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Phosphatidylinositol 3-Kinase Is an Early Intermediate in the Gβγ-mediated Mitogen-activated Protein Kinase Signaling Pathway

1996; Elsevier BV; Volume: 271; Issue: 21 Linguagem: Inglês

10.1074/jbc.271.21.12133

ISSN

1083-351X

Autores

Brian E. Hawes, Louis M. Luttrell, Tim van Biesen, Robert J. Lefkowitz,

Tópico(s)

PI3K/AKT/mTOR signaling in cancer

Resumo

The βγ-subunit of Gi mediates mitogen-activated protein (MAP) kinase activation through a signaling pathway involving Shc tyrosine phosphorylation, subsequent formation of a multiprotein complex including Shc, Grb2, and Sos, and sequential activation of Ras, Raf, and MEK. The mechanism by which Gβγ mediates tyrosine phosphorylation of Shc, however, is unclear. This study assesses the role of phosphatidylinositol 3-kinase (PI-3K) in Gβγ-mediated MAP kinase activation. We show that Gi-coupled receptor- and Gβγ-stimulated MAP kinase activation is attenuated by the PI-3K inhibitors wortmannin and LY294002 or by overexpression of a dominant negative mutant of the p85 subunit of PI-3K. Wortmannin and LY294002 also inhibit Gi-coupled receptor-stimulated Ras activation. The PI-3K inhibitors do not affect MAP kinase activation stimulated by overexpression of Sos, a constitutively active mutant of Ras, or a constitutively active mutant of MEK. These results demonstrate that PI-3K activity is required in the Gβγ-mediated MAP kinase signaling pathway at a point upstream of Sos and Ras activation. The βγ-subunit of Gi mediates mitogen-activated protein (MAP) kinase activation through a signaling pathway involving Shc tyrosine phosphorylation, subsequent formation of a multiprotein complex including Shc, Grb2, and Sos, and sequential activation of Ras, Raf, and MEK. The mechanism by which Gβγ mediates tyrosine phosphorylation of Shc, however, is unclear. This study assesses the role of phosphatidylinositol 3-kinase (PI-3K) in Gβγ-mediated MAP kinase activation. We show that Gi-coupled receptor- and Gβγ-stimulated MAP kinase activation is attenuated by the PI-3K inhibitors wortmannin and LY294002 or by overexpression of a dominant negative mutant of the p85 subunit of PI-3K. Wortmannin and LY294002 also inhibit Gi-coupled receptor-stimulated Ras activation. The PI-3K inhibitors do not affect MAP kinase activation stimulated by overexpression of Sos, a constitutively active mutant of Ras, or a constitutively active mutant of MEK. These results demonstrate that PI-3K activity is required in the Gβγ-mediated MAP kinase signaling pathway at a point upstream of Sos and Ras activation. INTRODUCTIONThe cellular signaling pathways leading to receptor-tyrosine kinase- (RTK) ( (1)The abbreviations used are: RTKreceptor-tyrosine kinaseMAPmitogen-activated proteinPI-3Kphosphatidylinositol 3-kinaseMEKmitogen-activated protein kinase/Erk kinaseMEK+a constitutively active mutant of MEKIPinositol phosphatesGPCRG protein-coupled receptorEGFepidermal growth factorGβγthe βγ subunit of the G proteinMBPmyelin basic proteinDMEMDulbecco's modified Eagle's mediumLPAlysophosphatidic acidPMAphorbol 12-myristate 13-acetateFBSfetal bovine serumα2ARα2A adrenergic receptorT24Rasa constitutively active mutant of RasΔp85a dominant negative mutant of the p85 subunit of PI-3KPIP3phosphatidylinositol 3,4,5-trisphosphateSH2 and SH3Src homology domains.) and G protein-coupled receptor- (GPCR) stimulated mitogen-activated protein (MAP) kinase activation have recently been the subject of intense investigation (1.Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1152) Google Scholar, 2.Alblas 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, 3.van Corven E.J. Hordijk P.L. Medema R.H. Bos J.L. Moolenaar W.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1257-1261Crossref PubMed Scopus (334) Google Scholar, 4.Winitz S. Gupta S.K. Qian N.X. Heasley L.E. Nemenoff R.A. Johnson G.L. J. Biol. Chem. 1994; 269: 1889-1895Abstract Full Text PDF PubMed Google Scholar, 5.Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar, 6.Dhanasekaran N. Heasley L.E. Johnson G.L. Endocr. Rev. 1995; 16: 259-270Crossref PubMed Scopus (178) Google Scholar). The signaling pathway of RTK-mediated MAP kinase activation is the most clearly understood. Epidermal growth factor (EGF) stimulation, for example, produces activation and autophosphorylation of the EGF receptor leading to the formation of a multiprotein complex containing the phosphorylated receptor, the phosphoprotein Shc, the adaptor protein Grb2, and the Ras-GTP exchange factor Sos(7.Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (924) Google Scholar, 8.Ravichandran K.S. Lorenz U. Shoelson S.E. Burakoff S.J. Mol. Cell. Biol. 1995; 15: 593-600Crossref PubMed Google Scholar, 9.Sasaoka T. Langlois W.J. Leitner J.W. Draznin B. Olefsky J.M. J. Biol. Chem. 1994; 269: 32621-32625Abstract Full Text PDF PubMed Google Scholar). Sos catalyzes exchange of GTP for GDP on the small guanine nucleotide-binding protein, Ras, thereby stimulating Ras activation(10.Aronheim A. Engelberg D. Li N. al-Alawi N. Schlessinger J. Karin M. Cell. 1994; 78: 949-961Abstract Full Text PDF PubMed Scopus (422) Google Scholar). Ras-GTP activates a kinase cascade involving Raf, MEK, and MAP kinase(11.Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (877) Google Scholar, 12.Howe L.R. Leevers S.J. Gomez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (627) Google Scholar, 13.de Vries-Smits A.M. Burgering B.M. Leevers S.J. Marshall C.J. Bos J.L. Nature. 1992; 357: 602-604Crossref PubMed Scopus (303) Google Scholar). Activated MAP kinases phosphorylate and activate transcription factors involved in cell growth and proliferation(1.Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1152) Google Scholar).The signaling pathways utilized by Gi-, Gs-, Go-, and Gq-coupled receptors to stimulate MAP kinase activation have also been assessed and compared(14.Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (404) Google Scholar, 15.Faure M. Voyno Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, 16.Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (758) Google Scholar, 17.Hawes 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, 18.van Biesen T. Hawes B.E. Raymond J.R. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 1266-1269Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). In many cell types, Gi-coupled receptors mediate MAP kinase activation via the βγ-dependent activation of Ras (14.Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (404) Google Scholar, 15.Faure M. Voyno Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, 16.Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (758) Google Scholar). Several of the intermediate steps in the Gβγ-stimulated MAP kinase pathway are identical with the RTK-stimulated signaling cascade including Shc phosphorylation, Shc/Grb2 association, and Sos activation(19.van 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). Inhibitors of Src family tyrosine kinase activity abrogate Gi-coupled receptor- and Gβγ-mediated Shc phosphorylation and MAP kinase activation in COS-7 cells (3.van Corven E.J. Hordijk P.L. Medema R.H. Bos J.L. Moolenaar W.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1257-1261Crossref PubMed Scopus (334) Google Scholar, 17.Hawes 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, 20.Touhara K. Hawes B.E. van Biesen T. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9284-9287Crossref PubMed Scopus (120) Google Scholar, 21.Hordijk P.L. Verlaan I. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1994; 269: 645-651Abstract Full Text PDF PubMed Google Scholar) suggesting that a Src family tyrosine kinase may be involved in the Gβγ-mediated MAP kinase activation pathway at a point upstream of Ras activation. The mechanism by which Gβγ subunits mediate activation of a tyrosine kinase resulting in increased Shc phosphorylation, however, is unclear.Recent studies have suggested that phosphatidylinositol 3-kinase (PI-3K) activity may be involved in both RTK- and GPCR-mediated mitogenic signaling(22.Sakanaka C. Ferby I. Waga I. Bito H. Shimizu T. Biochem. Biophys. Res. Commun. 1994; 205: 18-23Crossref PubMed Scopus (54) Google Scholar, 23.Rodriguez Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1717) Google Scholar, 24.Ferby I.M. Waga I. Sakanaka C. Kume K. Shimizu T. J. Biol. Chem. 1994; 269: 30485-30488Abstract Full Text PDF PubMed Google Scholar, 25.Stephens L. Eguinoa A. Corey S. Jackson T. Hawkins P.T. EMBO J. 1993; 12: 2265-2273Crossref PubMed Scopus (136) Google Scholar, 26.Stephens L.R. Hughes K.T. Irvine R.F. Nature. 1991; 351: 33-39Crossref PubMed Scopus (386) Google Scholar, 27.Stephens L. Jackson T. Hawkins P.T. J. Biol. Chem. 1993; 268: 17162-17172Abstract Full Text PDF PubMed Google Scholar, 28.Thelen M. Wymann M.P. Langen H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4960-4964Crossref PubMed Scopus (203) Google Scholar, 29.Traynor-Kaplan A.E. Harris A.L. Thompson B.L. Taylor P. Sklar L.A. Nature. 1988; 334: 353-356Crossref PubMed Scopus (208) Google Scholar). However, the role of PI-3K in mitogenic signaling pathways has not been clearly elucidated. In this study, we assess the role of PI-3K in the Gβγ-mediated MAP kinase signaling pathway using two inhibitors of PI-3K activity, wortmannin (30.Ui M. Okada T. Hazeki K. Hazeki O. Trends Biochem. Sci. 1995; 20: 303-307Abstract Full Text PDF PubMed Scopus (518) Google Scholar) and LY294002, and a dominant negative mutant of the p85 subunit of PI-3K (Δp85).EXPERIMENTAL PROCEDURESMaterialsCOS-7 and CHO cells were from the American Type Culture Collection. Culture media and LipofectAMINE were from Life Technologies, Inc. Fetal bovine serum (FBS) and gentamicin were from Life Technologies Inc. LY294002 was from Bio-Mol. Monoclonal antibody 12CA5 was from Boehringer Mannheim, and anti-ERK2 polyclonal antibody was from Santa Cruz Biotechnology. Protein A-agarose was from Pharmacia Biotech Inc. [γ-32P]ATP was from DuPont NEN. Myelin basic protein (MBP) and wortmannin were from Sigma. UK-14304 was from Pfizer. The cDNAs for the human α2-C10 AR and βARK1 were cloned in our laboratory(31.Kobilka B.K. Matsui H. Kobilka T.S. Yang-Feng T.L. Francke U. Caron M.G. Regan J.W. Science. 1987; 238: 650-656Crossref PubMed Scopus (593) Google Scholar); cDNAs encoding Gβ1 and Gγ2 were provided by M. Simon; cDNA encoding hemagglutinin (HA)-tagged p44mapk (ERK1) was from J. Pouysségur; cDNA encoding Δp85 was from M. Sakaue. Constitutively active MEK was from R. Erickson.Cell Culture and TransfectionCOS-7 and CHO-K1 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) and F-12 medium, respectively, supplemented with 10% FBS and 50 μg/ml gentamicin. Cells were transiently transfected using LipofectAMINE as described previously(17.Hawes 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). Assays were performed 48 h after transfection. In MAP kinase activation assays, transfected cells were serum-starved in DMEM containing 0.5% serum for 16 to 20 h prior to simulation.Measurement of MAP Kinase ActivityActivity of epitope-tagged p44mapkmapk (HA-ERK1), or endogenous ERK2, was determined following immunoprecipitation, using MBP as substrate (32.Meloche S. Pages G. Pouysségur J. Mol. Biol. Cell. 1992; 3: 63-71Crossref PubMed Scopus (130) Google Scholar) with modifications as described(33.Luttrell 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). Quantitation of labeled MBP was performed using a Molecular Dynamics PhosphorImager.Measurement of Inositol Phosphate ProductionTransfected cells were labeled overnight with [3H]inositol (1-2 μCi/ml) in DMEM containing 10% FBS, washed once with phosphate-buffered saline, and incubated in phosphate-buffered saline containing 1.0 mM CaCl2, 10 mM LiCl, and the indicated agonist for 45 min. Cells were lysed in 0.4 M perchloric acid (1 ml/well), and 0.8-ml aliquots were neutralized with 0.4 ml of 0.72 M KOH, 0.6 M KHCO3. Total inositol phosphate accumulation was quantitated using Dowex anion exchange chromatography as described previously(34.Hawes B.E. Luttrell L.M. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 15776-15785Abstract Full Text PDF PubMed Google Scholar).Measurement of Ras ActivationRas activation was determined as described previously(14.Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (404) Google Scholar, 35.Gibbs J.B. Marshall M.S. Scolnick E.M. Dixon R.A. Vogel U.S. J. Biol. Chem. 1990; 265: 20437-20442Abstract Full Text PDF PubMed Google Scholar). Ras-bound GDP and GTP were quantitated using a Molecular Dynamic PhosphorImager. Ras activation is expressed as the amount of GTP bound to Ras as a percent of total guanine nucleotide bound to Ras.RESULTS AND DISCUSSIONThe βγ subunit of Gi mediates Ras-dependent MAP kinase activation produced by stimulation of both the lysophosphatidic acid (LPA) receptor and α2A adrenergic receptor (α2AR)(14.Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (404) Google Scholar). In order to determine whether PI-3K activity is required in the Gβγ-mediated mitogenic signaling pathway, we assessed the effect of two chemical PI-3K inhibitors (wortmannin and LY294002) and the effect of overexpression of a dominant negative mutant of the p85 subunit of PI-3K (Δp85) on Gi-coupled receptor- and Gβγ-mediated MAP kinase activation. As shown in Fig. 1A, pretreatment of COS-7 cells with wortmannin or LY294002 markedly inhibits MAP kinase activation produced by stimulation of the endogenously expressed LPA receptor. This inhibition of the LPA signal was also detectable at the level of endogenous MAP kinase (ERK2) (Fig. 1A, right panel). Similar inhibition by wortmannin and LY294002 is observed on MAP kinase activation provoked by stimulation of transiently overexpressed α2AR and direct transfection of Gβγ (Fig. 1B). Wortmannin and Ly294002 pretreatment also inhibit GiPCR- and Gβγ-mediated MAP kinase activation in CHO-K1 cells (data not shown). In contrast, wortmannin and LY294002 have a lesser effect on MAP kinase activation stimulated by phorbol 12-myristate 13-acetate (PMA), or epidermal growth factor (EGF) (Fig. 1B). The inhibition by wortmannin and LY294002 is limited to the MAP kinase signaling pathway, in that the PI-3K inhibitors do not affect α2AR- or Gβγ-mediated phosphoinositide hydrolysis (Fig. 1C). Both wortmannin and LY294002 inhibit LPA and Gβγ-stimulated MAP kinase activation in a concentration-dependent manner (Fig. 1, D and E), with IC50 values of 100 nM and 1.0 μM for wortmannin and LY294002, respectively.A recent study (36.Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (416) Google Scholar) reported that expression of a dominant negative mutant of p85α (Δp85), which lacks the p110 binding site, inhibits insulin-stimulated PI-3K activity and PIP3 production. The effect of Δp85 expression on Gi-coupled receptor- and Gβγ-stimulated MAP kinase activation is shown in Fig. 2. Expression of Δp85 inhibits LPA-, α2AR-, and Gβγ-stimulated MAP kinase activation without affecting MAP kinase activation stimulated by PMA. Thus, expression of a dominant negative mutant of PI-3K produces effects similar to wortmannin and LY294002. These results strongly suggest that PI-3K activity is an essential component of the Gβγ-mediated MAP kinase signaling pathway.Figure 2:Effect of Δp85 expression on Gi-coupled receptor- and Gβγ-mediated MAP kinase (HA-ERK1) activation. COS-7 cells were cotransfected with plasmid DNA encoding p44mapkHA−mapk (0.1 μg/well) and either pRK5 alone (2.0 μg/well), Gβ1 and Gγ2 (1.0 μg each/well), or α2AR (0.2 μg/well) or pRK5 (2.0). Cells were also cotransfected with Δp85 (2.0 μg/well) where indicated. Cells were stimulated with vehicle, LPA (10 μM), the α2AR agonist UK-14304 (10 μM), PMA (1.0 μM), or EGF (10 ng/ml) for 5 min, and MAP kinase activation was determined. The data are expressed as fold stimulation where basal MAP kinase activity is defined as 1.0. Values are mean ± S.E. from three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In PC12 cells, GTP-dependent association of Ras with the catalytic subunit of PI-3K has been described(23.Rodriguez Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1717) Google Scholar), suggesting that activation of the p85/p110 PI-3K occurs subsequent to Ras activation. In contrast, a constitutively active mutant of PI-3K stimulates Ras-dependent Xenopus oocyte maturation and fos transcription (37.Hu Q. Klippel A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (516) Google Scholar) suggesting PI-3K activation precedes Ras activation in this system. To determine whether PI-3K activation in the Gi-coupled receptor/Gβγ-mediated MAP kinase signaling pathway precedes or follows Ras activation, the effect of PI-3K inhibitors on Gi-coupled receptor-mediated Ras activation was determined. As shown in Fig. 3, LPA-stimulated Ras activation is abolished by wortmannin and LY294002 pretreatment. In contrast, EGF-stimulated Ras activation is not significantly affected. The striking sensitivity of the LPA-stimulated signal to inhibition of PI-3K activity suggests a crucial role for PI-3K activity early in the Gi-coupled receptor/Gβγ-mediated MAP kinase signaling pathway.Figure 3:Effect of PI-3K inhibitors on LPA- and EGF-mediated Ras activation. COS-7 cells were preincubated for 15 min with wortmannin (1.0 μM), LY294002 (20 μM), or vehicle. Cells were then treated for 2 min with vehicle, LPA (10 μM), or EGF (10 ng/ml), and Ras activation was determined. Data are shown as GTP bound to Ras as a percent of the total guanyl nucleotides bound to Ras. Values represent the mean ± S.E. from three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Further evidence that PI-3K activation is an early event in the Gβγ-mediated MAP kinase signaling pathway is provided by the results in Fig. 4. The effect of PI-3K inhibition on MAP kinase activation provoked by overexpression of Sos, constitutively active Ras (T24Ras), and constitutively active MEK (MEK+) in COS-7 cells was assessed. Pretreatment with wortmannin or LY294002 (Fig. 4A) or overexpression of Δp85 (data not shown) has no effect on the increase in MAP kinase activation stimulated by Sos, T24Ras, or MEK+ (Fig. 4A), suggesting PI-3K activity is upstream of these intermediates in the Gβγ-mediated MAP kinase activation pathway.Figure 4:Effect of PI-3K inhibitors on MAP kinase (HA-ERK1) activation stimulated by intermediates of the Gβγ-mediated mitogenic signaling pathway. COS-7 (A) and CHO-K1 (B) cells were cotransfected with plasmid DNA encoding p44mapkHA−mapk (0.1 μg/well) and 1.0 μg/well of Sos, T24Ras, or MEK+ (A) and Gβ1/Gγ2, Sos, or Gβ1/Gγ2 and Sos (B). Cells were pretreated for 15 min with vehicle, wortmannin (1.0 μM), or LY294002 (20 μM), and MAP kinase activity was determined. The data are expressed as fold MAP kinase activity in which the basal MAP kinase activity is defined as 1.0. Values are the mean ± S.E. from at least three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In CHO-K1 cells, coexpression of Gβγ with Sos results in a synergistic increase in MAP kinase activation(19.van 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). Expression of Gβγ or Sos alone stimulates a 2-3-fold and 5-fold increase, respectively, in MAP kinase activation (Fig. 4B). Coexpression of Gβγ and Sos results in a 15-20-fold increase in MAP kinase activation. As in COS-7 cells, Gβγ-mediated MAP kinase activation in CHO-K1 cells is abolished by wortmannin or LY294002 pretreatment, and Sos-stimulated MAP kinase activation is unaffected. The synergistic increase in MAP kinase activation produced by Gβγ and Sos coexpression is not observed in cells pretreated with wortmannin or LY294002 (Fig. 4B). Thus, the ability of Gβγ subunits to synergize with Sos is dependent on PI-3K activity, suggesting that PI-3K activity is required downstream of Gβγ, but upstream of Sos in the Gi-mediated MAP kinase activation signaling pathway.A role for PI-3K in mitogenic signaling has been suggested by previous studies showing that PI-3K can associate with activated RTKs and Src family kinases(38.Corey S. Eguinoa A. Puyana-Theall K. Bolen J.B. Cantley L. Mollinedo F. Jackson T.R. Hawkins P.T. Stephens L.R. EMBO J. 1993; 12: 2681-2690Crossref PubMed Scopus (170) Google Scholar, 39.Yamanashi Y. Fukui Y. Wongsasant B. Kinoshita Y. Ichimori Y. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1118-1122Crossref PubMed Scopus (184) Google Scholar, 40.Pleiman C.M. Hertz W.M. Cambier J.C. Science. 1994; 263: 1609-1612Crossref PubMed Scopus (394) Google Scholar). PI-3K association with the Grb2-Sos complex has been demonstrated following monocyte colony-stimulating factor stimulation of human peripheral blood monocytes (41.Saleem A. Kharbanda S. Yuan Z.M. Kufe D. J. Biol. Chem. 1995; 270: 10380-10383Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and increased PI-3K/Shc association in cells transformed by BCR/abl oncoprotein has been reported(42.Harrison-Findik D. Susa M. Varticovski L. Oncogene. 1995; 10: 1385-1391PubMed Google Scholar). Thus, PI-3K is capable of interacting with many mitogenic signaling intermediates.Several studies have suggested a role for PI-3K in GPCR-mediated signaling. Activation of neutrophils by formylated-Met-Leu-Phe involves pertussis toxin-sensitive increases in PI-3K activity and PIP3 production(25.Stephens L. Eguinoa A. Corey S. Jackson T. Hawkins P.T. EMBO J. 1993; 12: 2265-2273Crossref PubMed Scopus (136) Google Scholar, 26.Stephens L.R. Hughes K.T. Irvine R.F. Nature. 1991; 351: 33-39Crossref PubMed Scopus (386) Google Scholar, 27.Stephens L. Jackson T. Hawkins P.T. J. Biol. Chem. 1993; 268: 17162-17172Abstract Full Text PDF PubMed Google Scholar, 28.Thelen M. Wymann M.P. Langen H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4960-4964Crossref PubMed Scopus (203) Google Scholar, 29.Traynor-Kaplan A.E. Harris A.L. Thompson B.L. Taylor P. Sklar L.A. Nature. 1988; 334: 353-356Crossref PubMed Scopus (208) Google Scholar, 43.Traynor-Kaplan A.E. Thompson B.L. Harris A.L. Taylor P. Omann G.M. Sklar L.A. J. Biol. Chem. 1989; 264: 15668-15673Abstract Full Text PDF PubMed Google Scholar). Increased PI-3K activity is observed in anti-phosphotyrosine immunoprecipitates following activation of G protein-mediated systems(27.Stephens L. Jackson T. Hawkins P.T. J. Biol. Chem. 1993; 268: 17162-17172Abstract Full Text PDF PubMed Google Scholar). Wortmannin attenuates platelet-activating factor-stimulated MAP kinase activation in guinea pig neutrophils (24.Ferby I.M. Waga I. Sakanaka C. Kume K. Shimizu T. J. Biol. Chem. 1994; 269: 30485-30488Abstract Full Text PDF PubMed Google Scholar) and pertussis toxin-sensitive somatostatin receptor-stimulated MAP kinase activation in CHO-K1 cells(22.Sakanaka C. Ferby I. Waga I. Bito H. Shimizu T. Biochem. Biophys. Res. Commun. 1994; 205: 18-23Crossref PubMed Scopus (54) Google Scholar). The results of the present study demonstrate that PI-3K activity is required in the Gβγ-mediated MAP kinase signaling pathway and that the site of PI-3K activity in the pathway is upstream of Sos.We have previously observed that Gβγ-mediated Shc phosphorylation is sensitive to tyrosine kinase inhibitors and wortmannin(20.Touhara K. Hawes B.E. van Biesen T. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9284-9287Crossref PubMed Scopus (120) Google Scholar). Wortmannin-sensitive Gβγ-stimulated PI-3K activity has been described in platelets and neutrophils(44.Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (519) Google Scholar, 45.Thomason P.A. James S.R. Casey P.J. Downes C.P. J. Biol. Chem. 1994; 269: 16525-16528Abstract Full Text PDF PubMed Google Scholar, 46.Zhang J. Benovic J.L. Sugai M. Wetzker R. Gout I. J. Biol. Chem. 1995; 270: 6589-6594Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and a Gβγ-sensitive PI-3K (designated p110γ or PI-3Kγ) has been cloned(47.Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. Gierschik P. Seedorf K. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (639) Google Scholar). It is thus attractive to speculate that PI-3Kγ activity may be required for recruitment and activation of the tyrosine kinase(s) responsible for mediating Ras and MAP kinase activation.It has recently been reported that a product of PI-3K activity, phosphatidylinositol 3,4,5-trisphosphate (PIP3), is capable of binding with high affinity to the SH2 domains of proteins such as Src and the p85 subunit of PI-3K(48.Rameh L.E. Chen C.-S. Cantley L.C. Cell. 1995; 83: 821-830Abstract Full Text PDF PubMed Scopus (289) Google Scholar). Further, PIP3 can compete with tyrosine-phosphorylated proteins for binding to these sites. The PIP3/SH2 interaction may suggest a novel mechanism for regulating signaling pathways in PI-3K-dependent systems. PIP3 may block phosphoprotein binding to SH2 domain-containing proteins or even supplant phosphoproteins bound to an SH2 domain. It is therefore possible that PIP3 may serve as an intermediate in the Gβγ-mediated MAP kinase signaling pathway.The ability of Δp85 to inhibit Gβγ-mediated MAP kinase activation may indicate a requirement for the p85α-p110 complex in the pathway, possibly as part of a complex containing Shc and a Src family tyrosine kinase(49.Ptasznik A. Traynor-Kaplan A. Bokoch G.M. J. Biol. Chem. 1995; 270: 19969-19973Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Alternatively, Δp85 may inhibit the Gβγ signal by binding directly to PIP3. Binding of Δp85 to PIP3 may disrupt PI-3K-dependent signaling by preventing PIP3 from competing with phosphoproteins for binding to an SH2 domain. Therefore, it is possible that both p85-p110 PI-3K- and PI-3Kγ-dependent signaling can be inhibited by expression of Δp85. Further investigation is required to determine which PI-3K isotype is utilized in the Gβγ-mediated MAP kinase signaling pathway, the mechanism by which PI-3K activity provokes tyrosine kinase activation resulting in Shc phosphorylation and the identity of the tyrosine kinase utilized in this pathway. INTRODUCTIONThe cellular signaling pathways leading to receptor-tyrosine kinase- (RTK) ( (1)The abbreviations used are: RTKreceptor-tyrosine kinaseMAPmitogen-activated proteinPI-3Kphosphatidylinositol 3-kinaseMEKmitogen-activated protein kinase/Erk kinaseMEK+a constitutively active mutant of MEKIPinositol phosphatesGPCRG protein-coupled receptorEGFepidermal growth factorGβγthe βγ subunit of the G proteinMBPmyelin basic proteinDMEMDulbecco's modified Eagle's mediumLPAlysophosphatidic acidPMAphorbol 12-myristate 13-acetateFBSfetal bovine serumα2ARα2A adrenergic receptorT24Rasa constitutively active mutant of RasΔp85a dominant negative mutant of the p85 subunit of PI-3KPIP3phosphatidylinositol 3,4,5-trisphosphateSH2 and SH3Src homology domains.) and G protein-coupled receptor- (GPCR) stimulated mitogen-activated protein (MAP) kinase activation have recently been the subject of intense investigation (1.Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1152) Google Scholar, 2.Alblas 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, 3.van Corven E.J. Hordijk P.L. Medema R.H. Bos J.L. Moolenaar W.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1257-1261Crossref PubMed Scopus (334) Google Scholar, 4.Winitz S. Gupta S.K. Qian N.X. Heasley L.E. Nemenoff R.A. Johnson G.L. J. Biol. Chem. 1994; 269: 1889-1895Abstract Full Text PDF PubMed Google Scholar, 5.Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar, 6.Dhanasekaran N. Heasley L.E. Johnson G.L. Endocr. Rev. 1995; 16: 259-270Crossref PubMed Scopus (178) Google Scholar). The signaling pathway of RTK-mediated MAP kinase activation is the most clearly understood. Epidermal growth factor (EGF) stimulation, for example, produces activation and autophosphorylation of the EGF receptor leading to the formation of a multiprotein complex containing the phosphorylated receptor, the phosphoprotein Shc, the adaptor protein Grb2, and the Ras-GTP exchange factor Sos(7.Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (924) Google Scholar, 8.Ravichandran K.S. Lorenz U. Shoelson S.E. Burakoff S.J. Mol. Cell. Biol. 1995; 15: 593-600Crossref PubMed Google Scholar, 9.Sasaoka T. Langlois W.J. Leitner J.W. Draznin B. Olefsky J.M. J. Biol. Chem. 1994; 269: 32621-32625Abstract Full Text PDF PubMed Google Scholar). Sos catalyzes exchange of GTP for GDP on the small guanine nucleotide-binding protein, Ras, thereby stimulating Ras activation(10.Aronheim A. Engelberg D. Li N. al-Alawi N. Schlessinger J. Karin M. Cell. 1994; 78: 949-961Abstract Full Text PDF PubMed Scopus (422) Google Scholar). Ras-GTP activates a kinase cascade involving Raf, MEK, and MAP kinase(11.Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (877) Google Scholar, 12.Howe L.R. Leevers S.J. Gomez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (627) Google Scholar, 13.de Vries-Smits A.M. Burgering B.M. Leevers S.J. Marshall C.J. Bos J.L. Nature. 1992; 357: 602-604Crossref PubMed Scopus (303) Google Scholar). Activated MAP kinases phosphorylate and activate transcription factors involved in cell growth and proliferation(1.Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1152) Google Scholar).The signaling pathways utilized by Gi-, Gs-, Go-, and Gq-coupled receptors to stimulate MAP kinase activation have also been assessed and compared(14.Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (404) Google Scholar, 15.Faure M. Voyno Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, 16.Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (758) Google Scholar, 17.Hawes 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, 18.van Biesen T. Hawes B.E. Raymond J.R. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 1266-1269Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). In many cell types, Gi-coupled receptors mediate MAP kinase activation via the βγ-dependent activation of Ras (14.Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (404) Google Scholar, 15.Faure M. Voyno Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, 16.Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (758) Google Scholar). Several of the intermediate steps in the Gβγ-stimulated MAP kinase pathway are identical with the RTK-stimulated signaling cascade including Shc phosphorylation, Shc/Grb2 association, and Sos activation(19.van 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). Inhibitors of Src family tyrosine kinase activity abrogate Gi-coupled receptor- and Gβγ-mediated Shc phosphorylation and MAP kinase activation in COS-7 cells (3.van Corven E.J. Hordijk P.L. Medema R.H. Bos J.L. Moolenaar W.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1257-1261Crossref PubMed Scopus (334) Google Scholar, 17.Hawes 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, 20.Touhara K. Hawes B.E. van Biesen T. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9284-9287Crossref PubMed Scopus (120) Google Scholar, 21.Hordijk P.L. Verlaan I. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1994; 269: 645-651Abstract Full Text PDF PubMed Google Scholar) suggesting that a Src family tyrosine kinase may be involved in the Gβγ-mediated MAP kinase activation pathway at a point upstream of Ras activation. The mechanism by which Gβγ subunits mediate activation of a tyrosine kinase resulting in increased Shc phosphorylation, however, is unclear.Recent studies have suggested that phosphatidylinositol 3-kinase (PI-3K) activity may be involved in both RTK- and GPCR-mediated mitogenic signaling(22.Sakanaka C. Ferby I. Waga I. Bito H. Shimizu T. Biochem. Biophys. Res. Commun. 1994; 205: 18-23Crossref PubMed Scopus (54) Google Scholar, 23.Rodriguez Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1717) Google Scholar, 24.Ferby I.M. Waga I. Sakanaka C. Kume K. Shimizu T. J. Biol. Chem. 1994; 269: 30485-30488Abstract Full Text PDF PubMed Google Scholar, 25.Stephens L. Eguinoa A. Corey S. Jackson T. Hawkins P.T. EMBO J. 1993; 12: 2265-2273Crossref PubMed Scopus (136) Google Scholar, 26.Stephens L.R. Hughes K.T. Irvine R.F. Nature. 1991; 351: 33-39Crossref PubMed Scopus (386) Google Scholar, 27.Stephens L. Jackson T. Hawkins P.T. J. Biol. Chem. 1993; 268: 17162-17172Abstract Full Text PDF PubMed Google Scholar, 28.Thelen M. Wymann M.P. Langen H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4960-4964Crossref PubMed Scopus (203) Google Scholar, 29.Traynor-Kaplan A.E. Harris A.L. Thompson B.L. Taylor P. Sklar L.A. Nature. 1988; 334: 353-356Crossref PubMed Scopus (208) Google Scholar). However, the role of PI-3K in mitogenic signaling pathways has not been clearly elucidated. In this study, we assess the role of PI-3K in the Gβγ-mediated MAP kinase signaling pathway using two inhibitors of PI-3K activity, wortmannin (30.Ui M. Okada T. Hazeki K. Hazeki O. Trends Biochem. Sci. 1995; 20: 303-307Abstract Full Text PDF PubMed Scopus (518) Google Scholar) and LY294002, and a dominant negative mutant of the p85 subunit of PI-3K (Δp85).

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