From the Department of Biochemistry, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232, the
§ 2nd Department of Internal Medicine, Tokyo Medical and
Dental University, Tokyo 113, Japan, the ¶ Department of Anatomy
and Physiology, Meharry Medical College, Nashville, Tennessee 37208, and the
Institute of Molecular and Cellular Biology for
Pharmaceutical Sciences, Kyoto Pharmaceutical University,
Kyoto 607, Japan
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ABSTRACT |
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We have recently reported that angiotensin II (Ang II)-induced mitogen-activated protein kinase (MAPK) activation is mainly mediated by Ca2+-dependent activation of a protein tyrosine kinase through Gq-coupled Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMC). In the present study, we found Ang II rapidly induced the tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and its association with Shc and Grb2. These reactions were inhibited by the EGF receptor kinase inhibitor, AG1478. The Ang II-induced phosphorylation of the EGF receptor was mimicked by a Ca2+ ionophore and completely inhibited by an intracellular Ca2+ chelator. Thus, AG1478 abolished the MAPK activation induced by Ang II, a Ca2+ ionophore as well as EGF but not by a phorbol ester or platelet-derived growth factor-BB in the VSMC. Moreover, Ang II induced association of EGF receptor with catalytically active c-Src. This reaction was not affected by AG1478. These data indicate that Ang II induces Ca2+-dependent transactivation of the EGF receptor which serves as a scaffold for pre-activated c-Src and for downstream adaptors, leading to MAPK activation in VSMC.
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INTRODUCTION |
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Protein tyrosine phosphorylation and subsequent protein-protein interaction induced by growth factors is a prototypical pathway to transmit mitogenic signals to the nucleus (1). For example, tyrosine phosphorylation of growth factor receptors recruits the guanine nucleotide exchange factor, son-of-sevenless (Sos)1 through adaptor proteins, Shc and Grb2, thereby initiating a sequential cascade from p21ras (Ras) to mitogen-activated protein kinases (MAPKs), referred to as p44mapk (ERK1) and p42mapk (ERK2) (2-5). MAPKs in turn phosphorylate and activate several kinases and transcriptional factors, including TCF/Elk1, and stimulate the induction of c-fos (4, 6).
Angiotensin II (Ang II), a major effector peptide of the renin-angiotensin system, is now believed to play a critical role in the pathogenesis of cardiovascular remodeling associated with hypertension, heart failure, and atherosclerosis (7). We and others (8, 9) have previously cloned the Ang II type 1 receptor (AT1) which not only mediates diverse hemodynamic effects of Ang II (10) but also promotes hypertrophy and/or hyperplasia of vascular smooth muscle cells (VSMC) (11-13), cardiomyocytes (14), and cardiac fibroblasts (15). AT1 belongs to the superfamily of heterotrimeric G protein-coupled receptors (GPCR) (8, 9). In cultured VSMC, AT1 activates phospholipase C (PLC), which initiates the generation of inositol trisphosphate and diacylglycerol, causing intracellular calcium mobilization and protein kinase C activation, respectively (16, 17). In addition, Ang II induces several signaling events commonly evoked by growth factor receptors, such as the activation of MAPK (18, 19) and the ribosomal S6 kinase (20), and the expression of the nuclear proto-oncogenes, c-fos, c-jun, and c-myc (21-23) in VSMC.
Although AT1 lacks intrinsic tyrosine kinase activity, it
appeared to induce tyrosine phosphorylation of multiple signaling proteins (24) including Shc (25), focal adhesion kinase (26), paxillin
(27), PLC- (28), JAK2, and STAT1 (29) in VSMC, suggesting cross-talk
of AT1 and a tyrosine kinase. In fact, recent works with
various GPCR including AT1 suggest that GPCR-induced MAPK
activation requires Shc-Grb2·Sos and/or Grb2·Sos complex formation
and subsequent Ras activation mediated by several candidate tyrosine
kinases, such as proline-rich tyrosine kinase 2 (PYK2) (30),
platelet-derived growth factor (PDGF) receptor (25), epidermal growth
factor (EGF) receptor (31), and Src family tyrosine kinases
(32-35).
We have recently reported that Ang II-induced Ras and MAPK activation is mainly mediated by a calcium-dependent protein tyrosine kinase through Gq-mediated PLC activation via AT1 in cultured rat quiescent VSMC (36). However, the identity of the tyrosine kinase and its pathophysiological significance in the growth promoting signal of Ang II have remained unclear. In the present study, we found that Ang II induces Ca2+-dependent tyrosine phosphorylation of the EGF receptor to recruit Shc and Grb2, thereby activating MAPK in VSMC. The transactivation of the EGF receptor seems to be an essential point of convergence in this growth promoting cascade because it provides docking sites for the upstream tyrosine kinase c-Src and downstream adaptors at the plasma membrane, and because its activity is required for the MAPK activation induced by Ang II.
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EXPERIMENTAL PROCEDURES |
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Chemicals and Reagents-- Chemicals and reagents were obtained from the following sources: Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, penicillin, and streptomycin from Life Technologies, Inc.; Ang II from Peninsula Laboratories; recombinant human EGF and PDGF-BB from Upstate Biotechnology Inc.; AG1478, AG1295, A23187, and BAPTA-AM from Calbiochem; phorbol 12-myristate 13-acetate and EGTA from Sigma; an agarose-conjugated glutathione S-transferase (GST)-Grb2-(1-217) fusion protein and protein A/G-agarose from Santa Cruz Biotechnology. CV11974 was a generous gift of Takeda Pharmaceutical Co.
Antibodies and Their Specificities--
The rabbit polyclonal
phospho-specific MAPK antibody (9101) raised against a synthetic
phosphotyrosine peptide corresponding to amino acids 196-209
(DHTGFLTEY(P)VATRWC, where P indicates phosphate)
of human p44 MAPK (ERK1) was obtained from New England Biolabs that
detects only the catalytically active form of p42/44 MAPKs which are
phosphorylated at Tyr204. We have previously shown (36)
that this antibody specifically recognizes
Tyr204-phosphorylated p42/44 MAPK in cultured rat VSMC.
Anti-EGF receptor polyclonal antibody (pAb)(1005) raised against a
synthetic peptide corresponding to amino acid residues 1005-1016 of
human EGF receptor (identical to the corresponding mouse sequence) was
obtained from Santa Cruz Biotechnology that also specifically
recognizes rat EGF receptor in both immunoblotting and
immunoprecipitation. Anti-Shc pAb (06-203) and monoclonal antibody
(mAb) (S52420: clone 8) raised against a GST-tagged fusion protein
corresponding to the SH2 domain (amino acid residues 366-473) of the
human p46/p52 Shc was obtained from Upstate Biotechnology and
Transduction Laboratories, respectively. Anti-Shc pAb specifically
reacts with p46/52/66 Shc of rat origin by immunoblotting and
immunoprecipitation (34, 37), and anti-Shc mAb also reacts with
p46/52/66 Shc of rat origin by immunoblotting (34, 38). Anti-Grb2 pAb
(C-23) raised against a peptide corresponding to amino acid residues
195-217 of human Grb2 was obtained from Santa Cruz Biotechnology that
is also specific for Grb2 of the rat origin by immunoprecipitation (34,
38). Anti-Grb2 mAb (G16720: clone 24) raised against the entire 24-kDa
Grb2 protein from rat brain was obtained from Transduction Laboratories
that specifically reacts with rat Grb2 by immunoblotting (30). Anti-Sos pAbs (S15530) raised against a protein fragment of mouse Sos1 corresponding to amino acid residues 1-109 and a protein fragment of
mouse Sos2 corresponding to amino acid residues 1095-1297, respectively, were obtained from Transduction Laboratories that also
specifically react with Sos1 and Sos2 of rat origin by immunoblotting (34). Anti-PDGF receptor pAb (06-498) raised against a synthetic peptide corresponding to amino acid residues 1013-1025 of human PDGF
receptor was obtained from Upstate Biotechnology. It specifically reacts with PDGF
receptor by immunoblotting and
immunoprecipitation. We have previously confirmed that this antibody
also specifically reacts with rat PDGF
receptor in cultured rat
VSMC (39). The mAb directed to Tyr530-dephosphorylated
c-Src (clone 28) was prepared as described previously which selectively
recognizes the active form of rat c-Src (40). A horseradish
peroxidase-conjugated recombinant antibody fragment specific for
phosphotyrosine (RC20) and anti-phosphotyrosine mAb (4G10) were from
Transduction Laboratories and Upstate Biotechnology Inc., respectively.
Horseradish peroxidase-conjugated second antibodies were from Amersham
Pharmacia Biotech.
Cell Culture--
VSMC were prepared from the thoracic aorta of
12-week-old Sprague-Dawley rats (Charles River Breeding Laboratories)
by the explant method and cultured in DMEM containing 10% fetal calf serum, penicillin, and streptomycin as described previously (41). Subcultured VSMC from passages 3-15, used in the experiments, showed
>99% positive immunostaining of smooth muscle -actin antibody and
were negative for mycoplasma infection. The expression of AT1 but not AT2 receptors was confirmed on the
basis of binding studies with specific receptor antagonists (36). Cells
at ~80% confluence in culture wells were made quiescent by
incubation with serum-free DMEM for 3 days.
MAPK Activity--
VSMC grown on a 24-well plate were stimulated
with agonists at 37 °C in serum-free DMEM for specified durations.
The reaction was terminated by the replacement of medium with the
ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 20 mM NaCl, 2 mM EGTA, 2 mM
dithiothreitol, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and
10 µg/ml aprotinin). After brief sonication (10 s), the samples were
centrifuged for 5 min at 14,000 × g, and the
supernatant was assayed for MAPK activity with an assay kit (Amersham
Pharmacia Biotech) that measures the incorporation of
[33P]phosphate from [-33P]ATP into a
synthetic peptide (KRELVEPLTPAGEAPNQALLR) as a specific MAPK substrate
(36). The reaction was carried out with the cell lysate (~1 µg of
protein) in 75 mM HEPES buffer, pH 7.4, containing 1.2 mM MgCl2, 2 mM substrate peptide,
and 1.2 mM ATP, 1 µCi of [
-33P]ATP for
30 min at 30 °C. The resultant solution was applied to a
phosphocellulose membrane and extensively washed in 1% acetic acid and
then in deionized water. The radioactivity trapped on the membrane was
measured by liquid scintillation counting.
Immunoprecipitation and Immunoblotting-- Cells were lysed by adding ice-cold lysis buffer, pH 7.5, containing 50 mM HEPES, 50 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl2, 1 mM EDTA, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 100 mM NaF, 30 mM 2-(p-nitrophenyl) phosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin and centrifuged for 5 min at 14,000 × g. Supernatant was mixed with the immunoprecipitation antibody and rocked at 4 °C for 2-16 h, and then protein A/G-Sepharose was added for an additional 2 h to overnight. Immunoprecipitates were washed 3 times in the lysis buffer, solubilized in Laemmli sample buffer with 2-mercaptoethanol, resolved by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech). After blocking with 5% milk, the membrane was treated with a primary antibody followed by a secondary antibody conjugated with horseradish peroxidase. Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech). For repeated immunoblotting, membranes were stripped in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 0.1 M 2-mercaptoethanol for 30-45 min at 50 °C. For immunoblot analysis of MAPK phosphorylation, VSMC grown on a 6-well plate were directly lysed by Laemmli sample buffer with 2-mercaptoethanol, resolved by SDS-polyacrylamide gel electrophoresis, and subjected to immunoblotting. For immunoblot analysis of Grb2-associated proteins, agarose-conjugated GST-Grb2 fusion protein was rocked with Triton X-100 lysate of VSMC for 2 h to overnight at 4 °C and washed 3 times with lysis buffer. Bound proteins were solubilized, resolved by SDS-polyacrylamide gel electrophoresis, and subjected to immunoblotting, as described above.
Reproducibility of the Results-- Unless stated otherwise, results are representative of at least three experiments giving similar results.
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RESULTS |
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Inhibition of Ang II-induced MAPK Activation by AG1478, the EGF
Receptor Kinase Inhibitor--
In many MAPK activation systems, a
tyrosine-phosphorylated scaffold is needed to assemble adaptor proteins
(1-3). Highly likely candidates in VSMC for such a scaffold mediating
MAPK activation by Ang II are the PDGF or EGF receptors. In VSMC, Ang
II has been shown to cause PDGF receptor phosphorylation which
leads to recruitment of the Shc·Grb2 complex to the receptor (25).
This pathway may account for Ras and MAPK activation. A recent study revealed that several GPCRs use EGF receptor transactivation for MAPK
activation, c-fos induction, and DNA synthesis in Rat-1
fibroblasts (31). To clarify the role of these receptor tyrosine
kinases in Ang II-induced signal transduction in VSMC, we first tested the effect of selective receptor tyrosine kinase inhibitors (42) on the
MAPK activity in VSMC. As shown in Fig.
1A, the EGF receptor kinase
inhibitor tyrphostin AG1478 dose-dependently and completely blocked EGF-induced MAPK activation, whereas it had no effect on the
PDGF-induced activation confirming its stringent selectivity. We then
observed marked inhibition of Ang II-induced MAPK activation by AG1478
(Fig. 1B) but not by the PDGF receptor-selective tyrosine kinase inhibitor, AG1295 (Fig. 1C). AG1478 also inhibited
the tyrosine phosphorylation of MAPK by Ang II and EGF without
affecting that by PDGF-BB (Fig. 1D). Moreover, AG1478
inhibited Ca2+ ionophore (A23187)-induced MAPK activation,
whereas it had no effect on the phorbol ester-induced activation (Fig.
1C). This is in good agreement with our previous observation
that the Ang II-induced Ras and MAPK activation requires a
Ca2+-sensitive tyrosine kinase but not protein kinase C in
VSMC (36).
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Ang II Induces Calcium-dependent Tyrosine Phosphorylation of the EGF Receptor and Its Association with Shc, Grb2, and Sos-- The activated EGF receptor can recruit the Grb2·Sos complex directly and indirectly via tyrosine phosphorylation of Shc, thereby activating the Ras/MAPK signaling pathway (1-3). To examine the possibility that Ang II signaling utilizes the EGF receptor to provide a docking site for Grb2, we examined proteins that interact with a GST-Grb2 fusion protein in the lysate of VSMC upon stimulation by Ang II. As shown in Fig. 2A, Ang II transiently increased association of several tyrosine-phosphorylated proteins with the GST-Grb2 fusion protein as detected by anti-phosphotyrosine antibody. The association of tyrosine-phosphorylated proteins is specific to Grb2 because no band was seen when GST-agarose alone was used (Fig. 2D). A similar pattern of Grb2-associating tyrosine-phosphorylated proteins was observed following treatment of VSMC with the Ca2+ ionophore, A23187 (Fig. 2B). The major phosphoprotein (~170 kDa) associated with the GST-Grb2 fusion protein upon treatment with Ang II was identified as the EGF receptor because it was recognized by the anti-EGF receptor antibody (Fig. 2C) and was diminished by pretreatment with AG1478 (data not shown). We confirmed that the phosphorylated EGF receptor was coprecipitated with endogenous Grb2 upon Ang II treatment and was also diminished by AG1478 (Fig. 3). It should be noted that the tyrosine-phosphorylated bands of ~50 kDa were practically wiped out by AG1478, whereas the ~120-kDa band was not visibly affected (Fig. 3). In cultured rat VSMC, Ang II was shown to induce tyrosine phosphorylation of 46, 52, and 66 kDa Shc isoforms which subsequently form a complex with Grb2 (25). The ~50-kDa phosphoprotein associated with Grb2 upon treatment with Ang II shown in Fig. 3 should be p52 Shc. Indeed, the Ang II treatment resulted in association of 46-, 52-, and 66-kDa Shc isoforms to the GST-Grb2 fusion protein in which p52 Shc was the dominant form in VSMC (Fig. 2A). A23187 also increased p52 Shc association to the GST-Grb2 fusion protein in VSMC (Fig. 2B).
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Interaction of c-Src with Shc and EGF Receptor upon Ang II Stimulation-- The Src family tyrosine kinases have been implicated in the GPCR-induced MAPK activation (32-35). c-Src mediates Shc phosphorylation, Shc·Grb2 complex formation, and ensuing MAPK activation elicited by Gi-coupled receptors in COS-7 cells (35). In VSMC, Ang II was shown to activate c-Src (43) which may be required for the Ras activation by Ang II (44). Ca2+-dependent c-Src activation was also reported in epidermal keratinocytes (45) and neuronal cells (46). To examine whether c-Src is involved in the MAPK cascade initiated by Ang II, the proteins inducibly associated with the GST-Grb2 fusion protein were immunoblotted by a monoclonal antibody, clone 28, which selectively recognizes the active (Tyr530-dephosphorylated) form of c-Src (40). Ang II increased transient association of active c-Src with the GST-Grb2 fusion protein (Fig. 6A). Since Shc is known to be tyrosine-phosphorylated by Src kinases (47) presumably through the interaction with the SH3 domain of the kinases (48), we examined whether active c-Src forms a coprecipitable complex with Shc in response to Ang II. As shown in Fig. 6B, Ang II induced complex formation of active c-Src with Shc that was correlated with p52 Shc tyrosine phosphorylation in VSMC.
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DISCUSSION |
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VSMC in culture has proven to be a useful model to examine the molecular mechanisms whereby vasoactive substances such as Ang II contribute to abnormal vascular hypertrophy. Recently, we have reported (36) that in VSMC, Ras and MAPK activation through AT1 was mediated by a tyrosine kinase which may respond to Gq-coupled intracellular Ca2+ mobilization but not to protein kinase C activation. In the present study, we have demonstrated that an EGF receptor kinase inhibitor, AG1478, selectively inhibited MAPK activation induced by Ang II and the Ca2+ ionophore A23187, whereas it had no effect on the activation induced by a phorbol ester. Furthermore, Ang II and A23187 induced tyrosine phosphorylation of the EGF receptor, which was sufficient to recruit the adaptor proteins that are involved in Ras activation. Thus, it is reasonable to speculate that the Ca2+-dependent tyrosine phosphorylation of the EGF receptor may be a common mechanism to activate MAPK shared by several GPCRs coupled to Gq, such as AT1 in VSMC. This notion is supported by the recent findings that several GPCR agonists (31) as well as KCl-induced depolarization (38) elicited tyrosine phosphorylation of the EGF receptor and subsequent recruitment of the adaptor proteins to the receptor. In general, phosphorylation of the EGF receptor by GPCR agonists and Ca2+ agonists is relatively weaker than EGF itself as observed by us and others (31, 38) indicating that there may exist a threshold phosphorylation level of the receptor that is sufficient for the recruitment of the adaptors and subsequent MAPK activation.
AT1 has been shown to mobilize intracellular
Ca2+ by activating PLC- through Gq in
cultured VSMC (16, 17). However, recent reports by Marrero et
al. (28, 52) indicate that Ang II can mobilize intracellular
Ca2+ by PLC-
activation mediated by Src family tyrosine
kinases in cultured rat VSMC. Since the EGF receptor can recruit and
activate PLC-
through an autophosphorylation site at
Tyr992 (2), Ang II could elevate intracellular
Ca2+ through PLC-
activated by the EGF receptor thereby
activating the MAPK cascade in VSMC. However, AG1478 (250 nM) did not influence Ang II-induced intracellular
Ca2+ mobilization in
VSMC.2 We also showed that
intracellular but not extracellular Ca2+ chelation was
sufficient to inhibit EGF receptor phosphorylation induced by Ang II in
the present study. Thus, the EGF receptor should be functionally
downstream of the intracellular Ca2+ mobilization in the
Ras-MAPK signal cascade originating at AT1.
Although AG1478 is highly selective for the EGF receptor over other receptor tyrosine kinases (42), it is still possible that it affects other non-receptor kinases or signaling intermediates nonspecifically. This possibility is suspected by the fact that a higher dose of AG1478 was required for the inhibition of the MAPK activation by EGF than by Ang II. A similar phenomenon was also observed in Rat-1 fibroblasts which was attributed to the relatively weaker receptor phosphorylation by GPCR agonists than by EGF itself (31). In addition, pretreatment of VSMC with AG1478 tended to increase the amount of precipitated EGF receptor (see Fig. 6C). This may be due to the inhibition of basal level ubiquitination and subsequent proteolytic degradation of the receptor which requires the receptor tyrosine kinase activity (53). On the contrary, we found AG1478 had no effect on Ang II-induced association of c-Src to the EGF receptor nor on phorbol ester- or PDGF-BB-induced MAPK activation in the present study. Daub et al. (31) have reported that AG1478 inhibited the MAPK activation, c-fos mRNA expression, and DNA synthesis induced by endothelin-1 and thrombin, but it did not affect the tyrosine phosphorylation of focal adhesion kinase and paxillin by these agonists in Rat-1 fibroblasts. They have further demonstrated that these GPCR agonists failed to stimulate MAPK when Rat-1 cells were transfected with the dominant negative EGF receptor mutant. We have also found that AG1478 (250 nM) inhibits Ang II-induced c-Fos expression and protein synthesis but not its intracellular Ca2+ mobilization or c-Jun induction in cultured rat VSMC.2 These data further confirm the specificity of AG1478 and strongly support the general observations that AG1478 acts at the point of the EGF receptor transactivation induced by GPCRs, leading to specific inhibition of GPCR-coupled MAPK-dependent growth promoting signals, but does not interfere with functional coupling of GPCRs to other downstream kinases or signaling intermediates.
In addition, incomplete inhibition of Ang II-induced MAPK
activation by AG1478 indicates that the activation is not exclusively mediated by the AG1478-sensitive pathway. The alternative activation signal(s) of MAPK by Ang II may involve other upstream transducers such
as a novel Ca2+-sensitive tyrosine kinase, PYK2 (30)/CAK
(54)/RAFTK (55)/CADTK (56), Src family kinases (32-35) as discussed
below, ErbB2 (31, 38), or protein kinase C (57). Further studies are
required to determine relative contribution and possible cross-talks of these mechanisms leading to global growth promoting signaling.
Linseman et al. (25) showed that Ang II induced PDGF receptor phosphorylation and subsequent complex formation with Shc, Grb2, as well as c-Src in cultured rat VSMC. However, the contribution of the PDGF
receptor to the MAPK activation by Ang II in VSMC is
not likely. This view is supported by the observations that Ang
II-induced MAPK activation was minimally affected by the selective PDGF
receptor kinase inhibitor, AG1295, which almost completely abolished
the PDGF-BB-induced MAPK activation (Fig. 1C) and that we
could not detect the enhanced phosphotyrosine content of PDGF
receptor by Ang II in our VSMC (Fig. 5) during the time course in which
the Ang II-induced maximum Ras (3~4 min) and MAPK activation (5 min)
took place (36). However, Ang II-induced phosphorylation of the EGF
receptor and its complex formation with the adaptors are detectable
within 1 min. Given that the reported PDGF
receptor phosphorylation
(25) was detected in 5 min, plateaued in 10 min, and sustained up to
120 min, it may signal to different downstream transducers rather than
MAPK.
In cultured rat VSMC, Ang II has been shown to induce tyrosine phosphorylation of all three Shc isoforms of p46, p52, and p66 which subsequently form a complex with Grb2 (25). In the present study, tyrosine phosphorylation of Shc by Ang II was observed in the immunoprecipitates of the three Shc isoforms in which p52 phosphorylation was dominant (Fig. 6B). Furthermore, Ang II treatment resulted in association of these Shc isoforms to the GST-Grb2 fusion protein (Fig. 2A) and to the EGF receptor (Fig. 4A) in VSMC. In addition to Shc, Grb2 and Sos were also coprecipitated with the EGF receptor upon Ang II stimulation. Taken together with the recent findings that the expression of mutant Shc proteins defective in Grb2 binding displays a dominant negative effect on the pertussis toxin-insensitive MAPK activation induced by thrombin in fibroblasts (58), it is possible to speculate that the Ras and MAPK activation by Ang II may be at least partly mediated through Shc by linking the EGF receptor to a Grb2·Sos complex in VSMC. Some differences are noted in relative changes in the intensity of Shc bands immunoblotted by anti-phosphotyrosine mAb between Fig. 2A and Fig. 3 and between Fig. 2A blotted with anti-Shc pAb and Fig. 4A with anti-Shc mAb (the latter being needed to eliminate a thick rabbit IgG band). These may be due to a difference in efficiency of exogenous GST-Grb2 and endogenous Grb2 in binding Shc and also likely due to a difference in selectivity of anti-Shc pAb and mAb to each Shc isoform. It may also be possible that the relative difference in Shc band intensities is due to different affinity of each isoforms to Grb2 and the EGF receptor, respectively.
Ang II was reported to activate c-Src in cultured VSMC (43) which was proposed as an essential step for Ras activation by Ang II (44). Recently, Sadoshima and Izumo (34) reported that a Src family tyrosine kinase, Fyn, is activated by Ang II which recruits and phosphorylates Shc, leading to Ras activation in cardiac myocytes. We also found that Ang II increased transient association of the active c-Src with Shc which is contingent on Shc phosphorylation, suggesting a similar mechanism involving c-Src may operate the recruitment of Shc by Ang II in VSMC. Furthermore, the present study showed that Ang II and a Ca2+ ionophore enhanced the association of c-Src with the EGF receptor. Although the exact hierarchical order of activation of the kinases and adapters has yet to be clarified, given that c-Src has been shown to phosphorylate the EGF receptor (51) and that the enhanced association of c-Src with the EGF receptor by Ang II was not affected by AG1478 (Fig. 6C), we submit the scenario in which the active c-Src phosphorylates the EGF receptor.
In the case of pertussis toxin-sensitive GPCR, the subunits of G
protein play a crucial role in the Ras and MAPK activation which also
involve Src kinases (35). Recently, it has been proposed that Src
kinase is downstream of the wortmannin-sensitive phosphoinositide 3-kinase
in this cascade (59). However, as we reported, Ang II
induces pertussis toxin-insensitive Ras and MAPK activation in VSMC
(36), and wortmannin has no effect on the Ang II-induced MAPK
activation.3 In agreement
with our concept that c-Src phosphorylates the EGF receptor, a recent
report by Luttrel et al. (60) showed that Gi-coupled receptor utilizes c-Src to phosphorylate the EGF
receptor and for subsequent recruitment of the adaptors in MAPK
activation. Thus, Gi- and Gq-coupled
receptor-mediated MAPK cascades could converge on the Src
kinase-operated EGF receptor transactivation.
In conclusion, we have demonstrated several lines of evidence that Ang II induces Ca2+-dependent tyrosine phosphorylation of the EGF receptor which serves as docking sites for presumably pre-activated c-Src and downstream adaptors at the plasma membrane, leading to MAPK activation in cultured rat VSMC. The identification and characterization of the putative transducer(s) which directly sense intracellular Ca2+ mobilization to activate the kinases are under investigation.
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ACKNOWLEDGEMENTS |
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We thank Dr. G. Carpenter for helpful discussions; T. Fizgerald and E. Price for excellent technical assistance; and T. Stack for secretarial assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL-58205, HL-35323, HL-03320, and DK-20593.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-322-4347; Fax: 615-322-3201.
1 The abbreviations used are: Sos, son-of-sevenless; MAPK/ERK, mitogen-activated protein kinase/extracellular signal-regulated kinase; Ang II, angiotensin II; AT1, angiotensin II type 1 receptor; VSMC, vascular smooth muscle cells; GPCR, G protein-coupled receptor; PLC, phospholipase C; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; mAb, monoclonal antibody; pAb, polyclonal antibody.
2 S. Eguchi, unpublished data.
3 S. Eguchi, unpublished data.
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REFERENCES |
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