Ras-dependent Mitogen-activated Protein Kinase Activation by G Protein-coupled Receptors
1997; Elsevier BV; Volume: 272; Issue: 31 Linguagem: Inglês
10.1074/jbc.272.31.19125
ISSN1083-351X
AutoresGregory J. Della Rocca, Tim van Biesen, Yehia Daaka, Deirdre K. Luttrell, Louis M. Luttrell, Robert J. Lefkowitz,
Tópico(s)Receptor Mechanisms and Signaling
ResumoMany receptors that couple to heterotrimeric guanine-nucleotide binding proteins (G proteins) have been shown to mediate rapid activation of the mitogen-activated protein kinases Erk1 and Erk2. In different cell types, the signaling pathways employed appear to be a function of the available repertoire of receptors, G proteins, and effectors. In HEK-293 cells, stimulation of either α1B- or α2A-adrenergic receptors (ARs) leads to rapid 5–10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the α2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a Gβγ subunit complex sequestrant peptide (βARK1ct) and dominant-negative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (ΔN-Raf). Erk1/2 phosphorylation in response to α1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or ΔN-Raf, but not by coexpression of βARK1ct or by pretreatment with pertussis toxin. The α1B- and α2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca2+chelation, and inhibitors of protein-tyrosine kinases. Overexpression of a dominant-negative mutant of c-Src or of the negative regulator of c-Src function, Csk, results in attenuation of the α1B-AR- and α2A-AR-mediated Erk1/2 signals. Chemical inhibitors of calmodulin, but not of PKC, and overexpression of a dominant-negative mutant of the protein-tyrosine kinase Pyk2 also attenuate mitogen-activated protein kinase phosphorylation after both α1B- and α2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium-, and tyrosine kinase-dependent pathway for both Gi- and Gq/11-coupled receptors. These results indicate that in HEK-293 cells, the Gβγ subunit-mediated α2A-AR- and the Gαq/11-mediated α1B-AR-coupled Erk1/2 activation pathways converge at the level of phospholipase C. These data suggest that calcium-calmodulin plays a central role in the calcium-dependent regulation of tyrosine phosphorylation by G protein-coupled receptors in some systems. Many receptors that couple to heterotrimeric guanine-nucleotide binding proteins (G proteins) have been shown to mediate rapid activation of the mitogen-activated protein kinases Erk1 and Erk2. In different cell types, the signaling pathways employed appear to be a function of the available repertoire of receptors, G proteins, and effectors. In HEK-293 cells, stimulation of either α1B- or α2A-adrenergic receptors (ARs) leads to rapid 5–10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the α2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a Gβγ subunit complex sequestrant peptide (βARK1ct) and dominant-negative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (ΔN-Raf). Erk1/2 phosphorylation in response to α1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or ΔN-Raf, but not by coexpression of βARK1ct or by pretreatment with pertussis toxin. The α1B- and α2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca2+chelation, and inhibitors of protein-tyrosine kinases. Overexpression of a dominant-negative mutant of c-Src or of the negative regulator of c-Src function, Csk, results in attenuation of the α1B-AR- and α2A-AR-mediated Erk1/2 signals. Chemical inhibitors of calmodulin, but not of PKC, and overexpression of a dominant-negative mutant of the protein-tyrosine kinase Pyk2 also attenuate mitogen-activated protein kinase phosphorylation after both α1B- and α2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium-, and tyrosine kinase-dependent pathway for both Gi- and Gq/11-coupled receptors. These results indicate that in HEK-293 cells, the Gβγ subunit-mediated α2A-AR- and the Gαq/11-mediated α1B-AR-coupled Erk1/2 activation pathways converge at the level of phospholipase C. These data suggest that calcium-calmodulin plays a central role in the calcium-dependent regulation of tyrosine phosphorylation by G protein-coupled receptors in some systems. GTP-binding protein (G protein) 1The abbreviations used are: G protein, GTP-binding protein; MAP, mitogen-activated protein; GPCR, G protein-coupled receptor; PLC, phospholipase C; βARK1ct, the β-adrenergic receptor kinase 1 COOH-terminal peptide; Gα and Gβγ, the α and βγ subunits, respectively, of G proteins; PKC, Ca2+-dependent protein kinase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; PMA, phorbol 12-myristoyl 13-acetate; GFX, bisindolylmaleimide I; AR, adrenergic receptor; HEK, human embryonic kidney; PTX, pertussis toxin; LPA, lysophosphatidic acid; EGF, epidermal growth factor; SFLLRN, thrombin agonist peptide.1The abbreviations used are: G protein, GTP-binding protein; MAP, mitogen-activated protein; GPCR, G protein-coupled receptor; PLC, phospholipase C; βARK1ct, the β-adrenergic receptor kinase 1 COOH-terminal peptide; Gα and Gβγ, the α and βγ subunits, respectively, of G proteins; PKC, Ca2+-dependent protein kinase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; PMA, phorbol 12-myristoyl 13-acetate; GFX, bisindolylmaleimide I; AR, adrenergic receptor; HEK, human embryonic kidney; PTX, pertussis toxin; LPA, lysophosphatidic acid; EGF, epidermal growth factor; SFLLRN, thrombin agonist peptide.-coupled receptors (GPCRs) comprise a family of heptahelical membrane-bound receptors that mediate responses to a vast array of ligands (1Dhanasekaran N. 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In the mammalian Erk1/2 pathway, the proximal kinases Raf-1 and B-Raf phosphorylate and activate the dual function threonine/tyrosine kinases MAP/Erk kinases 1 and 2, which in turn phosphorylate Erk1/2. Once phosphorylated, activated Erk1/2 translocate to the cell nucleus, where they phosphorylate and activate nuclear transcription factors (6Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1156) Google Scholar). Many signals received at the cell surface, including those mediated by growth factor receptor tyrosine kinases (7Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2222) Google Scholar) and integrins, which mediate cell adhesion (8Chen Q. Kinch M.S. Lin T.H. Burridge K. Juliano R.L. J. Biol. Chem. 1994; 269: 26602-26605Abstract Full Text PDF PubMed Google Scholar), initiate the MAP kinase cascade via activation of the low molecular weight GTP-binding protein, p21 ras (9Boguski M.S. McCormick F. Nature. 1993; 366: 643-653Crossref PubMed Scopus (1755) Google Scholar). Association with GTP-bound p21 ras localizes Raf to the plasma membrane, which is sufficient to induce its activation (10Howe L.R. Leevers S.J. Gomez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (630) Google Scholar). Recently, receptors that couple to heterotrimeric G proteins, including the lysophosphatidic acid (LPA) (11Howe L.R. Marshall C.J. J. Biol. Chem. 1993; 268: 20717-20720Abstract Full Text PDF PubMed Google Scholar, 12Hordijk P.L. Verlaan I. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1994; 269: 645-651Abstract Full Text PDF PubMed Google Scholar), bombesin (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar), thromboxane A2/prostaglandin H2 (14Morinelli T.A. Zhang L.-M. Newman W.H. Meier K.E. J. Biol. 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Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, 19van Biesen T. Hawes B.E. Luttrell D.K. Krueger K.M. Touhara K. Porfiri E. Sakaue M. Luttrell L.M. Lefkowitz R.J. Nature. 1995; 376: 781-784Crossref PubMed Scopus (525) Google Scholar), M1 muscarinic acetylcholine (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar), D2 dopamine (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar), and A1 adenosine (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar) receptors, have been shown to activate MAP kinases (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, 20Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (409) Google Scholar, 21Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (764) Google Scholar). The signal transduction pathways employed by these receptors are heterogeneous. In Rat-1 and COS-7 cells, receptors coupled to pertussis toxin-sensitive G proteins mediate Erk1/2 activation via a Gβγ subunit complex-mediated pathway that is dependent upon tyrosine protein phosphorylation and p21 ras activation (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, 19van Biesen T. Hawes B.E. Luttrell D.K. Krueger K.M. Touhara K. Porfiri E. Sakaue M. Luttrell L.M. Lefkowitz R.J. Nature. 1995; 376: 781-784Crossref PubMed Scopus (525) Google Scholar, 22Alblas J. van Corven E.J. Hordijk P.L. Milligan G. Moolenaar W.H. J. Biol. Chem. 1993; 268: 22235-22238Abstract Full Text PDF PubMed Google Scholar). These signals are independent of receptor-mediated effects on phosphatidylinositol hydrolysis, calcium influx, or inhibition of adenylyl cyclase (22Alblas J. van Corven E.J. Hordijk P.L. Milligan G. Moolenaar W.H. J. Biol. Chem. 1993; 268: 22235-22238Abstract Full Text PDF PubMed Google Scholar, 23van Corven E.J. Groenink A. Jalink K. Eichholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (679) Google Scholar). In contrast, receptors coupled to pertussis toxin-insensitive G proteins mediate Erk1/2 activation via a Gα subunit pathway that is p21 ras-independent and may involve PKC (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar). Direct activators of PKC, such as phorbol esters, have been reported to stimulate activation of MAP kinases through both p21 ras-dependent and -independent pathways (4LaMorte V.J. Thorburn J. Absher D. Spiegel A. Brown J.H. Chien K.R. Feramisco J.R. Knowlton K.U. J. Biol. Chem. 1994; 269: 13490-13496Abstract Full Text PDF PubMed Google Scholar,9Boguski M.S. McCormick F. Nature. 1993; 366: 643-653Crossref PubMed Scopus (1755) Google Scholar). Significant heterogeneity may also exist between cell types. Activation of Erk1/2 by α1-adrenergic receptors in neonatal rat ventricular myocytes (4LaMorte V.J. Thorburn J. Absher D. Spiegel A. Brown J.H. Chien K.R. Feramisco J.R. Knowlton K.U. J. Biol. Chem. 1994; 269: 13490-13496Abstract Full Text PDF PubMed Google Scholar) and by prostaglandin F2α receptors in NIH-3T3 cells (15Watanabe T. Waga I. Honda Z. Kurokawa K. Shimizu T. J. Biol. Chem. 1995; 270: 8984-8990Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) is Gαq/11-mediated andp21ras-dependent, suggesting that Gαq/11 subunits also activate p21 ras in some cell types. This pathway differs markedly from the Gq/11-coupled receptor-mediated p21ras-independent MAP kinase activation that has been described in COS-7 cells (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar). In this paper, we characterize the mechanisms of Erk1/2 activation employed by the Gi-coupled α2A- and by the Gq/11-coupled α1B-adrenergic receptors, heterologously expressed in HEK-293 cells. We find that both receptors mediate p21 ras-dependent Erk1/2 activation via phospholipase C and calcium-dependent activation of Src family kinases. These data suggest that, in some cell types, the Gβγ subunit complex-dependent α2A-AR and Gαq/11 subunit-dependent α1B-AR signals converge at the level of PLC and proceed via a common, p21 ras-dependent, signaling pathway. Phorbol 12-myristate 13-acetate (PMA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) were from Sigma. UK-14304 was from Pfizer. Fluphenazine, calmidazolium, U73122, A23187, bisindolylmaleimide I (GFX), genistein, and herbimycin A were from Calbiochem. Ophiobolin A was from Biomol. Pertussis toxin was from List Biologicals. Rabbit polyclonal anti-Pyk2 IgG was a kind gift of J. Schlessinger. The cDNAs for the α1B- and the α2A-adrenergic receptors were cloned in our laboratory (24Cotecchia S. Schwinn D.A. Randall R.R. Lefkowitz R.J. Caron M.G. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7159-7163Crossref PubMed Scopus (483) Google Scholar, 25Kobilka B.K. Matsui H. Kobilka T.S. Yang-Feng T.L. Francke U. Caron M.G. Lefkowitz R.J. Regan J.W. Science. 1987; 238: 650-656Crossref PubMed Scopus (595) Google Scholar). The β-adrenergic receptor kinase 1 carboxyl-terminal (βARK1ct) peptide-encoding minigene, containing cDNA encoding the carboxyl-terminal 195 amino acids of βARK1, and the dominant-negative SOS-Pro construct, encompassing the proline-rich carboxyl-terminal fragment of mSOS1, were prepared in our laboratory as described previously (19van Biesen T. Hawes B.E. Luttrell D.K. Krueger K.M. Touhara K. Porfiri E. Sakaue M. Luttrell L.M. Lefkowitz R.J. Nature. 1995; 376: 781-784Crossref PubMed Scopus (525) Google Scholar, 26Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar). The cDNA encoding a constitutively active mutant of Gαq (Gαq-Q209L), as described previously (27Qian N.X. Winitz S. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4077-4081Crossref PubMed Scopus (57) Google Scholar), was prepared in our laboratory by R. Premont. The cDNA encoding a constitutively active mutant of Gαi2(Gαi2-Q204L) was from H. Bourne. The cDNAs encoding Gβ1 and Gγ2 were from M. Simon. The p21 N17ras dominant negative mutant was from D. Altschuler, the p74 raf-1 dominant negative mutant (ΔNRaf) was from L. T. Williams, the p112 pyk2 dominant negative mutant (PKM) was from J. Schlessinger, p60c- src was from D. Fujita, and p50 csk was from H. Hanafusa. The constitutively activated Y530F p60c- src (TAC(Y) → TTC(F)), in which the regulatory carboxyl-terminal tyrosine residue has been mutated, and catalytically inactive K298M p60c- src (AAA(K) → ATG(M)) were prepared as described (28Cartwright C.A. Eckhart W. Simon S. Kaplan P.L. Cell. 1987; 49: 83-91Abstract Full Text PDF PubMed Scopus (219) Google Scholar, 29Kmiecik T.E. Shalloway D. Cell. 1987; 49: 65-73Abstract Full Text PDF PubMed Scopus (409) Google Scholar, 30Piwnica-Worms H. Saunders K.B. Roberts T.M. Smith A.E. Cheng S.H. Cell. 1987; 49: 75-82Abstract Full Text PDF PubMed Scopus (312) Google Scholar, 31Snyder M.A. Bishop J.M. McGrath J.P. Levinson A.D. Mol. Cell. Biol. 1985; 5: 1772-1779Crossref PubMed Scopus (82) Google Scholar). All cDNAs were subcloned into pRK5, pcDNA, pCMV, or pRSα eukaryotic expression vectors for transient transfection. HEK-293, Rat-1, and PC12 cells were from the American Type Culture Collection. HEK-293 cells were maintained in minimum essential medium with Earle's salts (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies) and 100 μg/ml gentamicin (Life Technologies), at 37 °C in a humidified 5% CO2 atmosphere. Rat-1 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% fetal bovine serum and 100 μg/ml gentamicin under similar conditions. PC12 cells were maintained in RPMI medium 1640 (Life Technologies) supplemented with 10% heat-inactivated horse serum (Life Technologies), 5% fetal bovine serum, 100 μg/ml gentamicin, and 20 μg/ml l-glutamic acid (Life Technologies) under similar conditions. Transfections of HEK-293 cells were performed on 80–90% confluent monolayers in six-well dishes. Cells were transfected using the calcium phosphate coprecipitation method as described previously (32Didsbury J. Uhing R. Tomhave E. Gerard C. Gerard N. Snyderman R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11564-11568Crossref PubMed Scopus (129) Google Scholar). Empty pRK5 vector was added to transfections as needed to keep the total mass of DNA added per well constant within an experiment. Prior to stimulation, transfected monolayers were serum-starved in minimum essential medium with Earle's salts (HEK-293 cells) or Dulbecco's modified Eagle's medium (Rat-1 cells) supplemented with 0.1% bovine serum albumin (fraction V, protease-free) (Boehringer Mannheim), 100 μg/ml gentamicin, and 10 mm HEPES, pH 7.4, for approximately 24 h. Unstimulated PC12 and HEK-293 cell monolayers were lysed directly with 100 μl/well Laemmli sample buffer. Cell lysates were sonicated briefly, and approximately 30 μg of protein/lane were loaded for resolution via SDS-polyacrylamide gel electrophoresis. Pyk2 was detected by protein immunoblotting using a 1:1000 dilution of rabbit polyclonal anti-Pyk2 IgG with horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) as secondary antibody. Chemiluminescent detection of Pyk2 was performed after development of membranes with ECL reagent (Amersham Corp.), according to the manufacturer's instructions and exposure to Biomax XR scientific imaging film (Eastman Kodak Co.). Stimulations were carried out at 37 °C in serum-starving medium as described in the figure legends. After stimulation, monolayers were lysed directly with 100 μl/well Laemmli sample buffer. Cell lysates were sonicated briefly to disrupt DNA, and proteins (30 μg/lane) were resolved by SDS-polyacrylamide gel electrophoresis. Phosphorylation of Erk1/2 was detected by protein immunoblotting using a 1:1000 dilution of rabbit polyclonal phospho-specific MAP kinase IgG (New England Biolabs) with alkaline phosphatase-conjugated goat anti-rabbit IgG (Amersham) as secondary antibody. Quantitation of Erk1/2 phosphorylation was performed after development of membranes with Vistra ECF reagent (Amersham) by scanning on a Storm PhosphorImager (Molecular Dynamics). After scanning, membranes were treated for 30 min with 40% methanol to remove the Vistra ECF reagent, stripped by treatment with stripping buffer (62.5 mm Tris-Cl, pH 6.8, 2% SDS, 100 mm β-mercaptoethanol) for 30 min at 50 °C, and reprobed with rabbit polyclonal anti-Erk2 IgG (Santa Cruz Biotechnology) to quantitate total p42 mapk. LPA receptor-mediated Erk1/2 activation in Rat-1 fibroblasts is mediated by Gβγ subunits derived from PTX-sensitive G proteins (33Luttrell L.M. van Biesen T. Hawes B.E. Koch W.J. Touhara K. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 16495-16498Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) and is independent of changes in intracellular cAMP, calcium, or PKC (23van Corven E.J. Groenink A. Jalink K. Eichholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (679) Google Scholar). As shown in Fig.1 A, stimulation of endogenous LPA or thrombin receptors in these cells resulted in a 3–6-fold increase in Erk1/2 phosphorylation, which was completely inhibited by treatment with PTX. Acute activation of PKC by treatment with phorbol ester resulted in a less than 2-fold increase in Erk1/2 phosphorylation. Exposure to the calcium ionophore A23187 resulted in a less than 2-fold increase in Erk1/2 phosphorylation. HEK-293 cells exhibit a distinct pattern of Erk1/2 activation. As shown in Fig. 1 B, Erk1/2 phosphorylation via endogenous LPA and thrombin receptors is mediated via both PTX-sensitive and -insensitive G proteins in these cells. LPA and thrombin induce a similar 8–10-fold increase in Erk1/2 phosphorylation. Like Rat-1 cells, the LPA signal is PTX-sensitive. In contrast, the thrombin receptor mediates PTX-insensitive Erk1/2 phosphorylation, indicating that a distinct Erk1/2 activation pathway, mediated by PTX-insensitive G proteins, exists in these cells. Also, acute stimulation of HEK-293 cells with phorbol ester or with A23187 resulted in 24- and 15-fold stimulations of Erk1/2 phosphorylation, respectively, in stark contrast to the above results obtained in Rat-1 cells. To characterize the mechanisms of Erk1/2 activation via PTX-sensitive and insensitive G proteins in HEK-293 cells, we employed a transiently transfected model system in which Gi-coupled α2A-AR and Gq/11-coupled α1B-AR were heterologously expressed. As shown in Fig. 2 A, stimulation of the α2A-AR resulted in PTX-sensitive Erk1/2 phosphorylation, while α1B-AR-mediated Erk1/2 phosphorylation in response to stimulation was insensitive to pretreatment with PTX. Since Gβγ subunits mediate Erk1/2 activation by several GPCRs, we determined whether cellular expression of a Gβγ-sequestrant polypeptide derived from βARK1ct would inhibit α1B-AR- and α2A-AR-mediated MAP kinase activation. As shown in Fig. 2 B, expression of the βARK1ct peptide attenuated Erk1/2 phosphorylation in response to α2A-AR, but not α1B-AR, stimulation. EGF-stimulated Erk1/2 phosphorylation was not sensitive to pretreatment of cells with PTX or to overexpression of cDNA coding for the βARK1ct peptide. This suggests that the α2A-AR signals primarily via the Gβγ subunit complex from PTX-sensitive G proteins, whereas the α1B-AR signal is mediated by the Gα subunit from PTX-insensitive G proteins. As shown in Fig.2 C, overexpression of a constitutively active mutant of Gαq (Gαq-Q209L), but not of Gαi2 (Gαi2-Q204L), was sufficient to induce Erk1/2 phosphorylation. Overexpression of Gβ1γ2 resulted in a consistent 2-fold stimulation of Erk1/2 phosphorylation, unlike the 6–8-fold stimulations of MAP kinase activity observed previously in COS-7 cells (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, 34Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). In COS-7 cells, α2A-AR stimulation results in Erk1/2 activation by a p21 ras-dependent mechanism, whereas α1B-AR-mediated Erk1/2 activation is insensitive to overexpression of a p21 ras dominant-negative mutant and is inhibited by down-regulation of PKC (34Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). To determine the role of p21 ras in adrenergic receptor-mediated Erk1/2 phosphorylation in HEK-293 cells, cDNA coding for either the α1B-AR or α2A-AR was coexpressed with cDNA coding for dominant-negative mutant forms of p21 ras (N17-Ras), mSOS1 (SOS-Pro), or p74 raf-1 (ΔN-Raf). As shown in Fig. 3, phosphorylation of Erk1/2 in response to stimulation of both the α1B-AR and the α2A-AR was attenuated in cells coexpressing N17-Ras, SOS-Pro, or ΔN-Raf. Acute stimulation with phorbol esters was attenuated by overexpression of ΔN-Raf, but not by overexpression of N17-Ras or SOS-Pro, indicating that PKC-mediated Erk1/2 activation is Ras-independent. As expected, EGF-stimulated Erk1/2 phosphorylation was sensitive to the effects of overexpressed N17-Ras, SOS-Pro, and ΔN-Raf. Phosphorylation of Erk1/2 as a result of Gαq-Q209L expression was similarly attenuated in cells coexpressing N17-Ras (data not shown). These data suggest that in HEK-293 cells, stimulation of both Gq/11- and Gi-coupled receptors leads to Erk1/2 phosphorylation in a manner that is dependent upon mSOS, p21 ras, and p74 raf-1 activation. Pertussis toxin-sensitive Gβγ subunit-mediated activation of p21 ras in COS-7 cells is sensitive to inhibitors of tyrosine kinases and requires recruitment of the Ras guanine-nucleotide exchange factor, mSOS (19van Biesen T. Hawes B.E. Luttrell D.K. Krueger K.M. Touhara K. Porfiri E. Sakaue M. Luttrell L.M. Lefkowitz R.J. Nature. 1995; 376: 781-784Crossref PubMed Scopus (525) Google Scholar). The Gβγ subunit effectors responsible for these signals are unknown. Since PLCβ isoforms are regulated by both Gαq/11 and Gβγ subunits and PLCβ overexpression results in Erk1/2 activation in COS-7 cells (34Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar), we tested whether PLC activation was required for α1B-AR- and α2A-AR-mediated Erk1/2 phosphorylation in HEK-293 cells. As shown in Fig. 4, pretreatment of HEK-293 cells with the PLC inhibitor, U73122, markedly attenuated both α1B-AR- and α2A-AR-mediated Erk1/2 phosphorylation. Phorbol ester-mediated Erk1/2 phosphorylation was insensitive to the effects of U73122. The results suggest that one or more isoforms of PLC are required for both Gq/11- and Gi-coupled receptor-mediated MAP kinase activation in HEK-293 cells. Recent reports have suggested that Ras-dependent Erk1/2 activation in vascular smooth muscle cells (35Eguchi S. Matsumoto T. Motley E.D. Utsunomiya H. Inagami T. J. Biol. Chem. 1996; 271: 14169-14175Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) and in neuronal cells (36Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1247) Google Scholar) may be calcium-dependent. As shown in Fig.5, treatment of HEK-293 cells with the calcium ionophore A23187 resulted in 5–10-fold increases in Erk1/2 phosphorylation, similar to that observed after 5-min stimulation of cells expressing transfected α1B- and α2A-AR. Pretreatment of HEK-293 cells with the cell membrane-permeable Ca2+ chelating agent BAPTA abrogated α1B-AR- and α2A-AR-mediated, as well as A23187-induced, Erk1/2 phosphorylation. Erk1/2 phosphorylation after stimulation of BAPTA-pretreated cells with EGF was unaffected. These data suggest that increased intracellular Ca2+ concentration, resulting from Gβγ- or Gα-mediated PLC activation, is required for Erk1/2 activation in HEK-293 cells. Tyrosine phosphorylation of the Shc adaptor protein, which supports the SH2 domain-mediated recruitment of Grb2-Sos to the plasma membrane, has been implicated in both receptor-tyrosine kinase- and GPCR-mediated Erk1/2 activation in some cell types (37Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar). To test whether the calcium-dependent α1B- and α2A-AR signals in HEK-293 cells are also dependent upon tyrosine protein phosphorylation, we determined the effects of two tyrosine kinase inhibitors, genistein and herbimycin A, on α1B- and α2A-mediated Erk1/2 phosphorylation in HEK-293 cells. As shown in Fig. 6 A, pretreatment of HEK-293 cells with the tyrosine kinase inhibitors markedly attenuated α1B-AR-, α2A-AR-, and EGF-R-mediated Erk1/2 phosphorylation. Erk1/2 phosphorylation induced by the calcium ionophore A23187 was also tyrosine kinase inhibitor-sensitive, suggesting that elevation of intracellular Ca2+ levels is sufficient to induce tyrosine phosphorylation in these cells. Both pertussis toxin-sensitive (38Chen Y. Pouyssegur J. Courtneidge S.A. Van Obberghen-Schilling E. J. Biol. Chem. 1994; 269: 27372-27377Abstract Full Text PDF PubMed Google Scholar) and -insensitive (39Wan Y. Kurosaki T. Huang X.-Y. Nature. 1996; 380: 541-544Crossref PubMed Scopus (258) Google Scholar) activation of Src family nonreceptor tyrosine kinases have been described by several laboratories, and Src activity appears to be required for Gβγ subunit-mediated Erk1/2 activation in COS-7 cells (40Luttrell L.M. Della Rocca G.J. van Biesen T. Luttrell D.K. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 4637-4644Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). To determine whether Src family tyrosine kinases are involved in GPCR-mediated Erk1/2 activation in HEK-293 cells, we measured α1B-AR- and α2A-AR-mediated Erk1/2 phosphorylation in cells coexpressing either a catalytically inactive mutant of p60c- src, Src-K298M, or a negative regulatory protein of p60c- src, p50 csk, which phosphorylates and inactivates p60c- src (41Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-72Crossref PubMed Scopus (510) Google Scholar). As shown in Fig.6 B, overexpression of either a constitutively activated mutant form of p60c- src (Src-Y530F) or of wild-type p60c- src in HEK-293 cells was sufficient to induce Erk1/2 phosphorylation. As shown in Fig. 6 C, overexpression of either Src-K298M or p50 csk significantly attenuated Erk1/2 phosphorylation induced by either α1B-AR and α2A-AR stimulation or treatment with calcium ionophore. Erk1/2 phosphorylation induced by acute stimulation with phorbol ester or by overexpression of wild-type c-Src was unaffected. The inability of overexpressed p50 csk to significantly inhibit Erk1/2 phosphorylation mediated by overexpressed wild-type c-Src probably reflects ineffective competition between the two overexpressed proteins. These data suggest that both α1B- and α2A-AR signals in HEK-293 cells are calcium-dependent and mediated by Src family tyrosine kinase activity. Acute stimulation of PKC with phorbol ester is sufficient to induce Erk1/2 phosphorylation in HEK-293 cells. Unlike the α1B-AR- and α2A-AR-mediated signals, the acute PMA signal is insensitive to the effects of N17-Ras, SOS-Pro, overexpressed p50 csk, Src-K298M, and tyrosine kinase inhibitors. As shown in Fig.7, the PKC inhibitor GFX, which abolished acute PMA-stimulated Erk1/2 phosphorylation, had no effect on Erk1/2 phosphorylation after stimulation of cells with adrenergic agonists, EGF, or calcium ionophore. Similar results were obtained through down-regulation of endogenous PKC expression after chronic treatment of cells with phorbol ester. These data suggest that PKC activation mediates Erk1/2 phosphorylation via a pathway that is distinct from the calcium- and tyrosine kinase-dependent pathway employed by α1B- and α2A-ARs. Calcium-dependent activation of a novel focal adhesion kinase family protein-tyrosine kinase, Pyk2, has been shown to mediate calcium ionophore-, phorbol ester-, and Gq/11-coupled receptor-stimulated Erk1/2 activation in neuronal cells (36Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1247) Google Scholar) via a direct interaction with c-Src (42Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (877) Google Scholar). Although Pyk2 is expressed at high levels only in cells of neuronal origin (36Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1247) Google Scholar), it is possible that this or a related kinase might link calcium flux to tyrosine kinase signaling pathways in other cell types. However, as shown in Fig.8 A, protein immunoblots of HEK-293 cell lysates using anti-Pyk2 antisera detect only low levels of Pyk2 expression. To determine whether Pyk2 is involved in the calcium-dependent activation of Erk1/2 in these cells, α1B-AR- and α2A-AR-mediated Erk1/2 phosphorylation was assayed in cells expressing a dominant negative mutant of Pyk2 (PKM; Ref. 42Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (877) Google Scholar). As shown in Fig. 8 B, Erk1/2 phosphorylation in response to adrenergic receptor stimulation or treatment with calcium ionophore was significantly attenuated, with no effect on EGF- or phorbol ester-induced signals. The mechanism whereby calcium influx regulates the activity of the focal adhesion kinase family member Pyk2 is unknown. Neither Ca2+ nor PKC directly activate Pyk2 in vitro(36Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1247) Google Scholar). Recently, calmodulin inhibitors have been shown to inhibit p21 ras-dependent Erk1/2 activation in cultured rat vascular smooth muscle cells (35Eguchi S. Matsumoto T. Motley E.D. Utsunomiya H. Inagami T. J. Biol. Chem. 1996; 271: 14169-14175Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). To determine whether calmodulin might play a role in AR-mediated Erk1/2 activation in HEK-293 cells, we determined the effect of three different calmodulin inhibitors on α1B- and α2A-AR-stimulated Erk1/2 phosphorylation. As shown in Fig.9, pretreatment of HEK-293 cells with fluphenazine, calmidazolium, or ophiobolin resulted in marked attenuation of the phospho-MAP kinase signal, compared with Me2SO-pretreated controls. Erk1/2 phosphorylation resulting from stimulation of endogenous EGF receptors was unaffected. These data suggest that calcium-calmodulin may directly or indirectly contribute to the regulation of Pyk2 kinases and the Src-dependent activation of Erk1/2 by GPCRs. These data suggest a model for α1B-AR- and α2A-AR-mediated Erk1/2 activation that is mediated by calcium-dependent regulation of protein-tyrosine kinases. In HEK-293 cells, as in COS-7 cells (34Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar), the α1B-AR-mediated signal is dependent upon the α subunit of a pertussis toxin-insensitive G protein, while the α2A-AR-mediated signal is sensitive to PTX treatment and is dependent upon the release of free Gβγ subunit complexes. Fig.10 depicts a model of GPCR-mediated Erk1/2 activation in HEK-293 cells that is consistent with our data. Gαq/11- and Gβγ-dependent activation of PLC increases cytoplasmic levels of inositol 1,4,5-trisphosphate, resulting in an increase in cytoplasmic calcium concentration. High intracellular concentrations of calcium, perhaps through calmodulin, lead to activation of Pyk2 or a closely related tyrosine kinase, which regulates the activity of p60c- src. Src-dependent tyrosine phosphorylation of adaptor proteins, such as Shc, results in recruitment of the Grb2-SOS complex to the plasma membrane, where it catalyzes p21 ras guanine nucleotide exchange. Ras-dependent recruitment of p74 raf-1 kinase to the membrane initiates the phosphorylation cascade leading to activation of Erk1/2. In this system the Gβγ subunit- and Gαq/11 subunit-mediated pathways each require the PLCβ-dependent stimulation of calcium influx. This early convergence is distinct from findings in COS-7 and CHO cells (34Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar) and more closely resembles the calcium- and Ras-dependent activation of Erk1/2, which has been reported in primary cultures of vascular smooth muscle cells and ventricular myocytes (35Eguchi S. Matsumoto T. Motley E.D. Utsunomiya H. Inagami T. J. Biol. Chem. 1996; 271: 14169-14175Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 43Cartwright C.A. Eckhart W. Simon S. Kaplan P.L. Cell. 1987; 49: 83-91Abstract Full Text PDF PubMed Scopus (227) Google Scholar, 44Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (931) Google Scholar). The observation that dominant interfering mutants of p21 ras, p74 raf-1, and SOS do not fully attenuate α1B-AR- and α2A-AR-mediated Erk1/2 phosphorylation may reflect incomplete inhibition of receptor-mediated p74 raf-1 activation. Alternatively, these data may be indicative of another, p21 ras-independent, mechanism of Erk1/2 phosphorylation, as has been described for Gq- and Go-coupled MAP kinase activation in Chinese hamster ovary cells (34Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 45van Biesen T. Hawes B.E. Raymond J.R. Luttrell L.M. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 1266-1269Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Activation of p60c- src is required for Gi-coupled receptor-mediated, Gβγ subunit-dependent activation of Erk1/2 in COS-7 cells (37Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar), and Gi- and Gq/11-coupled receptor-stimulated Erk1/2 activation in PC12 cells (42Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (877) Google Scholar). Src family kinase recruitment into Shc-containing protein complexes has been demonstrated following stimulation of formyl-methionyl peptide receptors in human neutrophils (46Ptasznik A. Prossnitz E.R. Yoshikawa D. Smrcka A. Traynor-Kaplan A.E. Bokoch G.M. J. Biol. Chem. 1996; 271: 25204-25207Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and following stimulation of LPA and α2-adrenergic receptors in COS-7 cells (37Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar). Our data indicate that Src kinases function as key intermediates in calcium-dependent regulation of Erk1/2, mediated by both Gβγ and Gαq/11 subunits in some cell types. Collectively, these findings indicate that regulation of Src family protein-tyrosine kinase activity, potentially via multiple mechanisms, is a common requirement for GPCR-mediated Erk1/2 activation. In neuronal cells, association of p60c- src with the calcium-regulated focal adhesion kinase family member Pyk2 mediates both Shc phosphorylation and Erk1/2 activation (42Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (877) Google Scholar). Pyk2 was previously thought to be active only in neuronal cells. The detection of Pyk2 in HEK-293 cell lysates as well as the sensitivity of α1B-AR- and α2A-AR-mediated Erk1/2 activation in HEK-293 cells to both the dominant negative mutant of Pyk2 and specific inhibitors of p60c- src suggest that a Pyk2-mediated Src-dependent mechanism of p21 ras activation may represent a paradigm for mitogenic signaling in a variety of non-neuronal cell types. The mechanism of Ca2+-mediated Pyk2 activation remains unclear, however, since calcium does not directly modulate Pyk2 activity (36Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1247) Google Scholar). Perhaps significantly, both adrenergic receptor- and calcium ionophore-mediated Erk1/2 phosphorylation in HEK-293 cells is sensitive to chemical inhibitors of calmodulin. Eguchi et al. (35Eguchi S. Matsumoto T. Motley E.D. Utsunomiya H. Inagami T. J. Biol. Chem. 1996; 271: 14169-14175Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) have suggested that calmodulin regulates Erk1/2 activation in cultured rat vascular smooth muscle cells. In NG108 cells, depolarization induces calcium-dependent Erk1/2 activation, which is mediated by calmodulin-dependent kinase IV (47Enslen H. Tokomitsu H. Stork P.J.S. Davis R.J. Soderling T.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10803-10808Crossref PubMed Scopus (260) Google Scholar). Our data suggest that the calcium-mediated regulation of Src family tyrosine kinases proceeds through a calmodulin-dependent mechanism. These data also suggest that, if Pyk2 directly activates p60c- src in HEK-293 cells, then perhaps calcium/calmodulin is involved in activation of Pyk2, either directly or through a calcium/calmodulin effector protein. The elucidation of GPCR-mediated mitogenic signaling pathways has revealed significant degrees of heterogeneity between cell types. In Rat-1 fibroblasts, Gi-coupled, but not Gq/11-coupled, receptors mediate tyrosine kinase-dependent Erk1/2 activation via a calcium- and PLC-independent mechanism (23van Corven E.J. Groenink A. Jalink K. Eichholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (679) Google Scholar). In Chinese hamster ovary cells, Gq/11-coupled receptor stimulation leads to Gαq/11-mediated activation of PKC, p74 raf-1, and Erk1/2 in a tyrosine kinase- and p21 ras-independent manner (34Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). In PC12 neuroblastoma cells, both Gi- and Gq/11-coupled receptors have been shown to activate Erk1/2 via calcium-dependent regulation of p112 pyk2, p60c- src, and p21 ras (42Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (877) Google Scholar). Our data suggest that calcium-dependent regulation of Ras by both Gi- and Gq/11-coupled receptors may represent a common mechanism of GPCR-mediated Erk1/2 activation in many non-neuronal cell types. Indeed, Gq/11-coupled receptors mediate calmodulin inhibitor-sensitive, Ras-dependent Erk1/2 activation in cultured vascular smooth muscle cells (35Eguchi S. Matsumoto T. Motley E.D. Utsunomiya H. Inagami T. J. Biol. Chem. 1996; 271: 14169-14175Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar), a calcium-sensitive tyrosine kinase has been cloned from calf uterus (48Castoria G. Migliaccio A. Green S. Di Domenico M. Chambon P. Auricchio F. Biochemistry. 1993; 32: 1740-1750Crossref PubMed Scopus (89) Google Scholar), and Gq/11-coupled receptor-mediated hypertrophy of cultured rat ventricular myocytes is reportedly Ras-dependent (43Cartwright C.A. Eckhart W. Simon S. Kaplan P.L. Cell. 1987; 49: 83-91Abstract Full Text PDF PubMed Scopus (227) Google Scholar). Characterization of receptor- and kinase-specific differences in the mechanisms of GPCR-mediated mitogenic signal transduction may permit the development of strategies for selective antagonism of distinct G protein-coupled receptor-mediated mitogenic signaling pathways, which ultimately may permit selective modulation of cell proliferation in a variety of pathophysiologic states. We thank Donna Addison and Mary Holben for expert secretarial assistance, Sameena Rahman for technical assistance, and Dr. John Raymond for insightful discussion.
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