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Gβγ Subunits Mediate Src-dependent Phosphorylation of the Epidermal Growth Factor Receptor

Gβγ Subunits Mediate Src-dependent Phosphorylation of the Epidermal Growth Factor Receptor THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 7, Issue of February 14, pp. 4637–4644, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Gbg Subunits Mediate Src-dependent Phosphorylation of the Epidermal Growth Factor Receptor A SCAFFOLD FOR G PROTEIN-COUPLED RECEPTOR-MEDIATED Ras ACTIVATION* (Received for publication, August 26, 1996, and in revised form, November 18, 1996) ¶ i Louis M. Luttrell‡, Gregory J. Della Rocca§, Tim van Biesen , Deirdre K. Luttrell , and Robert J. Lefkowitz** 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 pro- monoclonal antibody. Lysophosphatidic acid stimulates tein kinases by both receptor tyrosine kinases and re- binding of EGF receptor to a GST fusion protein con- ceptors that couple to pertussis toxin-sensitive hetero- taining the c-Src SH2 domain, and this too is blocked by trimeric G proteins proceed via convergent signaling Csk expression. These data suggest that Gbg subunit- pathways. Both signals are sensitive to inhibitors of ty- mediated activation of Src family nonreceptor tyrosine rosine protein kinases and require Ras activation via kinases can account for the G -coupled receptor-medi- phosphotyrosine-dependent recruitment of Ras guanine ated tyrosine phosphorylation events that direct re- nucleotide exchange factors. Receptor tyrosine kinase cruitment of the Shc and Grb2 adapter proteins to the stimulation mediates ligand-induced receptor auto- membrane. phosphorylation, which creates the initial binding sites for SH2 domain-containing docking proteins. However, the mechanism whereby G protein-coupled receptors The low molecular weight G protein Ras functions as a sig- mediate the phosphotyrosine-dependent assembly of a naling intermediate in many pathways involved in the regula- mitogenic signaling complex is poorly understood. We tion of cellular mitogenesis and differentiation. Ras activation have studied the role of Src family nonreceptor tyrosine by growth factor receptors that possess intrinsic tyrosine ki- kinases in G protein-coupled receptor-mediated tyro- nase activity follows ligand-induced phosphorylation of specific sine phosphorylation in a transiently transfected COS-7 docking sites on the receptor itself or adapter proteins, such as cell system. Stimulation of G -coupled lysophosphatidic Shc and insulin receptor substrate-1, which serve to recruit acid and a2A adrenergic receptors or overexpression of Ras guanine nucleotide exchange factors to the plasma mem- Gb1g2 subunits leads to tyrosine phosphorylation of the brane (1, 2). Recently, several receptors that couple to hetero- Shc adapter protein, which then associates with tyro- trimeric G proteins, including the lysophosphatidic acid (LPA) sine phosphoproteins of approximately 130 and 180 kDa, (3, 4), a-thrombin (5), angiotensin II (6, 7), a2A adrenergic (AR) as well as Grb2. The 180-kDa Shc-associated tyrosine (8, 9), M2 muscarinic acetylcholine, D2 dopamine, and A1 aden- phosphoprotein band contains both epidermal growth neu osine receptors (10), have been shown to mediate Ras-depend- factor (EGF) receptor and p185 . 3–5-fold increases in neu ent mitogenic signals. In COS-7 cells, Ras-dependent activation EGF receptor but not p185 tyrosine phosphorylation of mitogen-activated protein kinases via the a2A AR, M2 mus- occur following G -coupled receptor stimulation. Inhibi- carinic acetylcholine, D2 dopamine, and A1 adenosine recep- tion of endogenous Src family kinase activity by cellular tors is mediated largely by Gbg subunits released from pertus- expression of a dominant negative kinase-inactive mu- tant of c-Src inhibits Gb1g2 subunit-mediated and G - sis toxin-sensitive G proteins (8, 9). These Gbg subunit- coupled receptor-mediated phosphorylation of both mediated signals are sensitive to inhibitors of tyrosine protein EGF receptor and Shc. Expression of Csk, which inacti- kinases (8), associated with increased tyrosine protein phos- vates Src family kinases by phosphorylating the regula- phorylation, and dependent upon recruitment of Ras guanine tory carboxyl-terminal tyrosine residue, has the same nucleotide exchange factors to the membrane (9), indicating effect. The G -coupled receptor-mediated increase in i that the pathway converges with the receptor tyrosine kinase EGF receptor phosphorylation does not reflect in- pathway at an early point. creased EGF receptor autophosphorylation, assayed us- G protein-coupled receptors have been shown to mediate ing an autophosphorylation-specific EGF receptor rapid tyrosine phosphorylation of several proteins that partic- ipate in mitogenic signal transduction. The thyrotropin-releas- ing hormone (11), endothelin 1 (12), LPA, and a2A AR recep- * This work was supported in part by National Institutes of Health Grant HL16037 (to R. J. L.). The costs of publication of this article were tors (9) stimulate tyrosine phosphorylation of the Shc adapter defrayed in part by the payment of page charges. This article must protein. This effect can be mimicked by the transient overex- therefore be hereby marked “advertisement” in accordance with 18 pression of Gbg subunits (9, 13) and correlates with Shc-Grb2 U.S.C. Section 1734 solely to indicate this fact. complex formation (9, 12) and the recruitment of Ras guanine ‡ Recipient of a National Institutes of Health Clinical Investigator nucleotide exchange factor activity (9). In addition, recent re- Development Award. § Supported by National Institutes of Health Medical Scientist Train- ing Program Grant T32GM-07171. ¶ Present address: Neuroscience Research, Abbott Laboratories, The abbreviations used are: LPA, lysophosphatidic acid; AR, adre- D-4PM, AP10, 100 Abbott Park Rd., Abbott Park, IL 60064. nergic receptor; PDGF, platelet-derived growth factor; EGF, epidermal ** To whom correspondence should be addressed: Howard Hughes growth factor; PAGE, polyacrylamide gel electrophoresis; GST, gluta- Medical Inst., Box 3821, Duke University Medical Center, Durham, NC thione S-transferase; MAP, mitogen-activated protein; PI3K, phos- 27710. Tel.: 919-684-2974; Fax: 919-684-8875. phatidylinositol 3-kinase. This paper is available on line at http://www-jbc.stanford.edu/jbc/ 4637 This is an Open Access article under the CC BY license. 4638 Gbg Subunit-mediated Tyrosine Phosphorylation of EGF Receptor immunoblotting of transfected whole cell lysates using commercially ports have described G protein-coupled receptor-mediated ty- available antisera. rosine phosphorylation of insulin receptor substrate-1 (14), fo- Immunoprecipitation and Immunoblotting—Stimulations were car- cal adhesion kinase (4, 15), and several receptor tyrosine ried out at 37 °C in serum-free medium as described in the figure kinases, including the platelet-derived growth factor (PDGF) legends. After stimulation, monolayers were washed once with ice-cold neu receptor (16), EGF receptor, p185 (17), and insulin-like phosphate-buffered saline and lysed in RIPA buffer (150 mM NaCl, 50 growth factor-1 receptor (14). mM Tris-HCl, pH 7.5, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 100 mM NaVO ,1mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 The mechanism whereby G protein-coupled receptors stim- 4 mg/ml aprotonin, 10 mg/ml leupeptin) for immunoprecipitation under ulate tyrosine protein phosphorylation is poorly understood. nondenaturing conditions or RIPA/SDS buffer (RIPA buffer containing The observation that the receptors for PDGF (16) and EGF (17) 0.1% SDS) for immunoprecipitation under denaturing conditions. Cell undergo tyrosine phosphorylation following G protein-coupled lysates were sonicated briefly, clarified by centrifugation, and diluted to receptor activation has led to the hypothesis that the intrinsic a protein concentration of 2 mg/ml. Immunoprecipitations from 1 ml of lysate were performed using the appropriate primary antibody plus 50 tyrosine kinase activity of these receptors becomes activated by ml of a 50% slurry of protein G plus/protein A agarose (Oncogene an unknown mechanism. G protein-coupled receptor-mediated Science) agitated for1hat4 °C. Immune complexes were washed twice activation of nonreceptor tyrosine kinases has also been re- with ice-cold RIPA buffer and once with phosphate-buffered saline, ported. Recently, activation of Src family kinases by the denatured in Laemmli sample buffer, and resolved by SDS-polyacryl- a-thrombin (18), LPA (19), angiotensin II (20), N-formyl me- amide gel electrophoresis (PAGE). Shc immunoprecipitations were per- thionyl peptide chemoattractant (21), a2A AR (18, 19), and M1 formed using rabbit polyclonal anti-Shc antibody (Transduction Labo- ratories). EGF receptor was immunoprecipitated using monoclonal muscarinic acetylcholine (18) receptors has been reported. Fur- neu anti-EGF receptor antibody (Transduction Laboratories), and p185 thermore, inhibition of Src family kinases has been shown to was immunoprecipitated using rabbit polyclonal anti-HER2 (Santa inhibit angiotensin II-stimulated Ras (22) and phospholipase Cruz Biotechnology). C-g1 (23) activation in rat aortic smooth muscle cells, LPA and Tyrosine phosphorylation or the presence of coprecipitated proteins a2A AR-stimulated MAP kinase activation in COS-7 cells (19), was detected by protein immunoblotting. Phosphotyrosine was detected M1 and M2 muscarinic acetylcholine-stimulated MAP kinase using a 1:1000 dilution of horseradish peroxidase-conjugated anti-phos- photyrosine monoclonal antibody (Transduction Laboratories). Shc pro- activation in avian B cells (24), and endothelin-1-stimulated tein was detected using a 1:1000 dilution of rabbit polyclonal anti-Shc transcriptional activation in rat glomerular mesangial cells neu IgG (Transduction Laboratories), p185 was detected using a 1:1000 (25). dilution of rabbit polyclonal anti-HER2 IgG (Santa Cruz Biotechnolo- We have previously shown in transiently transfected COS-7 gy), and Grb2 was detected using a 1:1000 dilution of rabbit polyclonal cells that pertussis toxin-sensitive G protein-coupled receptors anti-Grb2 IgG (Santa Cruz Biotechnology), each with horseradish per- oxidase-conjugated donkey anti-rabbit IgG (Amersham Corp.) as sec- mediate Gbg subunit-dependent activation of c-Src and that ondary antibody. C-Src was detected using 1:500 dilution of mouse inhibition of Src family kinases by cellular expression of Csk monoclonal anti-c-Src antibody 327 with horseradish peroxidase-conju- antagonizes G protein-coupled receptor-mediated MAP kinase gated donkey anti-mouse IgG (Jackson Laboratories) as secondary an- activation (19). Here, we examine the role of Src family nonre- tibody. EGF receptor was detected using 1:1000 dilution of sheep anti- ceptor tyrosine kinases in mediating Gbg subunit-dependent human EGF receptor IgG with horseradish peroxidase-conjugated tyrosine phosphorylation of receptor tyrosine kinases and Shc. donkey anti-sheep IgG (Jackson Laboratories) as secondary antibody. Immunoblots for autophosphorylated EGF receptor were performed Our data suggest that activation of Src family kinases by G using mouse monoclonal anti-activated EGF receptor IgG (26) with protein-coupled receptors can account for the G -coupled recep- horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson tor-mediated tyrosine phosphorylation events that direct re- Laboratories) as secondary antibody. Immune complexes on nitrocellu- cruitment of the Shc and Grb2 adapter proteins to the mem- lose were visualized by enzyme-linked chemiluminescence (Amersham brane using the EGF receptor as a scaffold. Corp.) and quantified by scanning laser densitometry. GST Fusion Proteins Containing the c-Src SH2 and SH3 Domains— EXPERIMENTAL PROCEDURES GST fusion proteins containing the human c-Src SH2 (amino acids 144–249), SH3 (amino acids 87–143), or SH2 and SH3 (amino acids DNA Constructs—The cDNA encoding the a2A AR was cloned in our 87–249) domains were prepared as GST Sepharose conjugates as de- laboratory. The cDNAs encoding Gb1 and Gg2 were provided by M. scribed previously (27). For the detection of c-Src SH2 or SH3 domain- Simon (California Institute of Technology, Pasadena, CA). The cDNA c-src binding proteins, appropriately transfected and stimulated COS-7 cells encoding human p60 was provided by D. Fujita (University of Cal- csk M dithiothreitol, soni- were lysed in RIPA/SDS buffer containing 5 m was provided by gary, Alberta, Canada), and the cDNA encoding p50 cated, clarified by centrifugation, precleared with 6 mg/ml GST Sepha- H. Hanafusa (Rockefeller University, New York, NY). The constitu- c-src rose for 1 h and incubated with 6 mg/ml of the GST fusion protein tively activated Y530F p60 (TAC(Y) 3 TTC(F)), in which the regu- Sepharose for3hat4 °C. After incubation, fusion protein complexes latory carboxyl-terminal tyrosine residue has been mutated, and cata- c-src were washed twice with ice-cold RIPA buffer and once with phosphate- lytically inactive K298M p60 (AAA(K) 3 ATG(M)) mutants were buffered saline, denatured in Laemmli sample buffer, and resolved by prepared as described (19). All cDNAs were subcloned into pRK5 or SDS-PAGE. Coprecipitated tyrosine phosphoproteins and EGF receptor pcDNA eukaryotic expression vectors for transient transfection. were detected by protein immunoblotting as described. Cell Culture and Transfection—COS-7 cells were maintained in Dul- becco’s modified Eagle’s medium supplemented with 10% fetal bovine RESULTS serum and 100 mg/ml gentamicin at 37 °C in a humidified 5% CO atmosphere. Transfections were performed on 80–90% confluent mono- G -coupled Receptors and Gbg Subunits Mediate Formation layers in 100-mm dishes. For transient transfection, cells were incu- of a Mitogenic Signaling Complex Containing EGF Receptor, bated at 37 °C in 4 ml serum-free Dulbecco’s modified Eagle’s medium Shc, and Grb2—As shown in Fig. 1A, stimulation of endoge- containing 6–10 mg of DNA/100-mm dish plus 6 ml of LipofectAMINE nous LPA receptors in COS-7 cells leads to a rapid 3–5-fold reagent (Life Technologies, Inc.)/mg of DNA. Empty pRK5 vector was increase in tyrosine phosphorylation of each of the three Shc added to transfections as needed to keep the total mass of DNA added isoforms. The phosphorylation is maximal within 2 min of per dish constant within an experiment. After 3–5 h of exposure to the transfection medium, monolayers were refed with growth medium and stimulation and declines slowly thereafter (9, 19). Under non- incubated overnight. Transfected monolayers were serum starved in denaturing conditions, Shc coprecipitates with two major tyro- Dulbecco’s modified Eagle’s medium supplemented with 0.1% bovine sine phosphoprotein bands of approximately 130 and 180 kDa serum albumin and 10 mM Hepes, pH 7.4, for 16–20 h prior to stimu- and with the adapter protein Grb2. The association of Shc with lation. Assays were performed 48 h after transfection. LipofectAMINE the p130 and p180 phosphoproteins is modulated with a time transfection of COS-7 cells consistently resulted in transfection efficien- course that parallels the time course of Shc phosphorylation cies of greater than 80% (data not shown). Transient expression of Gb1 and Gg2 subunits, Csk, and mutant c-Src proteins was confirmed by and Shc-Grb2 complex formation, suggesting that LPA stimu- Gbg Subunit-mediated Tyrosine Phosphorylation of EGF Receptor 4639 FIG.1. G -coupled receptor- and Gbg subunit-stimulated association of Shc with p130 and p180 tyrosine phosphoproteins and Grb2 in COS-7 cells. A, time course of LPA-stimulated Shc tyrosine phosphorylation and association with p130 and p180 tyrosine phosphopro- teins and Grb2. Serum-starved cells were stimulated for the indicated times with LPA (10 mM). Immunoprecipitates of Shc from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine (upper panel) or anti-Grb2 (lower panel)as described. The position of tyrosine phosphorylated Shc isoforms, Shc-associated p130 and p180 phosphoproteins, and Grb2 are as indicated. B, c-src effect of a2A AR, LPA, or EGF receptor stimulation and Gbg subunit or Y530F p60 expression on Shc tyrosine phosphorylation and association with p130 and p180 tyrosine phosphoproteins and Grb2. Cells were transiently transfected with empty pRK5 vector, Gb1 and Gg2, a2A AR, or c-src Y530F p60 as described. Duplicate plates of serum-starved cells were stimulated for 2 min with the a2A AR agonist UK14304 (UK,10 mM), LPA (10 mM), or EGF (10 ng/ml) as indicated. Immunoprecipitates of Shc from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine (upper panel) or anti-Grb2 (lower panel) as described. C, pertussis toxin-sensitivity of a2A AR- and LPA-stimulated Shc, p130, and p180 tyrosine phosphorylation. Cells were transiently transfected with empty pRK5 vector or a2A AR and serum-starved overnight in the presence or the absence of pertussis toxin (Ptx, 100 ng/ml) prior to stimulation with LPA, UK14304, or EGF as indicated. Immunoprecipitates of Shc from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti- shc phosphotyrosine. D, quantitation of the effects of pertussis toxin treatment on a2A AR- and LPA-stimulated tyrosine phosphorylation of p52 . UK14304-, LPA-, and EGF-stimulated p52shc phosphorylation was determined as described. 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 6 S.E. for three separate experiments. NS, nonstimulated. neu lation induces association of these proteins. As shown in Fig. receptor coprecipitation. In contrast, Shc-p185 complexes 1B, similar increases in Shc phosphorylation and Shc-p180 are present in nonstimulated cells and do not increase detect- association result from transient expression of Gb1g2 subunits ably following LPA or a2A AR receptor stimulation. As ex- or stimulation of endogenous LPA or transiently expressed a2A pected, EGF stimulation results in both Shc-EGF receptor and neu AR receptors. Stimulation of endogenous EGF receptors or Shc-p185 association, which may reflect heterodimerization transient overexpression of a constitutively activated mutant and transphosphorylation of the two related receptor tyrosine c-src human c-Src (Y530F p60 ; Refs. 28–30) has similar, al- kinases (32). As shown in Fig. 2B, the tyrosine phosphorylation neu though more robust effects, indicating that activation of either states of Shc, EGF receptor, and p185 , determined following the receptor tyrosine kinase or nonreceptor Src kinase can direct immunoprecipitation of each protein, reflect the changes mimic the G protein-mediated effects. As shown in Fig. 1 (C in Shc-receptor tyrosine kinase association. Shc and EGF re- and D), G -coupled receptor-mediated but not EGF receptor- ceptor phosphorylation is increased following LPA, a2A AR, or neu mediated Shc phosphorylation and Shc-p180 association are EGF receptor stimulation. P185 exhibits significant basal pertussis toxin-sensitive in these cells. tyrosine phosphorylation, consistent with the detection of Shc- neu Because G protein-coupled receptor-mediated tyrosine phos- p185 complexes in nonstimulated cells, which detectably neu phorylation of PDGF receptor (16), EGF receptor, and p185 increases only following EGF receptor stimulation. (17) has been reported, we performed immunoblots for EGF To confirm that Shc, Grb2, and EGF receptor directly asso- neu receptor and p185 on Shc immunoprecipitates from nonde- ciate following G -coupled receptor stimulation, EGF receptor natured cell lysates following stimulation of LPA, a2A AR, or immunoprecipitates were assayed for the presence of Shc and EGF receptors to determine whether these receptor tyrosine Grb2 following LPA stimulation. As shown in Fig. 2C, stimu- kinases are present in the Shc-associated p180 phosphoty- lation with either LPA or EGF resulted in the association of rosine band. COS-7 cells lack detectable expression of PDGF Shc and Grb2 with EGF receptor. G -coupled receptor-induced receptor (31). As shown in Fig. 2A, EGF receptor is not detect- association of Src family nonreceptor tyrosine kinases with Shc able in Shc immunoprecipitates from nonstimulated cells, but has been reported (19, 21). As shown, c-Src can also be detected stimulation of either G -coupled receptor results in Shc-EGF in EGF receptor immunoprecipitates from LPA- or EGF-stim- i 4640 Gbg Subunit-mediated Tyrosine Phosphorylation of EGF Receptor neu FIG.2. Correlation between G -coupled receptor-stimulated EGF receptor and p185 tyrosine phosphorylation with Shc com- neu plex formation. A, coprecipitation of endogenous EGF receptor and p185 with Shc following G -coupled receptor or EGF receptor stimulation. Serum-starved cells, transiently transfected with empty pRK5 vector or a2A AR, were stimulated for 2 min with UK14304 (UK), LPA, or EGF as indicated. Immunoprecipitates of Shc from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-EGF neu neu receptor (top panel), anti-p185 (HER2, center panel), or anti-Shc (bottom panel) as described. The position of EGF receptor, p185 , and Shc neu isoforms are as indicated. B, tyrosine phosphorylation of endogenous EGF receptor and p185 following G -coupled receptor or EGF receptor stimulation. Serum-starved cells, transiently transfected with empty pRK5 vector or a2A AR, were stimulated with UK14304, LPA, or EGF as neu indicated. Immunoprecipitates of EGF receptor (top panel), p185 (center panel), or Shc (bottom panel) from RIPA/SDS buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine as described. C, coprecipitation of Grb2, Shc, and c-Src with endogenous EGF receptor following LPA receptor or EGF receptor stimulation. Serum-starved cells were stimulated for 2 min with LPA or EGF as indicated. Immunoprecipitates of EGF receptor from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-EGF receptor, anti-Grb2, anti-Shc, or anti-c-Src as described. The position of EGF receptor, Grb2, Shc isoforms, and c-Src are as indicated. NS, nonstimulated. ulated cell lysates, suggesting that activation of the G -coupled autophosphorylation resulting from activation of the intrinsic receptor results in association of EGF receptor, c-Src, Shc, and tyrosine kinase activity of the EGF receptor or inhibition of a Grb2 in a multiprotein complex. phosphotyrosine phosphatase versus phosphorylation of the Src Family Kinase Activity Is Required for Both G -coupled EGF receptor mediated by the c-Src nonreceptor tyrosine ki- Receptor and Gbg Subunit-mediated Tyrosine Phosphorylation nase. As shown, this antibody does not detect EGF receptor of EGF Receptor and Shc—G -coupled receptor-mediated in- autophosphorylation following stimulation of LPA or a2A AR creases in EGF receptor phosphotyrosine might result from receptors, despite a 3–5-fold increase in total EGF receptor ligand-independent activation of the receptor tyrosine kinase, phosphotyrosine, suggesting that the increase in receptor tyro- phosphorylation by an activated nonreceptor tyrosine kinase, sine phosphorylation does not reflect activation of the intrinsic or inhibition of a phosphotyrosine phosphatase. To distinguish tyrosine kinase. between these alternative mechanisms, we employed a mono- Because expression of activated mutant c-Src is sufficient to clonal anti-EGF receptor antibody specific for autophosphoryl- mediate EGF receptor phosphorylation in the absence of li- ated EGF receptor. This antibody selectively recognizes acti- gand, we tested the hypothesis that G -coupled receptor-medi- vated EGF receptor via an epitope distal to amino acid 1052 ated activation of Src family kinases can account for the ob- (26), which is distinct from the major in vitro c-Src phospho- served tyrosine phosphorylation of Shc and EGF receptor. To rylation sites (33). As shown in Fig. 3 (A and B), antiphospho- inhibit endogenous Src family kinases, cells were transiently tyrosine immunoblots of EGF receptor immunoprecipitated transfected with cDNA encoding either Csk or a kinase-inac- c-src from EGF-stimulated cells, from cells transiently expressing tive dominant negative mutant c-Src (K298M p60 ; Ref. 34). c-src Y530F p60 , and from cells in which phosphotyrosine phos- Csk is a cytoplasmic tyrosine protein kinase (35) that inacti- phatase activity is inhibited by incubation with sodium or- vates Src family kinases by phosphorylating the regulatory thovanadate, each exhibit increased total receptor phosphoryl- carboxyl-terminal tyrosine residue. Csk overexpression has ation. Identical immunoblots probed with the anti-activated been shown to impair G protein-coupled receptor-mediated EGF receptor antibody give increased signals from EGF-stim- MAP kinase activation in COS-7 cells (19) and c-fos transcrip- ulated and sodium orthovanadate-treated cells but not from tion in rat glomerular mesangial cells (25). As shown in Fig. 4 c-src c-src Y530F p60 -transfected cells. Thus, the anti-activated EGF (A and B), coexpression of either Csk or K298M p60 mark- receptor antibody is able to discriminate between increased edly inhibits Gb1g2 subunit-, a2A AR-, and LPA receptor- Gbg Subunit-mediated Tyrosine Phosphorylation of EGF Receptor 4641 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 do- main 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 recep- tor has suggested that phosphorylation of the putative c-Src SH2 domain recognition site (Tyr ) 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 G -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 follow- ing 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 attenu- ated. These data suggest that G -coupled receptor stimulation results in both c-Src mediated phosphorylation of the EGF receptor and SH2 domain-dependent c-Src-EGF receptor com- plex formation. DISCUSSION 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 FIG.3. Discrimination between Src kinase-mediated EGF re- cell types has been reported, and inhibition of Src family ki- ceptor phosphorylation and EGF receptor autophosphorylation using anti-activated EGF receptor antibody. A, comparison of nases has been shown to block G protein-coupled receptor- anti-phosphotyrosine immunoblots of endogenous EGF receptor with mediated Ras and phospholipase C-g1 activation, MAP kinase anti-activated EGF receptor immunoblots following G -coupled receptor activation, and c-fos transcription (19, 22–24). Our data dem- and EGF receptor stimulation, expression of Y530F, and inhibition of onstrate that in COS-7 cells, G -coupled receptor-stimulated phosphotyrosine phosphatase activity. Serum-starved cells, transiently c-src tyrosine phosphorylation of the EGF receptor results in forma- transfected with empty pRK5 vector, a2A AR, or Y530F p60 were stimulated for 2 min with UK14304 (UK), LPA, or EGF or incubated for tion of a complex between the membrane-associated EGF re- 20 min in the presence of sodium orthovanadate (VO ,10 mM)as ceptor and the cytosolic adapter proteins Shc and Grb2, thus indicated. Duplicate immunoprecipitates of EGF receptor from RIPA/ providing a scaffold for the assembly of a mitogenic signaling SDS buffer lysates were resolved by SDS-PAGE and immunoblotted complex. The G -coupled receptor effects can be mimicked by with either anti-phosphotyrosine (upper panel) or anti-activated EGF receptor antibody (lower panel) as described. B, quantitation of anti- cellular overexpression of Gbg subunits, suggesting that the phosphotyrosine immunoblots of EGF receptor and anti-activated EGF process is Gbg subunit-mediated. Because inhibition of endog- receptor immunoblots. UK14304-, LPA-, EGF-, sodium orthovanadate-, enous Src kinases blocks both G protein-coupled receptor-me- c-src and Y530F p60 -stimulated EGF receptor total tyrosine phosphoryl- diated EGF receptor phosphorylation and binding of the EGF ation and autophosphorylation were determined as described. Autora- diographs were quantified by scanning laser densitometry, and the data receptor to the c-Src SH2 domain, the data also suggest that were presented as fold increase over nonstimulated or empty pRK5 Src family kinases directly associate with and phosphorylate vector transfected controls. The data shown represent the means 6 S.E. the EGF receptor following G -coupled receptor stimulation. for three separate experiments. NS, nonstimulated. Fig. 6 depicts a model of Gbg subunit-mediated, Ras-depend- ent MAP kinase activation that is consistent with these data. mediated tyrosine phosphorylation of both Shc and EGF recep- Gbg subunit-dependent activation of endogenous Src family tor. EGF-stimulated Shc and EGF receptor phosphorylation nonreceptor tyrosine kinases is an early event following G - were less dramatically effected. Shc and EGF receptor phos- coupled receptor stimulation (19). Once activated, the Src ki- phorylation mediated by Y530F, which is not a substrate for nases mediate phosphorylation of several intracellular targets, Csk, is not significantly attenuated by Csk overexpression. including receptor tyrosine kinases, adapter proteins such as c-Src SH2 Domain GST Fusion Proteins Bind EGF Receptor Shc and insulin receptor substrate-1, and possibly cytoskel- Following G -coupled Receptor Activation—To determine etally associated Src substrates such as focal adhesion kinase whether G -coupled receptor-stimulated EGF receptor phos- and paxillin. Once phosphorylated, membrane-associated pro- phorylation can induce binding of Src kinases directly to the teins such as the receptor tyrosine kinases and focal adhesion EGF receptor, GST-fusion proteins containing either the c-Src kinase would provide docking sites for the SH2 domains of the SH2, SH3, or SH2-SH3 domains (27) were assayed for the Shc and Grb2 adapter molecules, resulting in the recruitment ability to precipitate phosphorylated EGF receptor from lysates of Ras guanine nucleotide exchange factors, and potentially of of LPA-stimulated cells. As shown in Fig. 5A, the c-Src SH2 and other components of the mitogenic signaling complex, to the SH2-SH3 domain GST fusion proteins but not the c-Src SH3 plasma membrane. The ensuing activation of Ras would recruit domain GST fusion protein precipitate a 180-kDa tyrosine the Raf kinase to the membrane and initiate the phosphoryla- 4642 Gbg Subunit-mediated Tyrosine Phosphorylation of EGF Receptor FIG.5. Src kinase-dependent association of EGF receptor with c-Src SH2 domain GST fusion proteins. A, association of p180 with c-Src SH2 domain and SH2-SH3 domain GST fusion proteins following LPA and EGF receptor stimulation. RIPA/SDS lysates of serum-starved cells, stimulated for 2 min with LPA or EGF, were incubated with GST-Sepharose (GST) or GST-Src SH2, SH3, or SH2-SH3-Sepharose as described. Precipitated proteins were resolved by SDS-PAGE and im- munoblotted with anti-phosphotyrosine monoclonal antibody. The po- sition of the c-Src SH2 domain binding p180 protein is as indicated. B, association of EGF receptor with the c-Src SH2 domain GST fusion protein following LPA and EGF receptor stimulation. RIPA/SDS lysates of stimulated serum-starved cells were incubated with GST-Src SH2 sepharose as described. Precipitated proteins were resolved by SDS- PAGE and immunoblotted with anti-EGF receptor. The position of the EGF receptor from GST-Src SH2 Sepharose precipitates and COS-7 whole cell lysates is as indicated. C, quantitation of the effect of Csk expression on LPA- and EGF-stimulated association of EGF receptor with the c-Src SH2 domain GST fusion protein. Cells were transiently cotransfected with empty vector (Control) or expression plasmid encod- ing Csk (CSK), and LPA- and EGF-stimulated association of EGF receptor with the c-Src SH2 domain GST fusion protein was deter- mined. Autoradiographs were quantified by scanning laser densitome- try, and the data were presented as fold increase over nonstimulated or empty pRK5 vector transfected controls. The data shown represent the means 6 S.E. for three separate experiments. NS, nonstimulated. c-src FIG.4. Effect of Csk and K298M p60 expression on G -cou- tion cascade leading to MAP kinase activation. pled receptor-mediated Shc and EGF receptor tyrosine phos- Depending upon cell type, the G protein-coupled receptors phorylation. A, Immunoblots of Shc phosphotyrosine following a2A AR, LPA or EGF receptor stimulation and Gbg subunit or Y530F for angiotensin II, LPA, and a-thrombin have been shown to c-src p60 expression. Cells were transiently cotransfected with empty stimulate ligand-independent tyrosine phosphorylation of c-src vector (Control) or expression plasmid encoding Csk or K298M p60 , c-src PDGF receptor (16), insulin-like growth factor-1 receptor b plus empty pRK5 vector, Gb1 and Gg2, a2A AR, or Y530F p60 . Serum-starved cells were stimulated for 2 min with UK14304 (UK), LPA, or EGF as indicated, and immunoprecipitates of Shc from RIPA/ c-src SDS buffer lysates were immunoblotted with anti-phosphotyrosine as p60 coexpression on Gbg subunit-, a2A AR-, LPA-, EGF-, and Y530F c-src described. The position of tyrosine phosphorylated Shc isoforms are as p60 -stimulated Shc and EGF receptor tyrosine phosphorylation. Shc indicated. B, immunoblots of EGF receptor phosphotyrosine following and EGF receptor phosphotyrosine were determined as described fol- a2A AR, LPA, or EGF receptor stimulation and Gbg subunit or Y530F lowing a2A AR, LPA, or EGF receptor stimulation and Gbg subunit or c-src c-src p60 expression. Serum-starved, transiently cotransfected cells were Y530F p60 expression. Autoradiographs were quantified by scan- stimulated as described and immunoprecipitates of EGF receptor from ning laser densitometry, and the data were presented as fold increase RIPA/SDS buffer lysates were immunoblotted with anti-phosphoty- over nonstimulated or empty pRK5 vector transfected controls. The rosine as described. The position of tyrosine phosphorylated EGF re- data shown represent the means 6 S.E. for three to five separate ceptor is as indicated. C, quantitation of the effects of Csk and K298M experiments. NS, nonstimulated. Gbg Subunit-mediated Tyrosine Phosphorylation of EGF Receptor 4643 diated phosphorylation and EGF receptor autophosphoryla- tion, we have been unable to detect increased EGF receptor autophosphorylation following either overexpression of Y530F c-src p60 or G -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. Gbg subunit-mediated PI3K activity has been described in neutrophils and platelets (36, 37), a Gbg subunit-regulated isoform of p110 PI3K has been cloned, and Gbg subunits may contribute to the regulation of the conventional p85/p110 PI3K (38). We have previously reported that G -coupled receptor- and Gbg 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 c-src 2 activation by transiently expressed Y530F p60 , , mSos, and constitutively activated mutants of Ras and MAP kinase/erk kinase(39)iswortmannin-insensitive,suggestingthatthePI3K- 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-trisphos- phate, the product of PI3K (40), may provide an explanation for this phenomenon. Interaction between Src kinases and novel Gbg subunit- FIG.6. Model of G -coupled receptor-mediated, Ras-dependent regulated nonreceptor tyrosine kinases might also contribute MAP kinase activation in COS-7 cells. Activation of Src family kinases following release of Gbg subunits from pertussis toxin-sensitive to the regulation of Src kinase activity. In neuronal cells, G - heterotrimeric G proteins precedes tyrosine phosphorylation of several coupled receptors have been shown to stimulate the Ca and putative scaffolding molecules, such as receptor tyrosine kinases (RTK) protein kinase C dependent tyrosine protein kinase, PYK2 (41). and focal adhesion kinase, leading to the phosphotyroine-dependent, PYK2 is a member of the focal adhesion kinase family of inte- SH2 domain-mediated recruitment of Ras guanine nucleotide exchange FAK factor (mSos) to the plasma membrane. The subsequent activation of grin receptor-associated tyrosine kinases and like p125 (42) Ras initiates the Raf, MAP kinase/erk kinase, MAP kinase phosphoryl- can complex with activated c-Src upon stimulation (43). How- ation cascade that leads to MAP kinase (ERK 1/2) activation. ever, phospholipase C activation and Ca mobilization are apparently unable to account for G protein-coupled receptor- neu subunit (14), EGF receptor, and p185 (17). The finding that mediated tyrosine phosphorylation in many noneuronal cells several receptor tyrosine kinases undergo G protein-coupled (4, 44, 45). Bruton’s tyrosine kinase (Btk) and Tsk, two mem- receptor-mediated phosphorylation suggests the existence of a bers of a family of pleckstrin homology domain-containing ty- common mechanism that is not receptor tyrosine kinase-spe- rosine protein kinases that includes Btk, Itk, Tsk and Tec A, cific, such as activation of a nonreceptor tyrosine kinase or are reportedly regulated by Gbg subunits (46). In hematopoi- inhibition of a phosphotyrosine phosphatase. Our data, dem- etic cells, Btk interacts with the Src family kinases Fyn, Lyn, onstrating inhibition of G -coupled receptor-mediated tyrosine and Hck (47), and Src-Btk interaction is associated with Btk phosphorylation of the EGF receptor by specific inhibitors of autoactivation (48). This is unlikely to be a general mechanism Src family kinases, support the hypothesis that activation of for G protein-coupled receptor regulation of Src kinases, how- Src kinases can account for the observed receptor tyrosine ever, because the pleckstrin homology domain-containing tyro- kinase phosphorylation. sine kinases appear to have limited tissue distribution and are The role of the intrinsic tyrosine kinase activity of receptor not known to be involved in the regulation of Ras. tyrosine kinases in G -coupled receptor-mediated tyrosine The data presented in this report suggest that both Src phosphorylation is unclear. Daub et al. (17) have reported that family kinases and receptor tyrosine kinases play central roles inhibition of EGF receptor function in Rat1 cells, by either an in directing the assembly of membrane-associated mitogenic EGF receptor-selective tyrphostin, AG1478, or expression of a signaling complexes in response to G -coupled receptor activa- dominant negative mutant EGF receptor, blocks endothelin-1, tion in some cells. An understanding of the mechanisms LPA, and a-thrombin receptor-mediated EGF receptor/HER2 whereby G protein-coupled receptors regulate tyrosine protein phosphorylation and MAP kinase activation. They conclude phosphorylation and of the basis for cross-talk between G pro- that a ligand-independent transactivation of the EGF receptor/ tein-coupled receptor and receptor tyrosine kinase signaling HER2 tyrosine kinase is responsible for G protein-coupled re- pathways may ultimately provide strategies for selective acti- ceptor-mediated tyrosine phosphorylation. 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Gβγ Subunits Mediate Src-dependent Phosphorylation of the Epidermal Growth Factor Receptor

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 7, Issue of February 14, pp. 4637–4644, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Gbg Subunits Mediate Src-dependent Phosphorylation of the Epidermal Growth Factor Receptor A SCAFFOLD FOR G PROTEIN-COUPLED RECEPTOR-MEDIATED Ras ACTIVATION* (Received for publication, August 26, 1996, and in revised form, November 18, 1996) ¶ i Louis M. Luttrell‡, Gregory J. Della Rocca§, Tim van Biesen , Deirdre K. Luttrell , and Robert J. Lefkowitz** 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 pro- monoclonal antibody. Lysophosphatidic acid stimulates tein kinases by both receptor tyrosine kinases and re- binding of EGF receptor to a GST fusion protein con- ceptors that couple to pertussis toxin-sensitive hetero- taining the c-Src SH2 domain, and this too is blocked by trimeric G proteins proceed via convergent signaling Csk expression. These data suggest that Gbg subunit- pathways. Both signals are sensitive to inhibitors of ty- mediated activation of Src family nonreceptor tyrosine rosine protein kinases and require Ras activation via kinases can account for the G -coupled receptor-medi- phosphotyrosine-dependent recruitment of Ras guanine ated tyrosine phosphorylation events that direct re- nucleotide exchange factors. Receptor tyrosine kinase cruitment of the Shc and Grb2 adapter proteins to the stimulation mediates ligand-induced receptor auto- membrane. phosphorylation, which creates the initial binding sites for SH2 domain-containing docking proteins. However, the mechanism whereby G protein-coupled receptors The low molecular weight G protein Ras functions as a sig- mediate the phosphotyrosine-dependent assembly of a naling intermediate in many pathways involved in the regula- mitogenic signaling complex is poorly understood. We tion of cellular mitogenesis and differentiation. Ras activation have studied the role of Src family nonreceptor tyrosine by growth factor receptors that possess intrinsic tyrosine ki- kinases in G protein-coupled receptor-mediated tyro- nase activity follows ligand-induced phosphorylation of specific sine phosphorylation in a transiently transfected COS-7 docking sites on the receptor itself or adapter proteins, such as cell system. Stimulation of G -coupled lysophosphatidic Shc and insulin receptor substrate-1, which serve to recruit acid and a2A adrenergic receptors or overexpression of Ras guanine nucleotide exchange factors to the plasma mem- Gb1g2 subunits leads to tyrosine phosphorylation of the brane (1, 2). Recently, several receptors that couple to hetero- Shc adapter protein, which then associates with tyro- trimeric G proteins, including the lysophosphatidic acid (LPA) sine phosphoproteins of approximately 130 and 180 kDa, (3, 4), a-thrombin (5), angiotensin II (6, 7), a2A adrenergic (AR) as well as Grb2. The 180-kDa Shc-associated tyrosine (8, 9), M2 muscarinic acetylcholine, D2 dopamine, and A1 aden- phosphoprotein band contains both epidermal growth neu osine receptors (10), have been shown to mediate Ras-depend- factor (EGF) receptor and p185 . 3–5-fold increases in neu ent mitogenic signals. In COS-7 cells, Ras-dependent activation EGF receptor but not p185 tyrosine phosphorylation of mitogen-activated protein kinases via the a2A AR, M2 mus- occur following G -coupled receptor stimulation. Inhibi- carinic acetylcholine, D2 dopamine, and A1 adenosine recep- tion of endogenous Src family kinase activity by cellular tors is mediated largely by Gbg subunits released from pertus- expression of a dominant negative kinase-inactive mu- tant of c-Src inhibits Gb1g2 subunit-mediated and G - sis toxin-sensitive G proteins (8, 9). These Gbg subunit- coupled receptor-mediated phosphorylation of both mediated signals are sensitive to inhibitors of tyrosine protein EGF receptor and Shc. Expression of Csk, which inacti- kinases (8), associated with increased tyrosine protein phos- vates Src family kinases by phosphorylating the regula- phorylation, and dependent upon recruitment of Ras guanine tory carboxyl-terminal tyrosine residue, has the same nucleotide exchange factors to the membrane (9), indicating effect. The G -coupled receptor-mediated increase in i that the pathway converges with the receptor tyrosine kinase EGF receptor phosphorylation does not reflect in- pathway at an early point. creased EGF receptor autophosphorylation, assayed us- G protein-coupled receptors have been shown to mediate ing an autophosphorylation-specific EGF receptor rapid tyrosine phosphorylation of several proteins that partic- ipate in mitogenic signal transduction. The thyrotropin-releas- ing hormone (11), endothelin 1 (12), LPA, and a2A AR recep- * This work was supported in part by National Institutes of Health Grant HL16037 (to R. J. L.). The costs of publication of this article were tors (9) stimulate tyrosine phosphorylation of the Shc adapter defrayed in part by the payment of page charges. This article must protein. This effect can be mimicked by the transient overex- therefore be hereby marked “advertisement” in accordance with 18 pression of Gbg subunits (9, 13) and correlates with Shc-Grb2 U.S.C. Section 1734 solely to indicate this fact. complex formation (9, 12) and the recruitment of Ras guanine ‡ Recipient of a National Institutes of Health Clinical Investigator nucleotide exchange factor activity (9). In addition, recent re- Development Award. § Supported by National Institutes of Health Medical Scientist Train- ing Program Grant T32GM-07171. ¶ Present address: Neuroscience Research, Abbott Laboratories, The abbreviations used are: LPA, lysophosphatidic acid; AR, adre- D-4PM, AP10, 100 Abbott Park Rd., Abbott Park, IL 60064. nergic receptor; PDGF, platelet-derived growth factor; EGF, epidermal ** To whom correspondence should be addressed: Howard Hughes growth factor; PAGE, polyacrylamide gel electrophoresis; GST, gluta- Medical Inst., Box 3821, Duke University Medical Center, Durham, NC thione S-transferase; MAP, mitogen-activated protein; PI3K, phos- 27710. Tel.: 919-684-2974; Fax: 919-684-8875. phatidylinositol 3-kinase. This paper is available on line at http://www-jbc.stanford.edu/jbc/ 4637 This is an Open Access article under the CC BY license. 4638 Gbg Subunit-mediated Tyrosine Phosphorylation of EGF Receptor immunoblotting of transfected whole cell lysates using commercially ports have described G protein-coupled receptor-mediated ty- available antisera. rosine phosphorylation of insulin receptor substrate-1 (14), fo- Immunoprecipitation and Immunoblotting—Stimulations were car- cal adhesion kinase (4, 15), and several receptor tyrosine ried out at 37 °C in serum-free medium as described in the figure kinases, including the platelet-derived growth factor (PDGF) legends. After stimulation, monolayers were washed once with ice-cold neu receptor (16), EGF receptor, p185 (17), and insulin-like phosphate-buffered saline and lysed in RIPA buffer (150 mM NaCl, 50 growth factor-1 receptor (14). mM Tris-HCl, pH 7.5, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 100 mM NaVO ,1mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 The mechanism whereby G protein-coupled receptors stim- 4 mg/ml aprotonin, 10 mg/ml leupeptin) for immunoprecipitation under ulate tyrosine protein phosphorylation is poorly understood. nondenaturing conditions or RIPA/SDS buffer (RIPA buffer containing The observation that the receptors for PDGF (16) and EGF (17) 0.1% SDS) for immunoprecipitation under denaturing conditions. Cell undergo tyrosine phosphorylation following G protein-coupled lysates were sonicated briefly, clarified by centrifugation, and diluted to receptor activation has led to the hypothesis that the intrinsic a protein concentration of 2 mg/ml. Immunoprecipitations from 1 ml of lysate were performed using the appropriate primary antibody plus 50 tyrosine kinase activity of these receptors becomes activated by ml of a 50% slurry of protein G plus/protein A agarose (Oncogene an unknown mechanism. G protein-coupled receptor-mediated Science) agitated for1hat4 °C. Immune complexes were washed twice activation of nonreceptor tyrosine kinases has also been re- with ice-cold RIPA buffer and once with phosphate-buffered saline, ported. Recently, activation of Src family kinases by the denatured in Laemmli sample buffer, and resolved by SDS-polyacryl- a-thrombin (18), LPA (19), angiotensin II (20), N-formyl me- amide gel electrophoresis (PAGE). Shc immunoprecipitations were per- thionyl peptide chemoattractant (21), a2A AR (18, 19), and M1 formed using rabbit polyclonal anti-Shc antibody (Transduction Labo- ratories). EGF receptor was immunoprecipitated using monoclonal muscarinic acetylcholine (18) receptors has been reported. Fur- neu anti-EGF receptor antibody (Transduction Laboratories), and p185 thermore, inhibition of Src family kinases has been shown to was immunoprecipitated using rabbit polyclonal anti-HER2 (Santa inhibit angiotensin II-stimulated Ras (22) and phospholipase Cruz Biotechnology). C-g1 (23) activation in rat aortic smooth muscle cells, LPA and Tyrosine phosphorylation or the presence of coprecipitated proteins a2A AR-stimulated MAP kinase activation in COS-7 cells (19), was detected by protein immunoblotting. Phosphotyrosine was detected M1 and M2 muscarinic acetylcholine-stimulated MAP kinase using a 1:1000 dilution of horseradish peroxidase-conjugated anti-phos- photyrosine monoclonal antibody (Transduction Laboratories). Shc pro- activation in avian B cells (24), and endothelin-1-stimulated tein was detected using a 1:1000 dilution of rabbit polyclonal anti-Shc transcriptional activation in rat glomerular mesangial cells neu IgG (Transduction Laboratories), p185 was detected using a 1:1000 (25). dilution of rabbit polyclonal anti-HER2 IgG (Santa Cruz Biotechnolo- We have previously shown in transiently transfected COS-7 gy), and Grb2 was detected using a 1:1000 dilution of rabbit polyclonal cells that pertussis toxin-sensitive G protein-coupled receptors anti-Grb2 IgG (Santa Cruz Biotechnology), each with horseradish per- oxidase-conjugated donkey anti-rabbit IgG (Amersham Corp.) as sec- mediate Gbg subunit-dependent activation of c-Src and that ondary antibody. C-Src was detected using 1:500 dilution of mouse inhibition of Src family kinases by cellular expression of Csk monoclonal anti-c-Src antibody 327 with horseradish peroxidase-conju- antagonizes G protein-coupled receptor-mediated MAP kinase gated donkey anti-mouse IgG (Jackson Laboratories) as secondary an- activation (19). Here, we examine the role of Src family nonre- tibody. EGF receptor was detected using 1:1000 dilution of sheep anti- ceptor tyrosine kinases in mediating Gbg subunit-dependent human EGF receptor IgG with horseradish peroxidase-conjugated tyrosine phosphorylation of receptor tyrosine kinases and Shc. donkey anti-sheep IgG (Jackson Laboratories) as secondary antibody. Immunoblots for autophosphorylated EGF receptor were performed Our data suggest that activation of Src family kinases by G using mouse monoclonal anti-activated EGF receptor IgG (26) with protein-coupled receptors can account for the G -coupled recep- horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson tor-mediated tyrosine phosphorylation events that direct re- Laboratories) as secondary antibody. Immune complexes on nitrocellu- cruitment of the Shc and Grb2 adapter proteins to the mem- lose were visualized by enzyme-linked chemiluminescence (Amersham brane using the EGF receptor as a scaffold. Corp.) and quantified by scanning laser densitometry. GST Fusion Proteins Containing the c-Src SH2 and SH3 Domains— EXPERIMENTAL PROCEDURES GST fusion proteins containing the human c-Src SH2 (amino acids 144–249), SH3 (amino acids 87–143), or SH2 and SH3 (amino acids DNA Constructs—The cDNA encoding the a2A AR was cloned in our 87–249) domains were prepared as GST Sepharose conjugates as de- laboratory. The cDNAs encoding Gb1 and Gg2 were provided by M. scribed previously (27). For the detection of c-Src SH2 or SH3 domain- Simon (California Institute of Technology, Pasadena, CA). The cDNA c-src binding proteins, appropriately transfected and stimulated COS-7 cells encoding human p60 was provided by D. Fujita (University of Cal- csk M dithiothreitol, soni- were lysed in RIPA/SDS buffer containing 5 m was provided by gary, Alberta, Canada), and the cDNA encoding p50 cated, clarified by centrifugation, precleared with 6 mg/ml GST Sepha- H. Hanafusa (Rockefeller University, New York, NY). The constitu- c-src rose for 1 h and incubated with 6 mg/ml of the GST fusion protein tively activated Y530F p60 (TAC(Y) 3 TTC(F)), in which the regu- Sepharose for3hat4 °C. After incubation, fusion protein complexes latory carboxyl-terminal tyrosine residue has been mutated, and cata- c-src were washed twice with ice-cold RIPA buffer and once with phosphate- lytically inactive K298M p60 (AAA(K) 3 ATG(M)) mutants were buffered saline, denatured in Laemmli sample buffer, and resolved by prepared as described (19). All cDNAs were subcloned into pRK5 or SDS-PAGE. Coprecipitated tyrosine phosphoproteins and EGF receptor pcDNA eukaryotic expression vectors for transient transfection. were detected by protein immunoblotting as described. Cell Culture and Transfection—COS-7 cells were maintained in Dul- becco’s modified Eagle’s medium supplemented with 10% fetal bovine RESULTS serum and 100 mg/ml gentamicin at 37 °C in a humidified 5% CO atmosphere. Transfections were performed on 80–90% confluent mono- G -coupled Receptors and Gbg Subunits Mediate Formation layers in 100-mm dishes. For transient transfection, cells were incu- of a Mitogenic Signaling Complex Containing EGF Receptor, bated at 37 °C in 4 ml serum-free Dulbecco’s modified Eagle’s medium Shc, and Grb2—As shown in Fig. 1A, stimulation of endoge- containing 6–10 mg of DNA/100-mm dish plus 6 ml of LipofectAMINE nous LPA receptors in COS-7 cells leads to a rapid 3–5-fold reagent (Life Technologies, Inc.)/mg of DNA. Empty pRK5 vector was increase in tyrosine phosphorylation of each of the three Shc added to transfections as needed to keep the total mass of DNA added isoforms. The phosphorylation is maximal within 2 min of per dish constant within an experiment. After 3–5 h of exposure to the transfection medium, monolayers were refed with growth medium and stimulation and declines slowly thereafter (9, 19). Under non- incubated overnight. Transfected monolayers were serum starved in denaturing conditions, Shc coprecipitates with two major tyro- Dulbecco’s modified Eagle’s medium supplemented with 0.1% bovine sine phosphoprotein bands of approximately 130 and 180 kDa serum albumin and 10 mM Hepes, pH 7.4, for 16–20 h prior to stimu- and with the adapter protein Grb2. The association of Shc with lation. Assays were performed 48 h after transfection. LipofectAMINE the p130 and p180 phosphoproteins is modulated with a time transfection of COS-7 cells consistently resulted in transfection efficien- course that parallels the time course of Shc phosphorylation cies of greater than 80% (data not shown). Transient expression of Gb1 and Gg2 subunits, Csk, and mutant c-Src proteins was confirmed by and Shc-Grb2 complex formation, suggesting that LPA stimu- Gbg Subunit-mediated Tyrosine Phosphorylation of EGF Receptor 4639 FIG.1. G -coupled receptor- and Gbg subunit-stimulated association of Shc with p130 and p180 tyrosine phosphoproteins and Grb2 in COS-7 cells. A, time course of LPA-stimulated Shc tyrosine phosphorylation and association with p130 and p180 tyrosine phosphopro- teins and Grb2. Serum-starved cells were stimulated for the indicated times with LPA (10 mM). Immunoprecipitates of Shc from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine (upper panel) or anti-Grb2 (lower panel)as described. The position of tyrosine phosphorylated Shc isoforms, Shc-associated p130 and p180 phosphoproteins, and Grb2 are as indicated. B, c-src effect of a2A AR, LPA, or EGF receptor stimulation and Gbg subunit or Y530F p60 expression on Shc tyrosine phosphorylation and association with p130 and p180 tyrosine phosphoproteins and Grb2. Cells were transiently transfected with empty pRK5 vector, Gb1 and Gg2, a2A AR, or c-src Y530F p60 as described. Duplicate plates of serum-starved cells were stimulated for 2 min with the a2A AR agonist UK14304 (UK,10 mM), LPA (10 mM), or EGF (10 ng/ml) as indicated. Immunoprecipitates of Shc from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine (upper panel) or anti-Grb2 (lower panel) as described. C, pertussis toxin-sensitivity of a2A AR- and LPA-stimulated Shc, p130, and p180 tyrosine phosphorylation. Cells were transiently transfected with empty pRK5 vector or a2A AR and serum-starved overnight in the presence or the absence of pertussis toxin (Ptx, 100 ng/ml) prior to stimulation with LPA, UK14304, or EGF as indicated. Immunoprecipitates of Shc from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti- shc phosphotyrosine. D, quantitation of the effects of pertussis toxin treatment on a2A AR- and LPA-stimulated tyrosine phosphorylation of p52 . UK14304-, LPA-, and EGF-stimulated p52shc phosphorylation was determined as described. 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 6 S.E. for three separate experiments. NS, nonstimulated. neu lation induces association of these proteins. As shown in Fig. receptor coprecipitation. In contrast, Shc-p185 complexes 1B, similar increases in Shc phosphorylation and Shc-p180 are present in nonstimulated cells and do not increase detect- association result from transient expression of Gb1g2 subunits ably following LPA or a2A AR receptor stimulation. As ex- or stimulation of endogenous LPA or transiently expressed a2A pected, EGF stimulation results in both Shc-EGF receptor and neu AR receptors. Stimulation of endogenous EGF receptors or Shc-p185 association, which may reflect heterodimerization transient overexpression of a constitutively activated mutant and transphosphorylation of the two related receptor tyrosine c-src human c-Src (Y530F p60 ; Refs. 28–30) has similar, al- kinases (32). As shown in Fig. 2B, the tyrosine phosphorylation neu though more robust effects, indicating that activation of either states of Shc, EGF receptor, and p185 , determined following the receptor tyrosine kinase or nonreceptor Src kinase can direct immunoprecipitation of each protein, reflect the changes mimic the G protein-mediated effects. As shown in Fig. 1 (C in Shc-receptor tyrosine kinase association. Shc and EGF re- and D), G -coupled receptor-mediated but not EGF receptor- ceptor phosphorylation is increased following LPA, a2A AR, or neu mediated Shc phosphorylation and Shc-p180 association are EGF receptor stimulation. P185 exhibits significant basal pertussis toxin-sensitive in these cells. tyrosine phosphorylation, consistent with the detection of Shc- neu Because G protein-coupled receptor-mediated tyrosine phos- p185 complexes in nonstimulated cells, which detectably neu phorylation of PDGF receptor (16), EGF receptor, and p185 increases only following EGF receptor stimulation. (17) has been reported, we performed immunoblots for EGF To confirm that Shc, Grb2, and EGF receptor directly asso- neu receptor and p185 on Shc immunoprecipitates from nonde- ciate following G -coupled receptor stimulation, EGF receptor natured cell lysates following stimulation of LPA, a2A AR, or immunoprecipitates were assayed for the presence of Shc and EGF receptors to determine whether these receptor tyrosine Grb2 following LPA stimulation. As shown in Fig. 2C, stimu- kinases are present in the Shc-associated p180 phosphoty- lation with either LPA or EGF resulted in the association of rosine band. COS-7 cells lack detectable expression of PDGF Shc and Grb2 with EGF receptor. G -coupled receptor-induced receptor (31). As shown in Fig. 2A, EGF receptor is not detect- association of Src family nonreceptor tyrosine kinases with Shc able in Shc immunoprecipitates from nonstimulated cells, but has been reported (19, 21). As shown, c-Src can also be detected stimulation of either G -coupled receptor results in Shc-EGF in EGF receptor immunoprecipitates from LPA- or EGF-stim- i 4640 Gbg Subunit-mediated Tyrosine Phosphorylation of EGF Receptor neu FIG.2. Correlation between G -coupled receptor-stimulated EGF receptor and p185 tyrosine phosphorylation with Shc com- neu plex formation. A, coprecipitation of endogenous EGF receptor and p185 with Shc following G -coupled receptor or EGF receptor stimulation. Serum-starved cells, transiently transfected with empty pRK5 vector or a2A AR, were stimulated for 2 min with UK14304 (UK), LPA, or EGF as indicated. Immunoprecipitates of Shc from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-EGF neu neu receptor (top panel), anti-p185 (HER2, center panel), or anti-Shc (bottom panel) as described. The position of EGF receptor, p185 , and Shc neu isoforms are as indicated. B, tyrosine phosphorylation of endogenous EGF receptor and p185 following G -coupled receptor or EGF receptor stimulation. Serum-starved cells, transiently transfected with empty pRK5 vector or a2A AR, were stimulated with UK14304, LPA, or EGF as neu indicated. Immunoprecipitates of EGF receptor (top panel), p185 (center panel), or Shc (bottom panel) from RIPA/SDS buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine as described. C, coprecipitation of Grb2, Shc, and c-Src with endogenous EGF receptor following LPA receptor or EGF receptor stimulation. Serum-starved cells were stimulated for 2 min with LPA or EGF as indicated. Immunoprecipitates of EGF receptor from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-EGF receptor, anti-Grb2, anti-Shc, or anti-c-Src as described. The position of EGF receptor, Grb2, Shc isoforms, and c-Src are as indicated. NS, nonstimulated. ulated cell lysates, suggesting that activation of the G -coupled autophosphorylation resulting from activation of the intrinsic receptor results in association of EGF receptor, c-Src, Shc, and tyrosine kinase activity of the EGF receptor or inhibition of a Grb2 in a multiprotein complex. phosphotyrosine phosphatase versus phosphorylation of the Src Family Kinase Activity Is Required for Both G -coupled EGF receptor mediated by the c-Src nonreceptor tyrosine ki- Receptor and Gbg Subunit-mediated Tyrosine Phosphorylation nase. As shown, this antibody does not detect EGF receptor of EGF Receptor and Shc—G -coupled receptor-mediated in- autophosphorylation following stimulation of LPA or a2A AR creases in EGF receptor phosphotyrosine might result from receptors, despite a 3–5-fold increase in total EGF receptor ligand-independent activation of the receptor tyrosine kinase, phosphotyrosine, suggesting that the increase in receptor tyro- phosphorylation by an activated nonreceptor tyrosine kinase, sine phosphorylation does not reflect activation of the intrinsic or inhibition of a phosphotyrosine phosphatase. To distinguish tyrosine kinase. between these alternative mechanisms, we employed a mono- Because expression of activated mutant c-Src is sufficient to clonal anti-EGF receptor antibody specific for autophosphoryl- mediate EGF receptor phosphorylation in the absence of li- ated EGF receptor. This antibody selectively recognizes acti- gand, we tested the hypothesis that G -coupled receptor-medi- vated EGF receptor via an epitope distal to amino acid 1052 ated activation of Src family kinases can account for the ob- (26), which is distinct from the major in vitro c-Src phospho- served tyrosine phosphorylation of Shc and EGF receptor. To rylation sites (33). As shown in Fig. 3 (A and B), antiphospho- inhibit endogenous Src family kinases, cells were transiently tyrosine immunoblots of EGF receptor immunoprecipitated transfected with cDNA encoding either Csk or a kinase-inac- c-src from EGF-stimulated cells, from cells transiently expressing tive dominant negative mutant c-Src (K298M p60 ; Ref. 34). c-src Y530F p60 , and from cells in which phosphotyrosine phos- Csk is a cytoplasmic tyrosine protein kinase (35) that inacti- phatase activity is inhibited by incubation with sodium or- vates Src family kinases by phosphorylating the regulatory thovanadate, each exhibit increased total receptor phosphoryl- carboxyl-terminal tyrosine residue. Csk overexpression has ation. Identical immunoblots probed with the anti-activated been shown to impair G protein-coupled receptor-mediated EGF receptor antibody give increased signals from EGF-stim- MAP kinase activation in COS-7 cells (19) and c-fos transcrip- ulated and sodium orthovanadate-treated cells but not from tion in rat glomerular mesangial cells (25). As shown in Fig. 4 c-src c-src Y530F p60 -transfected cells. Thus, the anti-activated EGF (A and B), coexpression of either Csk or K298M p60 mark- receptor antibody is able to discriminate between increased edly inhibits Gb1g2 subunit-, a2A AR-, and LPA receptor- Gbg Subunit-mediated Tyrosine Phosphorylation of EGF Receptor 4641 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 do- main 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 recep- tor has suggested that phosphorylation of the putative c-Src SH2 domain recognition site (Tyr ) 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 G -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 follow- ing 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 attenu- ated. These data suggest that G -coupled receptor stimulation results in both c-Src mediated phosphorylation of the EGF receptor and SH2 domain-dependent c-Src-EGF receptor com- plex formation. DISCUSSION 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 FIG.3. Discrimination between Src kinase-mediated EGF re- cell types has been reported, and inhibition of Src family ki- ceptor phosphorylation and EGF receptor autophosphorylation using anti-activated EGF receptor antibody. A, comparison of nases has been shown to block G protein-coupled receptor- anti-phosphotyrosine immunoblots of endogenous EGF receptor with mediated Ras and phospholipase C-g1 activation, MAP kinase anti-activated EGF receptor immunoblots following G -coupled receptor activation, and c-fos transcription (19, 22–24). Our data dem- and EGF receptor stimulation, expression of Y530F, and inhibition of onstrate that in COS-7 cells, G -coupled receptor-stimulated phosphotyrosine phosphatase activity. Serum-starved cells, transiently c-src tyrosine phosphorylation of the EGF receptor results in forma- transfected with empty pRK5 vector, a2A AR, or Y530F p60 were stimulated for 2 min with UK14304 (UK), LPA, or EGF or incubated for tion of a complex between the membrane-associated EGF re- 20 min in the presence of sodium orthovanadate (VO ,10 mM)as ceptor and the cytosolic adapter proteins Shc and Grb2, thus indicated. Duplicate immunoprecipitates of EGF receptor from RIPA/ providing a scaffold for the assembly of a mitogenic signaling SDS buffer lysates were resolved by SDS-PAGE and immunoblotted complex. The G -coupled receptor effects can be mimicked by with either anti-phosphotyrosine (upper panel) or anti-activated EGF receptor antibody (lower panel) as described. B, quantitation of anti- cellular overexpression of Gbg subunits, suggesting that the phosphotyrosine immunoblots of EGF receptor and anti-activated EGF process is Gbg subunit-mediated. Because inhibition of endog- receptor immunoblots. UK14304-, LPA-, EGF-, sodium orthovanadate-, enous Src kinases blocks both G protein-coupled receptor-me- c-src and Y530F p60 -stimulated EGF receptor total tyrosine phosphoryl- diated EGF receptor phosphorylation and binding of the EGF ation and autophosphorylation were determined as described. Autora- diographs were quantified by scanning laser densitometry, and the data receptor to the c-Src SH2 domain, the data also suggest that were presented as fold increase over nonstimulated or empty pRK5 Src family kinases directly associate with and phosphorylate vector transfected controls. The data shown represent the means 6 S.E. the EGF receptor following G -coupled receptor stimulation. for three separate experiments. NS, nonstimulated. Fig. 6 depicts a model of Gbg subunit-mediated, Ras-depend- ent MAP kinase activation that is consistent with these data. mediated tyrosine phosphorylation of both Shc and EGF recep- Gbg subunit-dependent activation of endogenous Src family tor. EGF-stimulated Shc and EGF receptor phosphorylation nonreceptor tyrosine kinases is an early event following G - were less dramatically effected. Shc and EGF receptor phos- coupled receptor stimulation (19). Once activated, the Src ki- phorylation mediated by Y530F, which is not a substrate for nases mediate phosphorylation of several intracellular targets, Csk, is not significantly attenuated by Csk overexpression. including receptor tyrosine kinases, adapter proteins such as c-Src SH2 Domain GST Fusion Proteins Bind EGF Receptor Shc and insulin receptor substrate-1, and possibly cytoskel- Following G -coupled Receptor Activation—To determine etally associated Src substrates such as focal adhesion kinase whether G -coupled receptor-stimulated EGF receptor phos- and paxillin. Once phosphorylated, membrane-associated pro- phorylation can induce binding of Src kinases directly to the teins such as the receptor tyrosine kinases and focal adhesion EGF receptor, GST-fusion proteins containing either the c-Src kinase would provide docking sites for the SH2 domains of the SH2, SH3, or SH2-SH3 domains (27) were assayed for the Shc and Grb2 adapter molecules, resulting in the recruitment ability to precipitate phosphorylated EGF receptor from lysates of Ras guanine nucleotide exchange factors, and potentially of of LPA-stimulated cells. As shown in Fig. 5A, the c-Src SH2 and other components of the mitogenic signaling complex, to the SH2-SH3 domain GST fusion proteins but not the c-Src SH3 plasma membrane. The ensuing activation of Ras would recruit domain GST fusion protein precipitate a 180-kDa tyrosine the Raf kinase to the membrane and initiate the phosphoryla- 4642 Gbg Subunit-mediated Tyrosine Phosphorylation of EGF Receptor FIG.5. Src kinase-dependent association of EGF receptor with c-Src SH2 domain GST fusion proteins. A, association of p180 with c-Src SH2 domain and SH2-SH3 domain GST fusion proteins following LPA and EGF receptor stimulation. RIPA/SDS lysates of serum-starved cells, stimulated for 2 min with LPA or EGF, were incubated with GST-Sepharose (GST) or GST-Src SH2, SH3, or SH2-SH3-Sepharose as described. Precipitated proteins were resolved by SDS-PAGE and im- munoblotted with anti-phosphotyrosine monoclonal antibody. The po- sition of the c-Src SH2 domain binding p180 protein is as indicated. B, association of EGF receptor with the c-Src SH2 domain GST fusion protein following LPA and EGF receptor stimulation. RIPA/SDS lysates of stimulated serum-starved cells were incubated with GST-Src SH2 sepharose as described. Precipitated proteins were resolved by SDS- PAGE and immunoblotted with anti-EGF receptor. The position of the EGF receptor from GST-Src SH2 Sepharose precipitates and COS-7 whole cell lysates is as indicated. C, quantitation of the effect of Csk expression on LPA- and EGF-stimulated association of EGF receptor with the c-Src SH2 domain GST fusion protein. Cells were transiently cotransfected with empty vector (Control) or expression plasmid encod- ing Csk (CSK), and LPA- and EGF-stimulated association of EGF receptor with the c-Src SH2 domain GST fusion protein was deter- mined. Autoradiographs were quantified by scanning laser densitome- try, and the data were presented as fold increase over nonstimulated or empty pRK5 vector transfected controls. The data shown represent the means 6 S.E. for three separate experiments. NS, nonstimulated. c-src FIG.4. Effect of Csk and K298M p60 expression on G -cou- tion cascade leading to MAP kinase activation. pled receptor-mediated Shc and EGF receptor tyrosine phos- Depending upon cell type, the G protein-coupled receptors phorylation. A, Immunoblots of Shc phosphotyrosine following a2A AR, LPA or EGF receptor stimulation and Gbg subunit or Y530F for angiotensin II, LPA, and a-thrombin have been shown to c-src p60 expression. Cells were transiently cotransfected with empty stimulate ligand-independent tyrosine phosphorylation of c-src vector (Control) or expression plasmid encoding Csk or K298M p60 , c-src PDGF receptor (16), insulin-like growth factor-1 receptor b plus empty pRK5 vector, Gb1 and Gg2, a2A AR, or Y530F p60 . Serum-starved cells were stimulated for 2 min with UK14304 (UK), LPA, or EGF as indicated, and immunoprecipitates of Shc from RIPA/ c-src SDS buffer lysates were immunoblotted with anti-phosphotyrosine as p60 coexpression on Gbg subunit-, a2A AR-, LPA-, EGF-, and Y530F c-src described. The position of tyrosine phosphorylated Shc isoforms are as p60 -stimulated Shc and EGF receptor tyrosine phosphorylation. Shc indicated. B, immunoblots of EGF receptor phosphotyrosine following and EGF receptor phosphotyrosine were determined as described fol- a2A AR, LPA, or EGF receptor stimulation and Gbg subunit or Y530F lowing a2A AR, LPA, or EGF receptor stimulation and Gbg subunit or c-src c-src p60 expression. Serum-starved, transiently cotransfected cells were Y530F p60 expression. Autoradiographs were quantified by scan- stimulated as described and immunoprecipitates of EGF receptor from ning laser densitometry, and the data were presented as fold increase RIPA/SDS buffer lysates were immunoblotted with anti-phosphoty- over nonstimulated or empty pRK5 vector transfected controls. The rosine as described. The position of tyrosine phosphorylated EGF re- data shown represent the means 6 S.E. for three to five separate ceptor is as indicated. C, quantitation of the effects of Csk and K298M experiments. NS, nonstimulated. Gbg Subunit-mediated Tyrosine Phosphorylation of EGF Receptor 4643 diated phosphorylation and EGF receptor autophosphoryla- tion, we have been unable to detect increased EGF receptor autophosphorylation following either overexpression of Y530F c-src p60 or G -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. Gbg subunit-mediated PI3K activity has been described in neutrophils and platelets (36, 37), a Gbg subunit-regulated isoform of p110 PI3K has been cloned, and Gbg subunits may contribute to the regulation of the conventional p85/p110 PI3K (38). We have previously reported that G -coupled receptor- and Gbg 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 c-src 2 activation by transiently expressed Y530F p60 , , mSos, and constitutively activated mutants of Ras and MAP kinase/erk kinase(39)iswortmannin-insensitive,suggestingthatthePI3K- 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-trisphos- phate, the product of PI3K (40), may provide an explanation for this phenomenon. Interaction between Src kinases and novel Gbg subunit- FIG.6. Model of G -coupled receptor-mediated, Ras-dependent regulated nonreceptor tyrosine kinases might also contribute MAP kinase activation in COS-7 cells. Activation of Src family kinases following release of Gbg subunits from pertussis toxin-sensitive to the regulation of Src kinase activity. In neuronal cells, G - heterotrimeric G proteins precedes tyrosine phosphorylation of several coupled receptors have been shown to stimulate the Ca and putative scaffolding molecules, such as receptor tyrosine kinases (RTK) protein kinase C dependent tyrosine protein kinase, PYK2 (41). and focal adhesion kinase, leading to the phosphotyroine-dependent, PYK2 is a member of the focal adhesion kinase family of inte- SH2 domain-mediated recruitment of Ras guanine nucleotide exchange FAK factor (mSos) to the plasma membrane. The subsequent activation of grin receptor-associated tyrosine kinases and like p125 (42) Ras initiates the Raf, MAP kinase/erk kinase, MAP kinase phosphoryl- can complex with activated c-Src upon stimulation (43). How- ation cascade that leads to MAP kinase (ERK 1/2) activation. ever, phospholipase C activation and Ca mobilization are apparently unable to account for G protein-coupled receptor- neu subunit (14), EGF receptor, and p185 (17). The finding that mediated tyrosine phosphorylation in many noneuronal cells several receptor tyrosine kinases undergo G protein-coupled (4, 44, 45). Bruton’s tyrosine kinase (Btk) and Tsk, two mem- receptor-mediated phosphorylation suggests the existence of a bers of a family of pleckstrin homology domain-containing ty- common mechanism that is not receptor tyrosine kinase-spe- rosine protein kinases that includes Btk, Itk, Tsk and Tec A, cific, such as activation of a nonreceptor tyrosine kinase or are reportedly regulated by Gbg subunits (46). In hematopoi- inhibition of a phosphotyrosine phosphatase. Our data, dem- etic cells, Btk interacts with the Src family kinases Fyn, Lyn, onstrating inhibition of G -coupled receptor-mediated tyrosine and Hck (47), and Src-Btk interaction is associated with Btk phosphorylation of the EGF receptor by specific inhibitors of autoactivation (48). This is unlikely to be a general mechanism Src family kinases, support the hypothesis that activation of for G protein-coupled receptor regulation of Src kinases, how- Src kinases can account for the observed receptor tyrosine ever, because the pleckstrin homology domain-containing tyro- kinase phosphorylation. sine kinases appear to have limited tissue distribution and are The role of the intrinsic tyrosine kinase activity of receptor not known to be involved in the regulation of Ras. tyrosine kinases in G -coupled receptor-mediated tyrosine The data presented in this report suggest that both Src phosphorylation is unclear. Daub et al. (17) have reported that family kinases and receptor tyrosine kinases play central roles inhibition of EGF receptor function in Rat1 cells, by either an in directing the assembly of membrane-associated mitogenic EGF receptor-selective tyrphostin, AG1478, or expression of a signaling complexes in response to G -coupled receptor activa- dominant negative mutant EGF receptor, blocks endothelin-1, tion in some cells. An understanding of the mechanisms LPA, and a-thrombin receptor-mediated EGF receptor/HER2 whereby G protein-coupled receptors regulate tyrosine protein phosphorylation and MAP kinase activation. They conclude phosphorylation and of the basis for cross-talk between G pro- that a ligand-independent transactivation of the EGF receptor/ tein-coupled receptor and receptor tyrosine kinase signaling HER2 tyrosine kinase is responsible for G protein-coupled re- pathways may ultimately provide strategies for selective acti- ceptor-mediated tyrosine phosphorylation. 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