · Grb2 complex formation. Thyrotropin-releasing hormone and
formyl methionyl peptide receptor-mediated Shc phosphorylation is not
mimicked by Ca2+ ionophore, suggesting that the signal is
not PKC-dependent. The 2A adrenergic and formyl
methionyl peptide receptor-mediated Shc phosphorylation is pertussis
toxin-sensitive and can be mimicked by transient expression of G
subunits (9, 14, 15). Furthermore, cellular expression of a specific
G subunit sequestrant peptide derived from the carboxyl-terminal
G subunit-binding domain of the adrenergic receptor kinase 1 (ARK1) (16, 17) inhibits LPA and 2A adrenergic receptor-mediated
Shc phosphorylation in COS-7 cells (9), indicating that the
phosphorylation is mediated largely via G subunits derived from
pertussis toxin-sensitive G proteins. These data suggest that G
subunit-mediated formation of tyrosine-phosphorylated intermediates is
one of the earliest events in a MAP kinase activation pathway cascade
used by a significant subset of G protein-coupled receptors.
subunit-mediated,
Ras-dependent MAP kinase activation in COS-7 cells.
1 (19) and G2
(20) were provided by M. Simon. The cDNA encoding human
p60c-src (21) was provided by D. Fujita, and the
cDNA encoding p50csk (22) was provided by H. Hanafusa. The
constitutively activated Y530F p60c-src (TAC(Y) 
TTC(F); Refs. 23, 24, 25) and kinase inactive K298M
p60c-src (AAA(K) ATG(M); Ref. 26) mutants were
constructed by oligonucleotide-directed mutagenesis using a Sculptor
kit (Amersham Corp.). The cDNA encoding mSos1 was provided by M. Sakaue. The dominant-negative Sos-Pro construct, encompassing the
proline-rich carboxyl-terminal fragment of mSos1, was prepared as
described (9). The cDNAs encoding constitutively activated T24
p21ras (27) and dominant-negative N17 p21ras (28) were
provided by D. Altschuler and M. Ostrowski. The cDNA encoding the
p74raf-1 (29) dominant-negative mutant was provided by L. T. Williams. The cDNA encoding hemagglutinin-tagged p44mapk
(30) was provided by J. Pouyssegur. All cDNAs were subcloned into
pRK5 or pcDNA eukaryotic expression vectors for transient
transfection.
1 and G2 subunits,
Csk, wild-type and mutant c-Src proteins, Sos-Pro, N17 Ras and T24Ras,
and 
NRaf were confirmed by immunoblotting of transfected whole-cell
lysates using commercially available antisera. Transfected monolayers
were serum-starved in Dulbecco's modified Eagle's medium supplemented
with 0.1% bovine serum albumin and 10 mM Hepes, pH 7.4, for 16-20 h prior to stimulation.
-32P]ATP). Reactions
were terminated by the addition of 10 µl of stop solution (6 mg/ml
bovine serum albumin and 200 mM EDTA) and briefly
centrifuged. Twenty-µl aliquots of each supernatant were added to 40 µl of ice cold 10% trichloroacetic acid, precipitated for 20 min,
and centrifuged. Forty-µl aliquots of each clarified supernatant were
spotted onto P81 paper and washed three times in 0.425% phosphoric
acid and once in acetone; then Val5-angiotensin II
phosphorylation was quantified by scintillation counting.
-32P]ATP. Reactions were terminated by the
addition of 2 × Laemmli sample buffer, and labeled MBP was
resolved by SDS-PAGE. Quantitation of labeled MBP was performed using a
Molecular Dynamics PhosphorImager. Equal expression of
p44HA-mapk in cotransfected cells was confirmed by
immunoblotting with anti-erk1 following immunoprecipitation of
p44HA-mapkfrom whole-cell lysates using 12CA5
monoclonal antibody.
Subunits
1 and G2
subunits, on Shc tyrosine phosphorylation and Shc·Grb2 complex
formation in COS-7 cells. LPA stimulation resulted in a transient
3-4-fold increase in Shc phosphotyrosine and Shc·Grb2 complex
formation, compared with a 10-12-fold increase resulting from
stimulation of the endogenous EGF receptor tyrosine kinase.
Overexpression of G12 subunits resulted in a sustained 2-fold
increase. As shown in Fig. 1B, LPA receptor-mediated Shc
tyrosine phosphorylation and Shc·Grb2 complex formation were maximal
after 2-5 min of stimulation. The responses were inhibited by
pretreatment of cells with pertussis toxin, as is LPA receptor-mediated
MAP kinase activation in these cells (6).
Fig. 1.
Stimulation of Shc tyrosine phosphorylation
and Shc·Grb2 complex formation in COS-7 cells following endogenous
LPA receptor activation or transient overexpression of G
subunits. A, immunoblots of Shc phosphotyrosine and Grb2
from Shc immunoprecipitates following LPA or EGF stimulation or
transient overexpression of G subunits. Serum-starved cells were
stimulated for the indicated times with LPA (10 µM) or
EGF (10 ng/ml) (left panel) or transiently cotransfected
with empty pRK5 vector (NT) or G1 and G2 expression
plasmids (right panel). Immunoprecipitates of Shc from
nondenatured RIPA buffer lysates were resolved by SDS-PAGE and
immunoblotted with antiphosphotyrosine (upper panel) or
anti-Grb2 (lower panel) as described. The position of
tyrosine phosphorylated Shc isoforms and Grb2 are as indicated.
B, time course of LPA-mediated p52shc tyrosine
phosphorylation and Shc·Grb2 complex formation. Cells were
serum-starved overnight in the presence or absence of pertussis toxin
(PTx) (List Biological Labs; 100 ng/ml) prior to stimulation
for the indicated times with LPA. Shc phosphotyrosine and Shc·Grb2
complex formation were determined as described. Data are presented as
fold increase over nonstimulated controls and represent the means
(bars, S.E.) for three separate experiments.
subunits lead to direct recruitment of
a tyrosine kinase into Shc-containing signaling complexes, we assayed
for tyrosine kinase activity in Shc immunoprecipitates from COS-7 cells
stimulated with LPA or transiently cotransfected with G12
subunits. As shown in Fig. 2A, LPA
stimulation resulted in the rapid appearance of tyrosine kinase
activity in Shc immunoprecipitates assessed by an in vitro
kinase assay using Val5-angiotensin II as exogenous
substrate. The kinase activity was maximal 1-2 min after stimulation
and declined subsequently. As shown in Fig. 2B, the
LPA-induced recruitment of tyrosine kinase activity was pertussis
toxin-sensitive. Cells transiently expressing G subunits showed a
similar 6-8-fold increase in Shc-associated tyrosine kinase activity,
suggesting that the presence of free G subunits alone was
sufficient for kinase recruitment.
Fig. 2.
Detection of tyrosine kinase activity in Shc
immunoprecipitates of COS-7 cells following endogenous LPA receptor
stimulation or transient overexpression of G subunits.
A, time course of recruitment of tyrosine kinase activity
into Shc immunoprecipitates following stimulation of endogenous LPA
receptors. Serum-starved cells were stimulated for the indicated times
with LPA (10 µM), and Shc immunoprecipitates from
nondenatured RIPA buffer lysates were prepared as described.
Shc-containing immune complexes were assayed in vitro for
the presence of coprecipitated tyrosine kinase activity using
Val5-angiotensin II as substrate as described. Data are
presented net [32P] dpm incorporated into
Val5-angiotensin II and represent the means
(bars, S.E.) for duplicate determinations in one of three
separate experiments. B, pertussis toxin-sensitive
recruitment of tyrosine kinase activity into Shc immunoprecipitates
following endogenous LPA receptor stimulation or transient
overexpression of G subunits. Cells were serum-starved in the
presence or absence of pertussis toxin (100 ng/ml) and stimulated for 1 min with LPA (10 µM) (left panel) or
transiently cotransfected with empty pRK5 vector (NT) or
G1 and G2 expression plasmids (right panel). Shc
immunoprecipitates were prepared and assayed in vitro for
coprecipitated tyrosine kinase activity as described. Data are
presented as fold increase over nonstimulated or vector-only
transfected controls. Data shown represent the means (bars,
S.E.) for four separate experiments.
Fig. 3.
Recruitment of c-Src into Shc
immunoprecipitates of COS-7 cells following stimulation of endogenous
LPA or EGF receptors. A, detection of c-Src in Shc
immunoprecipitates by immunoblotting. Serum-starved cells were
stimulated for the indicated times with LPA (10 µM) or
EGF (10 ng/ml) or transiently transfected with the constitutively
active c-Src mutant, Y530F. Immunoprecipitates of Shc from nondenatured
RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with
anti-Shc (upper panel) or anti-Src monoclonal antibody
(lower panel) as described. The position of Shc isoforms and
c-Src are as indicated. B, time course of recruitment of
c-Src into Shc immunoprecipitates following stimulation of endogenous
LPA receptors. Serum-starved cells were stimulated for the indicated
times with LPA (10 µM), and Shc immunoprecipitates were
assayed for the presence of coprecipitated p60c-src
as described. Data are presented as fold increase over nonstimulated
controls and represent the means (bars, S.E.) for three
separate experiments.
-thrombin, 2A adrenergic, M2 muscarinic (33), and angiotensin II
receptors (34). Because there is a correlation between
autophosphorylation of Y416 and activation of the c-Src
kinase (24), whole-cell lysates from stimulated cells were assayed for
c-Src activation by immunoblotting using antiserum specific for
autophosphorylated c-Src (anti-pY416 c-Src) (35). As shown
in Fig. 4A, in control immunoblots of c-Src
from cells expressing kinase-deficient, K298M (26) or constitutively
autophosphorylated, Y530F, c-Src mutants, anti-pY416 c-Src
exhibited high specificity for the autophosphorylated kinase. One min
stimulation with LPA or EGF, or transient expression of G12
subunits, resulted in increased Y416 phosphorylated c-Src,
consistent with c-Src kinase activation in response to stimulation.
Both LPA stimulation and expression of G12 subunits resulted in
2-3-fold increases in autophosphorylated c-Src, as shown in Fig.
4B. LPA-induced c-Src autophosphorylation, like
LPA-stimulated Shc phosphorylation and Shc·Grb2 association, was
pertussis toxin-sensitive (data not shown). Thus, LPA- and G
subunit-stimulated c-Src·Shc protein complex formation correlated
with activation of the kinase.
Fig. 4.
Activation of c-Src in COS-7 cells following
endogenous LPA or EGF receptor activation or transient overexpression
of G subunits. A, detection of increased c-Src
autophosphorylation following LPA or EGF receptor stimulation or
G subunit overexpression using antiserum specific for
Y416-phosphorylated Src. Serum-starved cells were
stimulated for 1 min with LPA (10 µM) or EGF (10 ng/ml)
or transiently cotransfected with empty pRK5 vector (NT) or
G1 and G2 expression plasmids. Whole cell lysates were resolved
by SDS-PAGE, and c-Src was detected by protein immunoblotting using
either anti-Src monoclonal antibody 327 (upper panel) or
polyclonal antiserum specific for Y416-phosphorylated Src
(lower panel). Control immunoblots for each antibody were
performed on lysates prepared from empty pRK5 transfected cells or
cells transiently expressing kinase-deficient mutant c-Src (K298M) or
constitutively active c-Src (Y530F) (right panels).
B, quantitation of c-Src autophosphorylation following LPA
receptor stimulation or G subunit overexpression.
Autophosphorylation of c-Src was determined as described following
stimulation with LPA or transient overexpression of G subunits.
Autoradiographs were quantified by scanning laser densitometry, and
data are presented as fold increase over nonstimulated or empty pRK5
vector-transfected controls. Data shown represent the means
(bars, S.E.) for three separate experiments.
Fig. 5.
Recruitment of c-Src into Shc
immunoprecipitates and enhanced tyrosine phosphorylation of Shc in
COS-7 cells transiently expressing wild-type and mutant c-Src.
A, detection of c-Src in Shc immunoprecipitates of cells
transiently expressing wild-type and mutant c-Src. Cells were
transiently transfected with empty pRK5 vector (NT) or
expression plasmids encoding wild-type (c-Src) constitutively active
mutant (Y530F) or kinase-inactive (K298M) c-Src. Whole-cell lysates
(upper panel) and Shc immunoprecipitates from nondenatured
RIPA buffer lysates (lower panel) were resolved by SDS-PAGE
and immunoblotted with anti-p60c-src monoclonal
antibody as described. B, detection of tyrosine kinase
activity in Shc immunoprecipitates of cells transiently expressing
wild-type and mutant c-Src. Immunoprecipitates of Shc from cells
transiently expressing wild-type (c-Src), constitutively active mutant
(Y530F), or kinase-inactive mutant (K298M) c-Src were prepared as
described. Shc-containing immune complexes were assayed in
vitro for the presence of coprecipitated tyrosine kinase activity
using Val5-angiotensin II as substrate. Data are presented
as fold increase over empty pRK5 vector-transfected controls. Data
shown represent the means (bars, S.E.) for three separate
experiments. C, immunoblots of Shc phosphotyrosine and Grb2
from Shc immunoprecipitates following transient overexpression of
wild-type and mutant c-Src. Immunoprecipitates of Shc from cells
transiently expressing wild-type (c-Src) or constitutively active
mutant (Y530F) c-Src were resolved by SDS-PAGE and immunoblotted with
antiphosphotyrosine (upper panel) or anti-Grb2 (lower
panel) as described. Nonstimulated and EGF-stimulated lanes are
shown as controls. The position of tyrosine-phosphorylated Shc isoforms
and Grb2 are as indicated.
subunit expression on MAP kinases, we
determined the effects of each on Ras-dependent MAP kinase
activation. As shown in Fig. 6, stimulation of
endogenous LPA receptors or transient overexpression of either
G12 subunits or c-Src resulted in MAP kinase activation, as
determined by an in vitro kinase assay following
immunoprecipitation of coexpressed epitope-tagged p44MapK
(30). In each case, MAP kinase activation was inhibited by coexpression
of dominant negatives of mSos1 (9, 37), p21ras (28), and
p74raf-1 (29), indicating that the activation was
Ras-dependent. Thus, overexpression of c-Src mimicked the
effects of LPA receptor activation and G subunit expression,
resulting in tyrosine phosphorylation of Shc, Shc·Grb2 complex
formation, and Ras-dependent activation of MAP kinase.
Fig. 6.
Effects of dominant-negative Sos, Ras, and
Raf proteins on LPA, G, and c-Src-mediated MAP kinase
activation. COS-7 cells were transiently cotransfected with
hemagglutinin-tagged p44Mapk (p44HA-mapk) and
either empty vector (Control) or dominant-negative mutants
of mSos (Sos-Pro), p21ras (N17ras) or
p74raf-1 (Nraf) plus expression plasmids encoding G1 and
G2 or wild-type c-Src as indicated. Basal or 5-min LPA-stimulated
(10 µM) p44HA-mapkactivity was
determined following immunoprecipitation of
p44HA-mapk using MBP as substrate as described.
Expression of p44HA-mapk was not significantly
affected by coexpression of Sos-Pro, N17ras, or DNraf, as determined in
anti-p44mapk immunoblots from cotransfected cells (data not
shown). Data are presented as fold increase in
p44HA-mapkactivity over nonstimulated, empty pRK5
vector cotransfected controls (NS). Data shown represent the
means (bars, S.E.) of duplicate determinations in one of
three separate experiments.
subunit-mediated Shc
Phosphorylation and MAP Kinase Activation by Csk
Overexpression
subunit-mediated
signaling, we determined the effects of cellular expression of the
c-Src kinase, Csk, on Shc phosphorylation and MAP kinase activation.
Csk is a cytoplasmic protein tyrosine kinase (22) that inactivates Src
family kinases by phosphorylating a carboxyl-terminal regulatory
tyrosine residue. Mouse embryos lacking csk exhibit
increased c-Src, Fyn, and Lyn activity and increased levels of tyrosine
protein phosphorylation (38). Overexpression of wild-type Csk
suppresses endogenous c-Src activity (39) and, in opossum kidney cells,
blocks acid-induced activation of Na+/H+
antiporter, a process associated with p60c-src
activation (40).
Fig. 7.
Effect of Csk expression on Shc tyrosine
phosphorylation and Shc·Grb2 complex formation in COS-7 cells
following endogenous LPA receptor activation. A, immunoblots
of Shc phosphotyrosine and Grb2 from Shc immunoprecipitates following
LPA stimulation. Cells were transiently transfected with empty vector
(Control) or expression plasmid encoding Csk. Serum-starved
cells were stimulated for 2 min with LPA (10 µM) or EGF
(10 ng/ml), and immunoprecipitates of Shc from nondenatured RIPA buffer
lysates were immunoblotted with antiphosphotyrosine (upper
panel) or anti-Grb2 (lower panel) as described. The
position of tyrosine-phosphorylated Shc isoforms and Grb2 are as
indicated. B, inhibition of LPA-mediated Shc tyrosine
phosphorylation and Shc·Grb2 complex formation in cells
overexpressing Csk. Serum-starved control and Csk-expressing cells were
stimulated for 2 min with LPA. Shc phosphotyrosine and Shc·Grb2
complex formation were determined as described. Data are presented as
fold increase over nonstimulated controls and represent the means
(bars, S.E.) for three separate experiments.
subunit-mediated
activation by greater than 90%, with no significant effect on basal
levels of MAP kinase activity. In contrast, EGF-stimulated MAP kinase
activation was impaired by only 25%, consistent with the less dramatic
effects of Csk expression on EGF-mediated Shc phosphorylation. Phorbol
ester-mediated MAP kinase activation and that resulting from
overexpression of constitutively activated p21ras (T24ras) (27)
were not significantly affected, suggesting that the Csk-sensitive step
lies at a point in the pathway upstream of Ras and is not involved with
PKC-dependent MAP kinase activation. The partial inhibition
of LPA-mediated MAP kinase activation by Csk overexpression, compared
to nearly complete inhibition of LPA-stimulated Shc phosphorylation,
probably reflects downstream signal amplification occurring in
subsequent steps of the pathway. Although LPA receptors have been
reported to couple both to Gi and Gq/11 family G proteins,
LPA-stimulated MAP kinase activation observed in COS-7 cells was
greater than 90% pertussis toxin-sensitive (data not shown). Thus, the
alternative pertussis toxin-insensitive, Ras-independent, PKC-mediated
MAP kinase activation pathway, used by M1 muscarinic and 1B
adrenergic receptors in COS-7 cells (2), probably does not account for
the residual signal in the CSK-expressing cells. The ability of Csk
expression to inhibit pertussis toxin-sensitive G protein-mediated Shc
phosphorylation, Shc·Grb2 complex formation, and MAP kinase
activation without affecting PKC- or T24ras-dependent MAP
kinase activation suggests that Src family kinases are required for the
G protein-coupled, receptor-mediated tyrosine phosphorylation events
that precede Ras activation.
Fig. 8.
Effect of Csk expression on MAP kinase
activation in COS-7 cells. Cells were transiently cotransfected
with p44HA-mapk and either empty vector (Control)
or an expression plasmid encoding Csk plus G1 and G2 or
constitutively active p21ras (T24ras) as indicated. Basal, LPA
(10 µM), EGF (10 ng/ml), or phorbol myristate acetate
(PMA; 1 µM) stimulated
p44HA-mapk activity was determined following
immunoprecipitation of p44HA-mapk using MBP as
substrate as described. Data are presented as fold increase in
p44HA-mapk activity over nonstimulated, empty pRK5
vector cotransfected controls. Data shown represent the means
(bars, S.E.) for four separate experiments.
subunits and activated
c-Src mutants. Furthermore, expression of Csk, which inactivates Src
kinases, inhibits both LPA receptor-mediated Shc tyrosine
phosphorylation and MAP kinase activation, indicating that Src family
kinase activity is an important intermediate in the signal transduction
pathway. These results support a model of MAP kinase activation wherein
stimulation of Gi-coupled receptors and release of free G
subunits leads to activation of c-Src- and Src
kinase-dependent tyrosine phosphorylation of Shc, followed
by Grb2-mediated recruitment of Ras guanine nucleotide exchange factor
and Ras activation.
subunit-mediated MAP
kinase activation in COS-7 cells. Although it is likely that other Src
family tyrosine kinases, such as Lyn, Fyn and Yes, function in an
analogous manner in other cell types, we were able to demonstrate only
c-Src in Shc immunoprecipitates from COS-7 cells. Since Csk
specifically phosphorylates and inactivates Src family kinases, such as
Src, Fyn, and Yes (41), its inhibition of LPA receptor and G
subunit-mediated MAP kinase activation in COS-7 cells supports a
requirement for c-Src in the pathway. Since transient overexpression of
c-Src was sufficient to cause Shc tyrosine phosphorylation and Grb2
recruitment, LPA receptor-mediated activation of Src kinase is probably
sufficient to account for the tyrosine phosphorylation events required
for Ras activation. In our system, coexpression of the kinase-deficient
K298M c-Src mutant also blocks LPA- and G subunit-stimulated MAP
kinase activation. Unlike CSK expression, however, this construct also
strongly inhibits EGF receptor-, T24Ras-, and PMA-stimulated MAP kinase
activation (data not shown). This apparently nonspecific effect on MAP
kinase activation probably results from overexpression of the c-Src SH2
domain, which at high levels of expression could function as a
relatively nonspecific phosphotyrosine-binding protein, blocking the
MAP kinase signal transduction pathway at some point downstream of the
initial c-Src-dependent phosphorylations.
subunit-regulated effector(s) promote Src kinase activation remains
unclear. G subunit-mediated phosphatidylinositol hydrolysis and
Ca2+ mobilization are unable to account for G
subunit-mediated tyrosine phosphorylation in COS-7 cells (4, 8).
G subunit-mediated phosphorylation of p52shc is inhibited
by the PI3K inhibitor, wortmannin, (15), suggesting that PI3K activity
is required for assembly of the Ras activation complex. G
subunit-sensitive PI3K activity has been described in neutrophils and
platelets (49, 50), and the recently cloned p110 PI3K can be
activated by G subunits (51). Association between c-Src and PI3K
has been reported in chicken embryo fibroblasts expressing activated
c-Src mutants (52). Direct interaction between phosphatidylinositol
3,4,5-trisphosphate and the Src SH2 domain has also been proposed (53)
and might contribute to the localization or activation of the
kinase.
ARK1 and ARK2, contain pleckstrin homology domains. The
pleckstrin homology domain of ARK is required for kinase regulation,
because it mediates G subunit- and
phosphatidylinositol-dependent translocation of the kinase
from cytosol to membrane (55, 56). The activation of Btk and Tsk by
G subunits has been reported (57). These findings raise the
interesting possibility that G subunits, possibly in conjunction
with the products of PI3K, might regulate a class of tyrosine protein
kinase in a manner analogous to the ARK kinases and provide the
initial signaling events leading to Src family kinase activation and a
program of tyrosine protein phosphorylation. The relevance of G
protein-coupled, receptor-regulated PI3K and pleckstrin homology
domain-containing tyrosine protein kinases to the pathway of
c-Src-dependent Ras and MAP kinase activation remains the
subject of further study.
§
Recipient of a National Institutes of Health Clinical Investigator
Development Award.
To whom correspondence should be addressed: Howard Hughes
Medical Institute, Duke University, Box 3821, Durham, NC 27710. Tel.:
919-684-2974; Fax: 919-684-8875.
1
The abbreviations used are: MAP,
mitogen-activated protein; LPA, lysophosphatidic acid; PKC, protein
kinase C; ARK, adrenergic receptor kinase; PAGE, polyacrylamide
gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; MBP,
myelin basic protein; EGF, epidermal growth factor; PI3K,
phosphatidylinositol 3-kinase; Btk, Bruton's tyrosine kinase.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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J. Liu, Z. Liao, J. Camden, K. D. Griffin, R. C. Garrad, L. I. Santiago-Perez, F. A. Gonzalez, C. I. Seye, G. A. Weisman, and L. Erb Src Homology 3 Binding Sites in the P2Y2 Nucleotide Receptor Interact with Src and Regulate Activities of Src, Proline-rich Tyrosine Kinase 2, and Growth Factor Receptors J. Biol. Chem., February 27, 2004; 279(9): 8212 - 8218. [Abstract] [Full Text] [PDF]
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Y. Zagar, G. Chaumaz, and M. Lieberherr Signaling Cross-talk from G{beta}4 Subunit to Elk-1 in the Rapid Action of Androgens J. Biol. Chem., January 23, 2004; 279(4): 2403 - 2413. [Abstract] [Full Text] [PDF]
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L. Davidson, A. J. Pawson, R. P. Millar, and S. Maudsley Cytoskeletal Reorganization Dependence of Signaling by the Gonadotropin-releasing Hormone Receptor J. Biol. Chem., January 16, 2004; 279(3): 1980 - 1993. [Abstract] [Full Text] [PDF]
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R. K. H. Lo, H. Cheung, and Y. H. Wong Constitutively Active G{alpha}16 Stimulates STAT3 via a c-Src/JAK- and ERK-dependent Mechanism J. Biol. Chem., December 26, 2003; 278(52): 52154 - 52165. [Abstract] [Full Text] [PDF]
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R. D. Minshall, W. C. Sessa, R. V. Stan, R. G. W. Anderson, and A. B. Malik Caveolin regulation of endothelial function Am J Physiol Lung Cell Mol Physiol, December 1, 2003; 285(6): L1179 - L1183. [Abstract] [Full Text] [PDF]
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H. Lahlou, N. Saint-Laurent, J.-P. Esteve, A. Eychene, L. Pradayrol, S. Pyronnet, and C. Susini sst2 Somatostatin Receptor Inhibits Cell Proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 Activation J. Biol. Chem., October 10, 2003; 278(41): 39356 - 39371. [Abstract] [Full Text] [PDF]
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M. J. Smit, P. Verdijk, E. M. H. van der Raaij-Helmer, M. Navis, P. J. Hensbergen, R. Leurs, and C. P. Tensen CXCR3-mediated chemotaxis of human T cells is regulated by a Gi- and phospholipase C-dependent pathway and not via activation of MEK/p44/p42 MAPK nor Akt/PI-3 kinase Blood, September 15, 2003; 102(6): 1959 - 1965. [Abstract] [Full Text] [PDF]
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S. Kraus, O. Benard, Z. Naor, and R. Seger c-Src Is Activated by the Epidermal Growth Factor Receptor in a Pathway That Mediates JNK and ERK Activation by Gonadotropin-releasing Hormone in COS7 Cells J. Biol. Chem., August 29, 2003; 278(35): 32618 - 32630. [Abstract] [Full Text] [PDF]
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M. Yang, H. Zhang, T. Voyno-Yasenetskaya, and R. D. Ye Requirement of G{beta}{gamma} and c-Src in D2 Dopamine Receptor-Mediated Nuclear Factor-{kappa}B Activation Mol. Pharmacol., August 1, 2003; 64(2): 447 - 455. [Abstract] [Full Text] [PDF]
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D. Cai, S. Dhe-Paganon, P. A. Melendez, J. Lee, and S. E. Shoelson Two New Substrates in Insulin Signaling, IRS5/DOK4 and IRS6/DOK5 J. Biol. Chem., July 3, 2003; 278(28): 25323 - 25330. [Abstract] [Full Text] [PDF]
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B.-H. Ahn, S. Y. Kim, E. H. Kim, K. S. Choi, T. K. Kwon, Y. H. Lee, J.-S. Chang, M.-S. Kim, Y.-H. Jo, and D. S. Min Transmodulation between Phospholipase D and c-Src Enhances Cell Proliferation Mol. Cell. Biol., May 1, 2003; 23(9): 3103 - 3115. [Abstract] [Full Text] [PDF]
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C. Guillard, S. Chretien, A.-S. Pelus, F. Porteu, O. Muller, P. Mayeux, and V. Duprez Activation of the Mitogen-activated Protein Kinases Erk1/2 by Erythropoietin Receptor via a Gi Protein beta gamma -Subunit-initiated Pathway J. Biol. Chem., March 21, 2003; 278(13): 11050 - 11056. [Abstract] [Full Text] [PDF]
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A. Sabri, J. Guo, H. Elouardighi, A. L. Darrow, P. Andrade-Gordon, and S. F. Steinberg Mechanisms of Protease-activated Receptor-4 Actions in Cardiomyocytes. ROLE OF Src TYROSINE KINASE J. Biol. Chem., March 21, 2003; 278(13): 11714 - 11720. [Abstract] [Full Text] [PDF]
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P. Derkinderen, E. Valjent, M. Toutant, J.-C. Corvol, H. Enslen, C. Ledent, J. Trzaskos, J. Caboche, and J.-A. Girault Regulation of Extracellular Signal-Regulated Kinase by Cannabinoids in Hippocampus J. Neurosci., March 15, 2003; 23(6): 2371 - 2382. [Abstract] [Full Text] [PDF]
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A. Piiper, R. Elez, S.-J. You, B. Kronenberger, S. Loitsch, S. Roche, and S. Zeuzem Cholecystokinin Stimulates Extracellular Signal-regulated Kinase through Activation of the Epidermal Growth Factor Receptor, Yes, and Protein Kinase C. SIGNAL AMPLIFICATION AT THE LEVEL OF Raf BY ACTIVATION OF PROTEIN KINASE Cepsilon J. Biol. Chem., February 21, 2003; 278(9): 7065 - 7072. [Abstract] [Full Text] [PDF]
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M. Razandi, A. Pedram, S. T. Park, and E. R. Levin Proximal Events in Signaling by Plasma Membrane Estrogen Receptors J. Biol. Chem., January 17, 2003; 278(4): 2701 - 2712. [Abstract] [Full Text] [PDF]
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E. J. Dell, J. Connor, S. Chen, E. G. Stebbins, N. P. Skiba, D. Mochly-Rosen, and H. E. Hamm The beta gamma Subunit of Heterotrimeric G Proteins Interacts with RACK1 and Two Other WD Repeat Proteins J. Biol. Chem., December 13, 2002; 277(51): 49888 - 49895. [Abstract] [Full Text] [PDF]
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J. Xu, B. Jian, R. Chu, Z. Lu, Q. Li, J. Dunlop, S. Rosenzweig-Lipson, P. McGonigle, R. J. Levy, and B. Liang Serotonin Mechanisms in Heart Valve Disease II: The 5-HT2 Receptor and Its Signaling Pathway in Aortic Valve Interstitial Cells Am. J. Pathol., December 1, 2002; 161(6): 2209 - 2218. [Abstract] [Full Text] [PDF]
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Y. Pak, N. Pham, and D. Rotin Direct Binding of the {beta}1 Adrenergic Receptor to the Cyclic AMP-Dependent Guanine Nucleotide Exchange Factor CNrasGEF Leads to Ras Activation Mol. Cell. Biol., November 15, 2002; 22(22): 7942 - 7952. [Abstract] [Full Text] [PDF]
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A. Pedram, M. Razandi, and E. R. Levin Deciphering Vascular Endothelial Cell Growth Factor/Vascular Permeability Factor Signaling to Vascular Permeability. INHIBITION BY ATRIAL NATRIURETIC PEPTIDE J. Biol. Chem., November 8, 2002; 277(46): 44385 - 44398. [Abstract] [Full Text] [PDF]
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J. M. Schmitt and P. J. S. Stork Galpha and Gbeta gamma Require Distinct Src-dependent Pathways to Activate Rap1 and Ras J. Biol. Chem., November 1, 2002; 277(45): 43024 - 43032. [Abstract] [Full Text] [PDF]
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 E. Shumay, X. Song, H.-y. Wang, and C. C. Malbon pp60Src Mediates Insulin-stimulated Sequestration of the beta 2-Adrenergic Receptor: Insulin Stimulates pp60Src Phosphorylation and Activation Mol. Biol. Cell, November 1, 2002; 13(11): 3943 - 3954. [Abstract] [Full Text] [PDF]
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 A. Sabri, J. Short, J. Guo, and S. F. Steinberg Protease-Activated Receptor-1-Mediated DNA Synthesis in Cardiac Fibroblast Is via Epidermal Growth Factor Receptor Transactivation: Distinct PAR-1 Signaling Pathways in Cardiac Fibroblasts and Cardiomyocytes Circ. Res., September 20, 2002; 91(6): 532 - 539. [Abstract] [Full Text] [PDF]
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 A. Ptasznik, E. Urbanowska, S. Chinta, M. A. Costa, B. A. Katz, M. A. Stanislaus, G. Demir, D. Linnekin, Z. K. Pan, and A. M. Gewirtz Crosstalk Between BCR/ABL Oncoprotein and CXCR4 Signaling through a Src Family Kinase in Human Leukemia Cells J. Exp. Med., September 2, 2002; 196(5): 667 - 678. [Abstract] [Full Text] [PDF]
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S. Dalle, T. Imamura, D. W. Rose, D. S. Worrall, S. Ugi, C. J. Hupfeld, and J. M. Olefsky Insulin Induces Heterologous Desensitization of G Protein-Coupled Receptor and Insulin-Like Growth Factor I Signaling by Downregulating {beta}-Arrestin-1 Mol. Cell. Biol., September 1, 2002; 22(17): 6272 - 6285. [Abstract] [Full Text] [PDF]
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J. Kim, A. D. Eckhart, S. Eguchi, and W. J. Koch beta -Adrenergic Receptor-mediated DNA Synthesis in Cardiac Fibroblasts Is Dependent on Transactivation of the Epidermal Growth Factor Receptor and Subsequent Activation of Extracellular Signal-regulated Kinases J. Biol. Chem., August 23, 2002; 277(35): 32116 - 32123. [Abstract] [Full Text] [PDF]
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 P. Robin, I. Boulven, C. Desmyter, S. Harbon, and D. Leiber ET-1 stimulates ERK signaling pathway through sequential activation of PKC and Src in rat myometrial cells Am J Physiol Cell Physiol, July 1, 2002; 283(1): C251 - C260. [Abstract] [Full Text] [PDF]
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 A. C. B. Cato, A. Nestl, and S. Mink Rapid Actions of Steroid Receptors in Cellular Signaling Pathways Sci. Signal., June 25, 2002; 2002(138): re9 - re9. [Abstract] [Full Text] [PDF]
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A. Yart, S. Roche, R. Wetzker, M. Laffargue, N. Tonks, P. Mayeux, H. Chap, and P. Raynal A Function for Phosphoinositide 3-Kinase beta Lipid Products in Coupling beta gamma to Ras Activation in Response to Lysophosphatidic Acid J. Biol. Chem., June 7, 2002; 277(24): 21167 - 21178. [Abstract] [Full Text] [PDF]
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D. Liu and J. S. Dillon Dehydroepiandrosterone Activates Endothelial Cell Nitric-oxide Synthase by a Specific Plasma Membrane Receptor Coupled to Galpha i2,3 J. Biol. Chem., June 7, 2002; 277(24): 21379 - 21388. [Abstract] [Full Text] [PDF]
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M. J. Smit, D. Verzijl, P. Casarosa, M. Navis, H. Timmerman, and R. Leurs Kaposi's Sarcoma-Associated Herpesvirus-Encoded G Protein-Coupled Receptor ORF74 Constitutively Activates p44/p42 MAPK and Akt via Gi and Phospholipase C-Dependent Signaling Pathways J. Virol., February 15, 2002; 76(4): 1744 - 1752. [Abstract] [Full Text] [PDF]
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 M. Mendez and M. C. LaPointe Trophic Effects of the Cyclooxygenase-2 Product Prostaglandin E2 in Cardiac Myocytes Hypertension, February 1, 2002; 39(2): 382 - 388. [Abstract] [Full Text] [PDF]
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 S. Paruchuri, B. Hallberg, M. Juhas, C. Larsson, and A. Sjolander Leukotriene D4 activates MAPK through a Ras-independent but PKC{epsilon}-dependent pathway in intestinal epithelial cells J. Cell Sci., January 5, 2002; 115(9): 1883 - 1893. [Abstract] [Full Text] [PDF]
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L. M. Luttrell and R. J. Lefkowitz The role of {beta}-arrestins in the termination and transduction of G-protein-coupled receptor signals J. Cell Sci., January 2, 2002; 115(3): 455 - 465. [Abstract] [Full Text] [PDF]
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C. A. Singer, S. Vang, and W. T. Gerthoffer Coupling of M2 muscarinic receptors to Src activation in cultured canine colonic smooth muscle cells Am J Physiol Gastrointest Liver Physiol, January 1, 2002; 282(1): G61 - G68. [Abstract] [Full Text] [PDF]
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R. D. Peavy, M. S. S. Chang, E. Sanders-Bush, and P. J. Conn Metabotropic Glutamate Receptor 5-Induced Phosphorylation of Extracellular Signal-Regulated Kinase in Astrocytes Depends on Transactivation of the Epidermal Growth Factor Receptor J. Neurosci., December 15, 2001; 21(24): 9619 - 9628. [Abstract] [Full Text] [PDF]
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 I. Faerge, B. Terry, J. Kalous, P. Wahl, M. Lessl, J.L. Ottesen, P. Hyttel, and C. Grondahl Resumption of Meiosis Induced by Meiosis-Activating Sterol Has a Different Signal Transduction Pathway than Spontaneous Resumption of Meiosis in Denuded Mouse Oocytes Cultured In Vitro Biol Reprod, December 1, 2001; 65(6): 1751 - 1758. [Abstract] [Full Text] [PDF]
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E. S. Gilmore, M. J. Stutts, and S. L. Milgram Src Family Kinases Mediate Epithelial Na+ Channel Inhibition by Endothelin J. Biol. Chem., November 2, 2001; 276(45): 42610 - 42617. [Abstract] [Full Text] [PDF]
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J. N. Hislop, H. M. Everest, A. Flynn, T. Harding, J. B. Uney, B. E. Troskie, R. P. Millar, and C. A. McArdle Differential Internalization of Mammalian and Non-mammalian Gonadotropin-releasing Hormone Receptors. UNCOUPLING OF DYNAMIN-DEPENDENT INTERNALIZATION FROM MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING J. Biol. Chem., October 19, 2001; 276(43): 39685 - 39694. [Abstract] [Full Text] [PDF]
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Y. Chen, Y. H. Zhao, and R. Wu Differential Regulation of Airway Mucin Gene Expression and Mucin Secretion by Extracellular Nucleotide Triphosphates Am. J. Respir. Cell Mol. Biol., October 1, 2001; 25(4): 409 - 417. [Abstract] [Full Text] [PDF]
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X. Zhen, J. Zhang, G. P. Johnson, and E. Friedman D4 Dopamine Receptor Differentially Regulates Akt/Nuclear Factor-kappa B and Extracellular Signal-Regulated Kinase Pathways in D4MN9D Cells Mol. Pharmacol., October 1, 2001; 60(4): 857 - 864. [Abstract] [Full Text] [PDF]
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 J. Smith, R. Yu, and P. M. Hinkle Activation of MAPK by TRH Requires Clathrin-Dependent Endocytosis and PKC but Not Receptor Interaction with {beta}-Arrestin or Receptor Endocytosis Mol. Endocrinol., September 1, 2001; 15(9): 1539 - 1548. [Abstract] [Full Text] [PDF]
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S. Faivre, K. Regnauld, E. Bruyneel, Q.-D. Nguyen, M. Mareel, S. Emami, and C. Gespach Suppression of Cellular Invasion by Activated G-Protein Subunits Galpha o, Galpha i1, Galpha i2, and Galpha i3 and Sequestration of Gbeta gamma Mol. Pharmacol., August 1, 2001; 60(2): 363 - 372. [Abstract] [Full Text] [PDF]
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 I. Shefler and R. Sagi-Eisenberg Gi-Mediated Activation of the Syk Kinase by the Receptor Mimetic Basic Secretagogues of Mast Cells: Role in Mediating Arachidonic Acid/Metabolites Release J. Immunol., July 1, 2001; 167(1): 475 - 481. [Abstract] [Full Text] [PDF]
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D.-W. Kim and B. H. Cochran JAK2 Activates TFII-I and Regulates Its Interaction with Extracellular Signal-Regulated Kinase Mol. Cell. Biol., May 15, 2001; 21(10): 3387 - 3397. [Abstract] [Full Text]
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F. Alderton, P. P. A. Humphrey, and L. A. Sellers High-Intensity p38 Kinase Activity Is Critical for p21cip1 Induction and the Antiproliferative Function of Gi Protein-Coupled Receptors Mol. Pharmacol., April 16, 2001; 59(5): 1119 - 1128. [Abstract] [Full Text]
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Y. Gao, S. Tang, S. Zhou, and J. A. Ware The Thromboxane A2 Receptor Activates Mitogen-Activated Protein Kinase via Protein Kinase C-Dependent Gi Coupling and Src-Dependent Phosphorylation of the Epidermal Growth Factor Receptor J. Pharmacol. Exp. Ther., April 13, 2001; 296(2): 426 - 433. [Abstract] [Full Text]
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 G. Pearson, F. Robinson, T. Beers Gibson, B.-e Xu, M. Karandikar, K. Berman, and M. H. Cobb Mitogen-Activated Protein (MAP) Kinase Pathways: Regulation and Physiological Functions Endocr. Rev., April 1, 2001; 22(2): 153 - 183. [Abstract] [Full Text]
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 Y.-M. Yang, W. C. Hatch, Z.-Y. Liu, B. Du, and J. E. Groopman {beta}-Chemokine Induction of Activation Protein-1 and Cyclic AMP Responsive Element Activation in Human Myeloid Cells Cell Growth Differ., April 1, 2001; 12(4): 211 - 221. [Abstract] [Full Text]
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O. Kifor, R. J. MacLeod, R. Diaz, M. Bai, T. Yamaguchi, T. Yao, I. Kifor, and E. M. Brown Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells Am J Physiol Renal Physiol, February 1, 2001; 280(2): F291 - F302. [Abstract] [Full Text] [PDF]
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Q. He and M. C. LaPointe Src and Rac Mediate Endothelin-1 and Lysophosphatidic Acid Stimulation of the Human Brain Natriuretic Peptide Promoter Hypertension, February 1, 2001; 37(2): 478 - 484. [Abstract] [Full Text] [PDF]
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 L. Xing, A. M. Venegas, A. Chen, L. Garrett-Beal, B. F. Boyce, H. E. Varmus, and P. L. Schwartzberg Genetic evidence for a role for Src family kinases in TNF family receptor signaling and cell survival Genes & Dev., January 15, 2001; 15(2): 241 - 253. [Abstract] [Full Text]
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 E. Feraille and A. Doucet Sodium-Potassium-Adenosinetriphosphatase-Dependent Sodium Transport in the Kidney: Hormonal Control Physiol Rev, January 1, 2001; 81(1): 345 - 418. [Abstract] [Full Text] [PDF]
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R. A. Blake, M. A. Broome, X. Liu, J. Wu, M. Gishizky, L. Sun, and S. A. Courtneidge SU6656, a Selective Src Family Kinase Inhibitor, Used To Probe Growth Factor Signaling Mol. Cell. Biol., December 1, 2000; 20(23): 9018 - 9027. [Abstract] [Full Text]
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V. P. Krymskaya, M. J. Orsini, A. J. Eszterhas, K. C. Brodbeck, J. L. Benovic, R. A. Panettieri Jr., and R. B. Penn Mechanisms of Proliferation Synergy by Receptor Tyrosine Kinase and G Protein-Coupled Receptor Activation in Human Airway Smooth Muscle Am. J. Respir. Cell Mol. Biol., October 1, 2000; 23(4): 546 - 554. [Abstract] [Full Text]
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E. J. Filardo, J. A. Quinn, K. I. Bland, and A. R. Frackelton Jr. Estrogen-Induced Activation of Erk-1 and Erk-2 Requires the G Protein-Coupled Receptor Homolog, GPR30, and Occurs via Trans-Activation of the Epidermal Growth Factor Receptor through Release of HB-EGF Mol. Endocrinol., October 1, 2000; 14(10): 1649 - 1660. [Abstract] [Full Text]
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