(Received for publication, August 26, 1996, and in revised form, November 18, 1996)
From 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
Biochemistry, Glaxo Wellcome Research and Development,
Research Triangle Park, North Carolina 27709
In many cells, stimulation of mitogen-activated
protein kinases by both receptor tyrosine kinases and receptors that
couple to pertussis toxin-sensitive heterotrimeric G proteins proceed via convergent signaling pathways. Both signals are sensitive to
inhibitors of tyrosine protein kinases and require Ras activation via
phosphotyrosine-dependent recruitment of Ras guanine
nucleotide exchange factors. Receptor tyrosine kinase stimulation
mediates ligand-induced receptor autophosphorylation, which creates the initial binding sites for SH2 domain-containing docking proteins. However, the mechanism whereby G protein-coupled receptors mediate the
phosphotyrosine-dependent assembly of a mitogenic signaling complex is poorly understood. We have studied the role of Src family
nonreceptor tyrosine kinases in G protein-coupled receptor-mediated tyrosine phosphorylation in a transiently transfected COS-7 cell system. Stimulation of Gi-coupled lysophosphatidic acid and
2A adrenergic receptors or overexpression of G
1
2 subunits
leads to tyrosine phosphorylation of the Shc adapter protein, which then associates with tyrosine phosphoproteins of approximately 130 and
180 kDa, as well as Grb2. The 180-kDa Shc-associated tyrosine phosphoprotein band contains both epidermal growth factor (EGF) receptor and p185neu. 3-5-fold increases in EGF receptor but
not p185neu tyrosine phosphorylation occur following
Gi-coupled receptor stimulation. Inhibition of endogenous
Src family kinase activity by cellular expression of a dominant
negative kinase-inactive mutant of c-Src inhibits G
1
2
subunit-mediated and Gi-coupled receptor-mediated
phosphorylation of both EGF receptor and Shc. Expression of Csk, which
inactivates Src family kinases by phosphorylating the regulatory
carboxyl-terminal tyrosine residue, has the same effect. The
Gi-coupled receptor-mediated increase in EGF receptor phosphorylation does not reflect increased EGF receptor
autophosphorylation, assayed using an autophosphorylation-specific EGF
receptor monoclonal antibody. Lysophosphatidic acid stimulates binding
of EGF receptor to a GST fusion protein containing the c-Src SH2
domain, and this too is blocked by Csk expression. These data suggest
that G
subunit-mediated activation of Src family nonreceptor
tyrosine kinases can account for the Gi-coupled
receptor-mediated tyrosine phosphorylation events that direct
recruitment of the Shc and Grb2 adapter proteins to the membrane.
The low molecular weight G protein Ras functions as a signaling
intermediate in many pathways involved in the regulation of cellular
mitogenesis and differentiation. Ras activation by growth factor
receptors that possess intrinsic tyrosine kinase activity follows
ligand-induced phosphorylation of specific docking sites on the
receptor itself or adapter proteins, such as Shc and insulin receptor
substrate-1, which serve to recruit Ras guanine nucleotide exchange
factors to the plasma membrane (1, 2). Recently, several receptors that
couple to heterotrimeric G proteins, including the lysophosphatidic
acid (LPA)1 (3, 4), -thrombin (5),
angiotensin II (6, 7),
2A adrenergic (AR) (8, 9), M2 muscarinic
acetylcholine, D2 dopamine, and A1 adenosine receptors (10), have been
shown to mediate Ras-dependent mitogenic signals. In COS-7
cells, Ras-dependent activation of mitogen-activated
protein kinases via the
2A AR, M2 muscarinic acetylcholine, D2
dopamine, and A1 adenosine receptors is mediated largely by G
subunits released from pertussis toxin-sensitive G proteins (8, 9).
These G
subunit-mediated signals are sensitive to inhibitors of
tyrosine protein kinases (8), associated with increased tyrosine
protein phosphorylation, and dependent upon recruitment of Ras guanine
nucleotide exchange factors to the membrane (9), indicating that the
pathway converges with the receptor tyrosine kinase pathway at an early
point.
G protein-coupled receptors have been shown to mediate rapid tyrosine
phosphorylation of several proteins that participate in mitogenic
signal transduction. The thyrotropin-releasing hormone (11), endothelin
1 (12), LPA, and 2A AR receptors (9) stimulate tyrosine
phosphorylation of the Shc adapter protein. This effect can be mimicked
by the transient overexpression of G
subunits (9, 13) and
correlates with Shc-Grb2 complex formation (9, 12) and the recruitment
of Ras guanine nucleotide exchange factor activity (9). In addition,
recent reports have described G protein-coupled receptor-mediated
tyrosine phosphorylation of insulin receptor substrate-1 (14), focal
adhesion kinase (4, 15), and several receptor tyrosine kinases,
including the platelet-derived growth factor (PDGF) receptor (16), EGF receptor, p185neu (17), and insulin-like growth factor-1
receptor (14).
The mechanism whereby G protein-coupled receptors stimulate tyrosine
protein phosphorylation is poorly understood. The observation that the
receptors for PDGF (16) and EGF (17) undergo tyrosine phosphorylation
following G protein-coupled receptor activation has led to the
hypothesis that the intrinsic tyrosine kinase activity of these
receptors becomes activated by an unknown mechanism. G protein-coupled
receptor-mediated activation of nonreceptor tyrosine kinases has also
been reported. Recently, activation of Src family kinases by the
-thrombin (18), LPA (19), angiotensin II (20), N-formyl
methionyl peptide chemoattractant (21),
2A AR (18, 19), and M1
muscarinic acetylcholine (18) receptors has been reported. Furthermore,
inhibition of Src family kinases has been shown to inhibit angiotensin
II-stimulated Ras (22) and phospholipase C-
1 (23) activation in rat
aortic smooth muscle cells, LPA and
2A AR-stimulated MAP kinase
activation in COS-7 cells (19), M1 and M2 muscarinic
acetylcholine-stimulated MAP kinase activation in avian B cells (24),
and endothelin-1-stimulated transcriptional activation in rat
glomerular mesangial cells (25).
We have previously shown in transiently transfected COS-7 cells that
pertussis toxin-sensitive G protein-coupled receptors mediate G
subunit-dependent activation of c-Src and that inhibition of Src family kinases by cellular expression of Csk antagonizes G
protein-coupled receptor-mediated MAP kinase activation (19). Here, we
examine the role of Src family nonreceptor tyrosine kinases in
mediating G
subunit-dependent tyrosine
phosphorylation of receptor tyrosine kinases and Shc. Our data suggest
that activation of Src family kinases by G protein-coupled receptors
can account for the Gi-coupled receptor-mediated tyrosine
phosphorylation events that direct recruitment of the Shc and Grb2
adapter proteins to the membrane using the EGF receptor as a
scaffold.
The cDNA encoding the 2A AR was
cloned in our laboratory. The cDNAs encoding G
1 and G
2 were
provided by M. Simon (California Institute of Technology, Pasadena,
CA). The cDNA encoding human p60c-src was provided by D. Fujita (University of Calgary, Alberta, Canada), and the cDNA
encoding p50csk was provided by H. Hanafusa (Rockefeller
University, New York, NY). The constitutively activated Y530F
p60c-src (TAC(Y)
TTC(F)), in which the regulatory
carboxyl-terminal tyrosine residue has been mutated, and catalytically
inactive K298M p60c-src (AAA(K)
ATG(M)) mutants were
prepared as described (19). All cDNAs were subcloned into pRK5 or
pcDNA eukaryotic expression vectors for transient transfection.
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 transient transfection,
cells were incubated at 37 °C in 4 ml serum-free Dulbecco's
modified Eagle's medium containing 6-10 µg of DNA/100-mm dish 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. 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. 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 G1 and G
2 subunits, Csk, and
mutant c-Src proteins was confirmed by immunoblotting of transfected
whole cell lysates using commercially available antisera.
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 and lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 100 µM NaVO4, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotonin, 10 µg/ml leupeptin) for immunoprecipitation under nondenaturing conditions or RIPA/SDS buffer (RIPA buffer containing 0.1% SDS) for immunoprecipitation under denaturing conditions. Cell lysates were sonicated briefly, clarified by centrifugation, and diluted to a protein concentration of 2 mg/ml. Immunoprecipitations from 1 ml of lysate were performed using the appropriate primary antibody 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, denatured in Laemmli sample buffer, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE). Shc immunoprecipitations were performed using rabbit polyclonal anti-Shc antibody (Transduction Laboratories). EGF receptor was immunoprecipitated using monoclonal anti-EGF receptor antibody (Transduction Laboratories), and p185neu was immunoprecipitated using rabbit polyclonal anti-HER2 (Santa Cruz Biotechnology).
Tyrosine phosphorylation or the presence of coprecipitated proteins was detected by protein immunoblotting. Phosphotyrosine was detected using a 1:1000 dilution of horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody (Transduction Laboratories). Shc protein was detected using a 1:1000 dilution of rabbit polyclonal anti-Shc IgG (Transduction Laboratories), p185neu was detected using a 1:1000 dilution of rabbit polyclonal anti-HER2 IgG (Santa Cruz Biotechnology), 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 Corp.) as secondary antibody. C-Src was detected using 1:500 dilution of mouse monoclonal anti-c-Src antibody 327 with horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson Laboratories) as secondary antibody. EGF receptor was detected using 1:1000 dilution of sheep anti-human EGF receptor IgG with horseradish peroxidase-conjugated donkey anti-sheep IgG (Jackson Laboratories) as secondary antibody. Immunoblots for autophosphorylated EGF receptor were performed using mouse monoclonal anti-activated EGF receptor IgG (26) with horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson Laboratories) as secondary antibody. Immune complexes on nitrocellulose were visualized by enzyme-linked chemiluminescence (Amersham Corp.) and quantified by scanning laser densitometry.
GST Fusion Proteins Containing the c-Src SH2 and SH3 DomainsGST fusion proteins containing the human c-Src SH2 (amino acids 144-249), SH3 (amino acids 87-143), or SH2 and SH3 (amino acids 87-249) domains were prepared as GST Sepharose conjugates as described previously (27). For the detection of c-Src SH2 or SH3 domain-binding proteins, appropriately transfected and stimulated COS-7 cells were lysed in RIPA/SDS buffer containing 5 mM dithiothreitol, sonicated, clarified by centrifugation, precleared with 6 µg/ml GST Sepharose for 1 h and incubated with 6 µg/ml of the GST fusion protein Sepharose for 3 h at 4 °C. After incubation, fusion protein complexes were washed twice with ice-cold RIPA buffer and once with phosphate-buffered saline, denatured in Laemmli sample buffer, and resolved by SDS-PAGE. Coprecipitated tyrosine phosphoproteins and EGF receptor were detected by protein immunoblotting as described.
As shown in Fig. 1A,
stimulation of endogenous LPA receptors in COS-7 cells leads to a rapid
3-5-fold increase in tyrosine phosphorylation of each of the three Shc
isoforms. The phosphorylation is maximal within 2 min of stimulation
and declines slowly thereafter (9, 19). Under nondenaturing conditions,
Shc coprecipitates with two major tyrosine phosphoprotein bands of
approximately 130 and 180 kDa and with the adapter protein Grb2. The
association of Shc with the p130 and p180 phosphoproteins is modulated
with a time course that parallels the time course of Shc
phosphorylation and Shc-Grb2 complex formation, suggesting that LPA
stimulation induces association of these proteins. As shown in Fig.
1B, similar increases in Shc phosphorylation and Shc-p180
association result from transient expression of G1
2 subunits or
stimulation of endogenous LPA or transiently expressed
2A AR
receptors. Stimulation of endogenous EGF receptors or transient
overexpression of a constitutively activated mutant human c-Src (Y530F
p60c-src; Refs. 28-30) has similar, although more robust
effects, indicating that activation of either the receptor tyrosine
kinase or nonreceptor Src kinase can mimic the G protein-mediated
effects. As shown in Fig. 1 (C and D),
Gi-coupled receptor-mediated but not EGF receptor-mediated
Shc phosphorylation and Shc-p180 association are pertussis
toxin-sensitive in these cells.
Because G protein-coupled receptor-mediated tyrosine phosphorylation of
PDGF receptor (16), EGF receptor, and p185neu (17) has been
reported, we performed immunoblots for EGF receptor and p185neu
on Shc immunoprecipitates from nondenatured cell lysates following stimulation of LPA, 2A AR, or EGF receptors to determine whether these receptor tyrosine kinases are present in the Shc-associated p180
phosphotyrosine band. COS-7 cells lack detectable expression of PDGF
receptor (31). As shown in Fig. 2A, EGF
receptor is not detectable in Shc immunoprecipitates from nonstimulated
cells, but stimulation of either Gi-coupled receptor
results in Shc-EGF receptor coprecipitation. In contrast,
Shc-p185neu complexes are present in nonstimulated cells and do
not increase detectably following LPA or
2A AR receptor stimulation.
As expected, EGF stimulation results in both Shc-EGF receptor and
Shc-p185neu association, which may reflect heterodimerization
and transphosphorylation of the two related receptor tyrosine kinases
(32). As shown in Fig. 2B, the tyrosine phosphorylation
states of Shc, EGF receptor, and p185neu, determined following
direct immunoprecipitation of each protein, reflect the changes in
Shc-receptor tyrosine kinase association. Shc and EGF receptor
phosphorylation is increased following LPA,
2A AR, or EGF receptor
stimulation. P185neu exhibits significant basal tyrosine
phosphorylation, consistent with the detection of Shc-p185neu
complexes in nonstimulated cells, which detectably increases only
following EGF receptor stimulation.
To confirm that Shc, Grb2, and EGF receptor directly associate following Gi-coupled receptor stimulation, EGF receptor immunoprecipitates were assayed for the presence of Shc and Grb2 following LPA stimulation. As shown in Fig. 2C, stimulation with either LPA or EGF resulted in the association of Shc and Grb2 with EGF receptor. Gi-coupled receptor-induced association of Src family nonreceptor tyrosine kinases with Shc has been reported (19, 21). As shown, c-Src can also be detected in EGF receptor immunoprecipitates from LPA- or EGF-stimulated cell lysates, suggesting that activation of the Gi-coupled receptor results in association of EGF receptor, c-Src, Shc, and Grb2 in a multiprotein complex.
Src Family Kinase Activity Is Required for Both Gi-coupled Receptor and GGi-coupled
receptor-mediated increases in EGF receptor phosphotyrosine might
result from ligand-independent activation of the receptor tyrosine
kinase, phosphorylation by an activated nonreceptor tyrosine kinase, or
inhibition of a phosphotyrosine phosphatase. To distinguish between
these alternative mechanisms, we employed a monoclonal anti-EGF
receptor antibody specific for autophosphorylated EGF receptor.
This antibody selectively recognizes activated EGF receptor via an
epitope distal to amino acid 1052 (26), which is distinct from the
major in vitro c-Src phosphorylation sites (33). As shown in
Fig. 3 (A and B),
antiphosphotyrosine immunoblots of EGF receptor immunoprecipitated from
EGF-stimulated cells, from cells transiently expressing Y530F
p60c-src, and from cells in which phosphotyrosine phosphatase
activity is inhibited by incubation with sodium orthovanadate, each
exhibit increased total receptor phosphorylation. Identical immunoblots probed with the anti-activated EGF receptor antibody give increased signals from EGF-stimulated and sodium orthovanadate-treated cells but
not from Y530F p60c-src-transfected cells. Thus, the
anti-activated EGF receptor antibody is able to discriminate between
increased autophosphorylation resulting from activation of the
intrinsic tyrosine kinase activity of the EGF receptor or inhibition of
a phosphotyrosine phosphatase versus phosphorylation of the
EGF receptor mediated by the c-Src nonreceptor tyrosine kinase. As
shown, this antibody does not detect EGF receptor autophosphorylation
following stimulation of LPA or 2A AR receptors, despite a 3-5-fold
increase in total EGF receptor phosphotyrosine, suggesting that the
increase in receptor tyrosine phosphorylation does not reflect
activation of the intrinsic tyrosine kinase.
Because expression of activated mutant c-Src is sufficient to mediate
EGF receptor phosphorylation in the absence of ligand, we tested the
hypothesis that Gi-coupled receptor-mediated activation of
Src family kinases can account for the observed tyrosine
phosphorylation of Shc and EGF receptor. To inhibit endogenous Src
family kinases, cells were transiently transfected with cDNA
encoding either Csk or a kinase-inactive dominant negative mutant c-Src
(K298M p60c-src; Ref. 34). Csk is a cytoplasmic tyrosine
protein kinase (35) that inactivates Src family kinases by
phosphorylating the regulatory carboxyl-terminal tyrosine residue. Csk
overexpression has been shown to impair G protein-coupled
receptor-mediated MAP kinase activation in COS-7 cells (19) and
c-fos transcription in rat glomerular mesangial cells (25).
As shown in Fig. 4 (A and B), coexpression of either Csk or K298M p60c-src markedly inhibits
G1
2 subunit-,
2A AR-, and LPA receptor-mediated tyrosine
phosphorylation of both Shc and EGF receptor. EGF-stimulated Shc and
EGF receptor phosphorylation were less dramatically effected. Shc and
EGF receptor phosphorylation mediated by Y530F, which is not a
substrate for Csk, is not significantly attenuated by Csk
overexpression.
Effect of Csk and K298M p60c-src
expression on Gi-coupled receptor-mediated Shc and EGF
receptor tyrosine phosphorylation. A, Immunoblots of Shc
phosphotyrosine following 2A AR, LPA or EGF receptor stimulation and
G
subunit or Y530F p60c-src expression. Cells were
transiently cotransfected with empty vector (Control) or
expression plasmid encoding Csk or K298M p60c-src, plus empty
pRK5 vector, G
1 and G
2,
2A AR, or Y530F p60c-src.
Serum-starved cells were stimulated for 2 min with UK14304
(UK), LPA, or EGF as indicated, and immunoprecipitates of
Shc from RIPA/SDS buffer lysates were immunoblotted with
anti-phosphotyrosine as described. The position of tyrosine
phosphorylated Shc isoforms are as indicated. B, immunoblots
of EGF receptor phosphotyrosine following
2A AR, LPA, or EGF
receptor stimulation and G
subunit or Y530F p60c-src
expression. Serum-starved, transiently cotransfected cells were stimulated as described and immunoprecipitates of EGF receptor from
RIPA/SDS buffer lysates were immunoblotted with anti-phosphotyrosine as
described. The position of tyrosine phosphorylated EGF receptor is as
indicated. C, quantitation of the effects of Csk and
K298M p60c-src coexpression on G
subunit-,
2A AR-,
LPA-, EGF-, and Y530F p60c-src-stimulated Shc and EGF receptor
tyrosine phosphorylation. Shc and EGF receptor phosphotyrosine were
determined as described following
2A AR, LPA, or EGF receptor
stimulation and G
subunit or Y530F p60c-src expression.
Autoradiographs were quantified by scanning laser densitometry, and the
data were presented as fold increase over nonstimulated or empty pRK5
vector transfected controls. The data shown represent the means ± S.E. for three to five separate experiments. NS,
nonstimulated.
c-Src SH2 Domain GST Fusion Proteins Bind EGF Receptor Following Gi-coupled Receptor Activation
To determine whether
Gi-coupled receptor-stimulated EGF receptor phosphorylation
can induce binding of Src kinases directly to the EGF receptor,
GST-fusion proteins containing either the c-Src SH2, SH3, or SH2-SH3
domains (27) were assayed for the ability to precipitate phosphorylated
EGF receptor from lysates of LPA-stimulated cells. As shown in Fig.
5A, the c-Src SH2 and SH2-SH3 domain GST
fusion proteins but not the c-Src SH3 domain GST fusion protein
precipitate a 180-kDa tyrosine phosphoprotein band that increases in
intensity following LPA or EGF receptor stimulation. Immunoblots of EGF
receptor in c-Src GST SH2 domain precipitates, shown in Fig.
5B, reveal increased association of EGF receptor with the
c-Src SH2 domain following LPA or EGF stimulation, suggesting that
LPA-stimulated tyrosine phosphorylation of the EGF receptor is
responsible for recognition of EGF receptor by the c-Src SH2
domain.
In vitro mapping of phosphorylation sites on the EGF receptor has suggested that phosphorylation of the putative c-Src SH2 domain recognition site (Tyr891) is mediated by the c-Src kinase rather than the intrinsic receptor tyrosine kinase (33). To determine if phosphorylation of this site is mediated by endogenous Src kinases in the intact cell following Gi-coupled receptor stimulation, we tested the effect of Csk overexpression on LPA-stimulated phosphorylation of the c-Src SH2 domain binding site. As shown in Fig. 5C, the ability of the c-Src SH2 domain GST fusion protein to precipitate EGF receptor following LPA stimulation is markedly attenuated in Csk-expressing cells. Because Src kinases also mediate phosphorylation of this site following receptor tyrosine kinase activation, EGF receptor precipitation by the c-Src SH2 domain GST fusion protein following stimulation with EGF is also significantly attenuated. These data suggest that Gi-coupled receptor stimulation results in both c-Src mediated phosphorylation of the EGF receptor and SH2 domain-dependent c-Src-EGF receptor complex formation.
Several lines of evidence suggest that Src family kinases play a
key role in the transduction of mitogenic signals by G protein-coupled receptors. Pertussis toxin-sensitive activation of the Src family kinases Src, Fyn, Yes, and Lyn (18-21) in various cell types has been
reported, and inhibition of Src family kinases has been shown to block
G protein-coupled receptor-mediated Ras and phospholipase C-1
activation, MAP kinase activation, and c-fos transcription (19, 22-24). Our data demonstrate that in COS-7 cells,
Gi-coupled receptor-stimulated tyrosine phosphorylation of
the EGF receptor results in formation of a complex between the
membrane-associated EGF receptor and the cytosolic adapter proteins Shc
and Grb2, thus providing a scaffold for the assembly of a mitogenic
signaling complex. The Gi-coupled receptor effects can be
mimicked by cellular overexpression of G
subunits, suggesting
that the process is G
subunit-mediated. Because inhibition of
endogenous Src kinases blocks both G protein-coupled receptor-mediated
EGF receptor phosphorylation and binding of the EGF receptor to the
c-Src SH2 domain, the data also suggest that Src family kinases
directly associate with and phosphorylate the EGF receptor following
Gi-coupled receptor stimulation.
Fig. 6 depicts a model of G subunit-mediated,
Ras-dependent MAP kinase activation that is consistent with
these data. G
subunit-dependent activation of
endogenous Src family nonreceptor tyrosine kinases is an early event
following Gi-coupled receptor stimulation (19). Once
activated, the Src kinases mediate phosphorylation of several
intracellular targets, including receptor tyrosine kinases, adapter
proteins such as Shc and insulin receptor substrate-1, and possibly
cytoskeletally associated Src substrates such as focal adhesion kinase
and paxillin. Once phosphorylated, membrane-associated proteins such as
the receptor tyrosine kinases and focal adhesion kinase would provide
docking sites for the SH2 domains of the Shc and Grb2 adapter
molecules, resulting in the recruitment of Ras guanine nucleotide
exchange factors, and potentially of other components of the mitogenic
signaling complex, to the plasma membrane. The ensuing activation of
Ras would recruit the Raf kinase to the membrane and initiate the
phosphorylation cascade leading to MAP kinase activation.
Depending upon cell type, the G protein-coupled receptors for
angiotensin II, LPA, and -thrombin have been shown to stimulate ligand-independent tyrosine phosphorylation of PDGF receptor (16), insulin-like growth factor-1 receptor
subunit (14), EGF receptor, and p185neu (17). The finding that several receptor tyrosine
kinases undergo G protein-coupled receptor-mediated phosphorylation
suggests the existence of a common mechanism that is not receptor
tyrosine kinase-specific, such as activation of a nonreceptor tyrosine kinase or inhibition of a phosphotyrosine phosphatase. Our data, demonstrating inhibition of Gi-coupled receptor-mediated
tyrosine phosphorylation of the EGF receptor by specific inhibitors of Src family kinases, support the hypothesis that activation of Src
kinases can account for the observed receptor tyrosine kinase phosphorylation.
The role of the intrinsic tyrosine kinase activity of receptor tyrosine
kinases in Gi-coupled receptor-mediated tyrosine
phosphorylation is unclear. Daub et al. (17) have reported
that inhibition of EGF receptor function in Rat1 cells, by either an
EGF receptor-selective tyrphostin, AG1478, or expression of a dominant
negative mutant EGF receptor, blocks endothelin-1, LPA, and
-thrombin receptor-mediated EGF receptor/HER2 phosphorylation and
MAP kinase activation. They conclude that a ligand-independent
transactivation of the EGF receptor/HER2 tyrosine kinase is responsible
for G protein-coupled receptor-mediated tyrosine phosphorylation. Our
data suggest that activation of Src family nonreceptor tyrosine kinases
by Gi-coupled receptors can account for tyrosine
phosphorylation of both the EGF receptor and the Shc adapter protein in
COS-7 cells. The finding that inhibition of endogenous Src kinase
activity blocks Gi-coupled receptor-mediated EGF receptor
phosphorylation suggests that Src kinase activation precedes receptor
tyrosine kinase phosphorylation but does not preclude the possibility
that Src-mediated phosphorylation modulates the activity of the
receptor tyrosine kinase. However, using a monoclonal antibody that can
discriminate between c-Src-mediated phosphorylation and EGF receptor
autophosphorylation, we have been unable to detect increased EGF
receptor autophosphorylation following either overexpression of Y530F
p60c-src or Gi-coupled receptor stimulation.
The mechanism whereby effectors of activated G protein-coupled
receptors stimulate Src family kinases is unknown. Stimulation of
phosphatidylinositol 3-kinase activity may play a role in
Ras-dependent MAP kinase activation in some cells. G
subunit-mediated PI3K activity has been described in neutrophils and
platelets (36, 37), a G
subunit-regulated isoform of p110 PI3K
has been cloned, and G
subunits may contribute to the regulation
of the conventional p85/p110 PI3K (38). We have previously reported
that Gi-coupled receptor- and G
subunit-mediated MAP
kinase activation in COS-7 and CHO cells is sensitive to the PI3K
inhibitors wortmannin and LY294002 and to expression of a dominant
negative form of the p85 regulatory subunit of PI3K (39).
Interestingly, MAP kinase activation by transiently expressed Y530F
p60c-src,2, mSos, and
constitutively activated mutants of Ras and MAP kinase/erk kinase (39) is wortmannin-insensitive, suggesting that the
PI3K-dependent step in the pathway may lie upstream of Src
kinase activation. The recent report that the c-Src SH2 domain can bind
with high affinity to phosphatidylinositol 3,4,5-trisphosphate, the
product of PI3K (40), may provide an explanation for this
phenomenon.
Interaction between Src kinases and novel G subunit-regulated
nonreceptor tyrosine kinases might also contribute to the regulation of
Src kinase activity. In neuronal cells, Gq-coupled receptors have been shown to stimulate the Ca2+ and protein
kinase C dependent tyrosine protein kinase, PYK2 (41). PYK2 is a member
of the focal adhesion kinase family of integrin receptor-associated
tyrosine kinases and like p125FAK (42) can complex with
activated c-Src upon stimulation (43). However, phospholipase C
activation and Ca2+ mobilization are apparently unable to
account for G protein-coupled receptor-mediated tyrosine
phosphorylation in many noneuronal cells (4, 44, 45). Bruton's
tyrosine kinase (Btk) and Tsk, two members of a family of pleckstrin
homology domain-containing tyrosine protein kinases that includes Btk,
Itk, Tsk and Tec A, are reportedly regulated by G
subunits (46).
In hematopoietic cells, Btk interacts with the Src family kinases Fyn,
Lyn, and Hck (47), and Src-Btk interaction is associated with Btk
autoactivation (48). This is unlikely to be a general mechanism for G
protein-coupled receptor regulation of Src kinases, however, because
the pleckstrin homology domain-containing tyrosine kinases appear to
have limited tissue distribution and are not known to be involved in
the regulation of Ras.
The data presented in this report suggest that both Src family kinases and receptor tyrosine kinases play central roles in directing the assembly of membrane-associated mitogenic signaling complexes in response to Gi-coupled receptor activation in some cells. An understanding of the mechanisms whereby G protein-coupled receptors regulate tyrosine protein phosphorylation and of the basis for cross-talk between G protein-coupled receptor and receptor tyrosine kinase signaling pathways may ultimately provide strategies for selective activation or inhibition of cellular proliferation.
We thank D. Addison and M. Holben for excellent secretarial assistance.