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• W1968091333 abstract "p70s6k has a role in cell cycle progression in response to specific extracellular stimuli. The signal transduction pathway leading to activation of p70s6k by fibroblast growth factor receptor-1 (FGFR-1) was examined in FGF-2-treated rat L6 myoblasts. p70s6k was activated in a biphasic and rapamycin-sensitive manner. Although phosphatidylinositol 3′-kinase was not activated in the FGF-2 treated cells, as judged fromin vitro and in vivo analyses, wortmannin and LY294002 treatment inhibited p70s6k activation. Inhibition of protein kinase C (PKC), by bisindolylmaleimide or by chronic phorbol ester treatment of the FGFR-1 cells, suppressed but did not block p70s6k activation. In cells expressing a point-mutated FGFR-1, Y766F, unable to mediate PKC activation, p70s6k was still activated, in a bisindolylmaleimide- and phorbol ester-resistant manner. The involvement of S6 kinase in FGFR-1-dependent biological responses was examined in murine brain endothelial cells. In response to FGF-2, these cells differentiate to form tube-like structures in collagen gel cultures and proliferate when cultured on fibronectin. p70s6k was not activated in endothelial cells on collagen, whereas activation was observed during proliferation on fibronectin. In agreement with this finding, rapamycin inhibited the proliferative but not the differentiation response. Our results indicate that FGFR-1 mediates p70s6k activation by a phosphatidylinositol 3′-kinase-independent mechanism that does not require PKC activation and, furthermore, proliferation, but not differentiation of endothelial cells in response to FGF-2, is associated with p70s6k activation. p70s6k has a role in cell cycle progression in response to specific extracellular stimuli. The signal transduction pathway leading to activation of p70s6k by fibroblast growth factor receptor-1 (FGFR-1) was examined in FGF-2-treated rat L6 myoblasts. p70s6k was activated in a biphasic and rapamycin-sensitive manner. Although phosphatidylinositol 3′-kinase was not activated in the FGF-2 treated cells, as judged fromin vitro and in vivo analyses, wortmannin and LY294002 treatment inhibited p70s6k activation. Inhibition of protein kinase C (PKC), by bisindolylmaleimide or by chronic phorbol ester treatment of the FGFR-1 cells, suppressed but did not block p70s6k activation. In cells expressing a point-mutated FGFR-1, Y766F, unable to mediate PKC activation, p70s6k was still activated, in a bisindolylmaleimide- and phorbol ester-resistant manner. The involvement of S6 kinase in FGFR-1-dependent biological responses was examined in murine brain endothelial cells. In response to FGF-2, these cells differentiate to form tube-like structures in collagen gel cultures and proliferate when cultured on fibronectin. p70s6k was not activated in endothelial cells on collagen, whereas activation was observed during proliferation on fibronectin. In agreement with this finding, rapamycin inhibited the proliferative but not the differentiation response. Our results indicate that FGFR-1 mediates p70s6k activation by a phosphatidylinositol 3′-kinase-independent mechanism that does not require PKC activation and, furthermore, proliferation, but not differentiation of endothelial cells in response to FGF-2, is associated with p70s6k activation. The p70 S6 kinase (p70s6k) 1The abbreviations used are: p70s6k, p70 S6 kinase; DAG, diacylglycerol; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; FBS, fetal bovine serum; FGF, fibroblast growth factor; FKBP-12, FK506 binding protein 12; IBE, immortomouse brain-derived endothelium; MOPS, 4-morpholinepropanesulfonic acid; PI3-kinase, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PLC-γ, phospholipase C-γ; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; TOR, target of rapamycin; BSA, bovine serum albumin; PDGF, platelet-derived growth factor; PIP, phosphatidylinositol phosphate. is a serine/threonine kinase that phosphorylates 40 S ribosomal protein S6, in response to a number of extracellular stimuli (1Jenö P. Ballou L.M. Novak-Hofer I. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 406-410Crossref PubMed Scopus (144) Google Scholar, 2Proud C.G. Trends Biochem. Sci. 1996; 21: 181-185Abstract Full Text PDF PubMed Scopus (199) Google Scholar). The two isoforms of p70s6k, the 70-kDa s6k αII (cytosolic form) and the 85-kDa s6k αI (nuclear form), are derived from alternatively spliced products (3Reinhard C. Thomas G. Kozma S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4052-4056Crossref PubMed Scopus (98) Google Scholar, 4Grove J.R. Banerjee P. Balasubramanyam A. Coffer P.J. Price D.J. Avruch J. Woodgett J.R. Mol. Cell. Biol. 1991; 11: 5541-5550Crossref PubMed Scopus (151) Google Scholar) from a single gene (3Reinhard C. Thomas G. Kozma S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4052-4056Crossref PubMed Scopus (98) Google Scholar). Extracellular stimuli induce acute phosphorylation on multiple serine and threonine residues within p70s6k, which are associated with its activation. Four of these residues are located in the carboxyl terminus of p70s6k (5Ferrari S. Bannwarth W. Morley S.J. Totty N.F. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7282-7286Crossref PubMed Scopus (134) Google Scholar). These sites are potential mitogen-activated protein kinase targets. However, mitogen-activated protein kinase fails to activate p70s6k after phosphorylation of these sitesin vitro (6Mukhopadhyay N.K. Price D.J. Kyriakis J.M. Pelech S. Sanghera J. Avruch J. J. Biol. Chem. 1992; 267: 3325-3335Abstract Full Text PDF PubMed Google Scholar), and p70s6k activation lies on a Ras-independent pathway (7Ballou L.M. Luther H. Thomas G. Nature. 1991; 349: 348-350Crossref PubMed Scopus (153) Google Scholar, 8Ming X.-F. Burgering B.M.T. Wennström S. Claesson-Welsh L. Heldin C.-H. Bos J.L. Kozma S.C. Thomas G. Nature. 1994; 371: 426-429Crossref PubMed Scopus (204) Google Scholar). Protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3-kinase), and protein kinase B have been implicated as upstream signaling molecules of p70s6kactivation in insulin-, platelet-derived growth factor (PDGF)-, epidermal growth factor (EGF)- and interleukin-2-treated cells (9Chou M.M. Blenis J. Curr. Opin. Cell Biol. 1995; 7: 806-814Crossref PubMed Scopus (245) Google Scholar). Recently, p70s6k was moreover shown to complex with and to be activated by the GTP-binding Rho family proteins Rac1 and Cdc42 (10Chou M.M. Blenis J. Cell. 1996; 85: 573-583Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). However, the relative contribution of these pathways to activation of p70S6K is unclear (8Ming X.-F. Burgering B.M.T. Wennström S. Claesson-Welsh L. Heldin C.-H. Bos J.L. Kozma S.C. Thomas G. Nature. 1994; 371: 426-429Crossref PubMed Scopus (204) Google Scholar). Rapamycin is a potent and specific inhibitor of p70s6k, preventing phosphorylation and activation of p70s6k by all known external stimuli (11Kuo C.J. Chung J. Fiorentino D.F. Flanagan W.M. Blenis J. Crabtree G.R. Nature. 1992; 358: 70-73Crossref PubMed Scopus (568) Google Scholar, 12Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1033) Google Scholar, 13Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (591) Google Scholar, 14Jayaraman T. Marks A.R. J. Biol. Chem. 1993; 268: 25385-25388Abstract Full Text PDF PubMed Google Scholar, 15Han J.-W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). After binding of rapamycin to its cellular receptor, the FK506-binding protein-12 (FKBP-12), this complex targets TOR kinases in Saccharomyces cerevisiae or the related protein FKBP-12-rapamycin-associated protein/rapamycin-FKBP target 1/mammalian TOR in mammalian cells (16Zheng X.-F. Fiorentino D. Chen J. Crabtree G.R. Schreiber S.L. Cell. 1995; 82: 121-130Abstract Full Text PDF PubMed Scopus (249) Google Scholar, 17Brown E.J. Albers M.W. Shin T.B. Ichikawa K. Keith C.T. Lane W.S. Schreiber S.L. Nature. 1994; 369: 756-758Crossref PubMed Scopus (1684) Google Scholar, 18Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (621) Google Scholar). Inactivation of p70s6k by rapamycin is associated with selective dephosphorylation of a unique set of serine and threonine sites, flanked by large aromatic residues, in p70s6k (19Pearson R.B. Dennis P.B. Han J.-W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 14: 5279-5287Crossref PubMed Scopus (389) Google Scholar). Rapamycin is known to inhibit growth of many types of cells; causing G1 arrest in T lymphocytes and delaying entry into S phase in fibroblasts (11Kuo C.J. Chung J. Fiorentino D.F. Flanagan W.M. Blenis J. Crabtree G.R. Nature. 1992; 358: 70-73Crossref PubMed Scopus (568) Google Scholar, 12Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1033) Google Scholar). Microinjection of a neutralizing antibody against p70s6k or p85s6k has also been shown to block the entry into S phase of injected cells (20Lane H.A. Fernandez A. Lamb N.J.C. Thomas G. Nature. 1993; 363: 170-172Crossref PubMed Scopus (318) Google Scholar, 21Reinhard C. Fernandez A. Lamb N.J.C. Thomas G. EMBO J. 1994; 13: 1557-1565Crossref PubMed Scopus (180) Google Scholar). These results indicate that p70s6k activation is important for cell cycle progression. Fibroblast growth factors (FGF) are heparin-binding polypeptide growth factors, which form a family of nine members (22Friesel R.E. Maciag T. FASEB J. 1995; 9: 919-925Crossref PubMed Scopus (406) Google Scholar). Extracellular signaling by FGFs is transduced via specific receptor tyrosine kinases, denoted FGF receptor-1 to -4 (23Partanen J. Vainikka S. Korhonen J. Armstrong E. Alitalo K. Prog. Growth Factor Res. 1992; 4: 69-83Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 24Givol D. Yayon A. FASEB J. 1992; 6: 3362-3369Crossref PubMed Scopus (400) Google Scholar). Heparin and heparan sulfate proteoglycans are known to modulate ligand binding to the receptor tyrosine kinase. Binding of FGFs to the receptor tyrosine kinase leads to receptor dimerization and activation of the kinase domain, followed by autophosphorylation of the receptor and association with downstream signaling components. Thus far, only one Src homology 2 (SH2) domain-containing protein, phospholipase C-γ (PLC-γ), has been shown to bind directly to FGF receptor-1, via a carboxyl-terminal autophosphorylation site at Tyr-766 in the receptor (25Mohammadi M. Honegger A.M. Rotin D. Fischer R. Bellot F. Li W. Dionne C.A. Jaye M. Rubinstein M. Schlessinger J. Mol. Cell. Biol. 1991; 11: 5068-5078Crossref PubMed Google Scholar). The FGF receptors are known to mediate a variety of cellular responses, such as cell proliferation, migration, and differentiation (23Partanen J. Vainikka S. Korhonen J. Armstrong E. Alitalo K. Prog. Growth Factor Res. 1992; 4: 69-83Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 24Givol D. Yayon A. FASEB J. 1992; 6: 3362-3369Crossref PubMed Scopus (400) Google Scholar). We have previously shown that murine brain capillary endothelial cells respond to FGF-1 and FGF-2 treatment either by proliferation or by differentiation, the latter visualized in vitro as tube formation of cells cultured in collagen gels (26Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth Differ. 1996; 7: 383-395PubMed Google Scholar). It is likely that several distinct signal transduction pathways, coupling directly or indirectly to the receptor, contribute to establish these responses. p70s6k has been shown to be activated in FGF-treated cells (27Kahan C. Seuwen K. Meloche S. Pouysségur J. J. Biol. Chem. 1992; 267: 13369-13375Abstract Full Text PDF PubMed Google Scholar). In this paper, we have used different inhibitors of signal transduction pathways, known to contribute to p70s6kactivation, as well as a mutant FGF receptor unable to bind PLC-γ, to characterize FGF-induced p70s6k activation biochemically and to investigate its function in cellular responses to FGF. Rat L6 myoblasts expressing wild-type FGF receptor-1 and Y766F point-mutated FGF receptor-1 (28Klint P. Kanda S. Claesson-Welsh L. J. Biol. Chem. 1995; 270: 23337-23344Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) were cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc., London, UK) supplemented with 10% fetal bovine serum, at 37 °C. The IBE cell line is a capillary endothelial cell line established from H-2Kb-tsA58 SV40 large T transgenic mouse (Immortomouse) brain (26Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth Differ. 1996; 7: 383-395PubMed Google Scholar). The parental IBE cells were cultured routinely in Ham's F12 medium containing 5 μg/ml insulin, 10 ng/ml epidermal growth factor (EGF), 20 units/ml mouse interferon-γ (IFN-γ), 75 μg/ml endothelial growth supplement, and 20% heat-inactivated fetal bovine serum. Dependent on the assay, as outlined below, IBE cells were cultured on dishes coated with 20 μg/ml human plasma fibronectin (Sigma), on dishes covered with type I collagen gels (denoted collagen-coated dishes), or, alternatively, in the middle of two layers of type I collagen gels (tube formation assay). To prepare collagen-coated dishes, 4 volumes of type I collagen solution (Vitrogen 100; Celtrix Pharmaceuticals, Inc. Santa Clara, CA), 4 volumes of 0.012m HCl, 1 volume of 10 × concentrated Ham's F-12, and 1 volume of concentrated buffer (260 mm NaHCO3, 200 mm HEPES, 50 mm NaOH) were mixed at 4 °C, and the mixture was poured into 6-cm dishes (1.5 ml per dish), which were incubated at 33 °C. Rapamycin (Biomol, Plymouth Meeting, PA) was dissolved in ethanol at a concentration of 20 mg/ml as a stock solution and kept at −20 °C. This solution was diluted using culture medium and added to the cells at indicated concentrations 30 min before FGF-2 stimulation. Wortmannin (Sigma) was dissolved in Me2SO at a concentration of 1 mm. After further dilution in Me2SO, 100 nm (final concentration) was added to the cells 30 min before FGF-2 stimulation. Bisindolylmaleimide (Calbiochem) was dissolved in Me2SO at a concentration of 2.4 mm and kept at −20 °C. Prior to use, this solution was diluted 20-fold in Me2SO, and 120 nm (final concentration) was added to the cells 30 min before FGF-2 treatment. Phorbol 12-myristate 13-acetate (PMA; Calbiochem) was dissolved in Me2SO at a concentration of 5 mm and kept at −70 °C. For down-regulation of PKC, 5 μm (final concentration) was added to the cells and the culture was continued for 24 h, at which point FGF-2 stimulation was performed. Receptor tyrosine kinase activity was measured as autophosphorylation of receptor protein in vitro as described previously (26Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth Differ. 1996; 7: 383-395PubMed Google Scholar). Briefly, cells were cultured in 10-cm dishes, and the culture medium was replaced by DMEM containing 0.1% FBS and cultured overnight. Cells were either unstimulated or stimulated by 100 ng/ml FGF-2 for 8 min, and cell lysate was prepared in 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 0.1 mm sodium orthovanadate, 100 units/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride (PMSF), 2.5 mmEDTA, and 1 mm dithiothreitol. After incubation of lysate with anti-FGF receptor-1 antiserum (29Wennström S. Sandström C. Claesson-Welsh L. Growth Factors. 1991; 4: 197-208Crossref PubMed Scopus (32) Google Scholar), receptor protein was precipitated followed by incubation with Protein A-Sepharose beads. After washing, beads were incubated with [γ-32P]ATP on ice for 10 min, and proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was fixed and treated with 1m KOH at 55 °C for 35 min and then dried and exposed on x-ray film (Fuji). The p70s6kactivity assay was performed as described previously (3Reinhard C. Thomas G. Kozma S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4052-4056Crossref PubMed Scopus (98) Google Scholar) with some modifications. L6 cells cultured on 6-cm dishes were either unstimulated or stimulated with 100 ng/ml ligand for 10 or 60 min. IBE cells were inoculated on either fibronectin- or collagen-coated 6-cm dishes with Ham's F-12 medium containing 0.25% BSA, cultured for 4 h at 33 °C, and then stimulated with 100 ng/ml FGF-2, for indicated times. Cells were washed with extraction buffer (EB), which is composed of 50 mm Tris-HCl, pH 8.0, 120 mmNaCl, 20 mm NaF, 1 mm benzamidine, 1 mm EDTA, 6 mm EGTA, 15 mmNa4P2O7, 30 mm4-nitrophenyl phosphate, 0.1 mm PMSF. After washing, cells were lysed in EB containing 1% Nonidet P-40 on ice. Collected cell lysate was frozen in liquid nitrogen and kept at −80 °C until assay. After thawing, the lysate was centrifuged at 10,000 ×g for 30 min and then 100 μg of lysed protein was incubated with the anti-p70s6k antiserum M5 (3Reinhard C. Thomas G. Kozma S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4052-4056Crossref PubMed Scopus (98) Google Scholar) at 4 °C for 2 h, followed by precipitation with Protein A-Sepharose beads. Beads were washed 3 times with EB containing Nonidet P-40, once with dilution buffer (DB; 50 mm MOPS, pH 7.2, 5 mmMgCl2, 0.2% Triton X-100, 1 mm dithiothreitol) and resuspended in 5 μl of DB. The kinase reaction was initiated by addition of 5 μl of DB containing 200 μm ATP, 10 mm 4-nitrophenyl phosphate, 10 μg of 40 S ribosomal protein, and 3 μCi of [γ-32P]ATP to the beads. The reaction proceeded at 37 °C for 30 min and was stopped by addition of SDS sample buffer, followed by electrophoresis in 15% SDS-polyacrylamide gels. After electrophoresis, the gel was fixed and dried and then exposed on an Image Analyzer screen (Fuji). Cells kept in DMEM containing 0.5% FBS, overnight, were either unstimulated or stimulated with 100 ng/ml FGF-2 or platelet-derived growth factor-BB (PDGF-BB) for indicated times, rinsed, and then lysed in 25 mm HEPES, pH 7.5, 150 mm NaCl, 0.1 mm sodium orthovanadate, 10% glycerol, 1% Nonidet P-40, 5 mm EDTA, 100 units/ml aprotinin, and 1 mm PMSF. Clarified cell lysates were immunoprecipitated with anti-phosphotyrosine monoclonal antibody, PY-20 (Transduction Laboratory, Lexington, KT). Protein A-Sepharose immune complexes were washed twice with phosphate-buffered saline containing 1% Nonidet P-40, once with phosphate-buffered saline, once with 0.1m Tris-HCl, pH 7.5, containing 0.5 m LiCl, once with distilled water, and once with 25 mm HEPES, pH 7.5, 100 mm NaCl, 1 mm EDTA. The beads were suspended in 50 μl of 25 mm HEPES, pH 7.5, 100 mm NaCl, 0.5 mm EGTA, and 10 μg of phosphatidylinositol (sonicated for 5 min at 20 °C; Sigma) and preincubated at 20 °C for 10 min. Twenty μCi of [γ-32P]ATP and MgCl2 (final concentration, 10 mm) were added, and samples were further incubated for 10 min at 20 °C. The reaction was stopped by addition of chloroform/methanol, 11.6 m HCl (50:100:1), phospholipids were extracted with chloroform, and the organic phase was washed with methanol, 1 m HCl (1:1). Reaction products were concentrated in vacuo, dissolved in chloroform, spotted on silica Gel-60 plates (Merck) impregnated with 1% potassium oxalate, and resolved by chromatography in chloroform/methanol, 28% ammonia/water (43:38:5:7) for 45 min. Phosphorylated products were detected by Image Analyzer (Fuji) and then exposed on x-ray films (Fuji). L6 cells expressing FGFR-1 were washed with phosphate-free DMEM containing 0.1% fatty acid-free BSA, 0.0375% sodium bicarbonate, and 20 mm HEPES, pH 7.4, and labeled for 90 min in medium containing 300 μCi/ml of [32P]Pi. Washed cells were stimulated with FGF-2 for 5 min, and the lipids were extracted into chloroform/methanol, deacylated using monomethylamine, and deglycerated using periodate as described (30Cross M.J. Stewart A. Hodgkin M.N. Kerr D.J. Wakelam M.J.O. J. Biol. Chem. 1995; 270: 25352-25355Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). The generated inositol phosphates (which corresponded to the inositol lipids) were separated by anion exchange high performance liquid chromatography on a 25-cm partisphere 5SAX column, eluted with a linear gradient of ammonium dihydrogen phosphate (0.5 m, pH 3.8) at 1 ml/min over 110 min. Fractions were collected every 0.5 min and [32P] determined by scintillation counting; peak retention times were compared with authentic 3H-labeled standards which were run every 5th injection. Changes in DAG mass were determined by the DAG kinase-linked assay as described (31Wakelam M.J.O. Hodgkin M. Martin A. Kendall D.A. Hill S.J. Receptor Transduction Protocols. Humana Press Inc., Totowa, NJ1995: 271-278Google Scholar). Briefly, cells were stimulated as above; the medium was aspirated, and incubations were terminated by the addition of ice-cold methanol. The lipids were extracted into chloroform/methanol and dried in vacuo. The solubilized lipids were then incubated withEscherichia coli DAG kinase and [γ32P]ATP; the generated [32P]phosphatidate was separated by thin layer chromatography and analyzed using a PhosphorImager, and the mass of DAG was determined by comparison to a standard curve generated in parallel. Mouse brain capillary endothelial cells (IBE cells) were inoculated into 24-well culture plates coated with human plasma fibronectin at a density of 1.5 × 104 cells/cm2 (3 × 104cells/well) in Ham's F-12 medium containing 5% FBS and cultured at 33 °C. The next day, medium was changed to Ham's F-12 medium containing 2% FBS, or 0.2% FBS, with either vehicle (ethanol) or rapamycin. Twenty minutes later, 1 ng/ml FGF-2 was added as indicated, and the culture was continued for 3 days. Cells were detached from the well by trypsinization, and the cell number was counted by a hemocytometer. Tube formation assays were performed as described previously (26Kanda S. Landgren E. Ljungström M. Claesson-Welsh L. Cell Growth Differ. 1996; 7: 383-395PubMed Google Scholar). In brief, IBE cells were inoculated on the first layer of collagen gels made in wells of 12-well plates at a density of 8 × 105 cells/well in Ham's F-12 medium containing 0.25% BSA with or without rapamycin. Thirty minutes later, 5 ng/ml FGF-2 was added to indicated cells. Culture was continued for additional 3.5 h, at which point the medium was removed and the second layer of collagen was added onto the cells. After gelation of the second layer of collagen, Ham's F-12 medium containing 0.25% BSA and rapamycin or vehicle with or without FGF-2 was added and cells were cultured overnight. Photographs were taken under phase-contrast microscopic examination. p70s6k is known to be activated in response to various mitogens, including insulin, PDGF, EGF, and serum (9Chou M.M. Blenis J. Curr. Opin. Cell Biol. 1995; 7: 806-814Crossref PubMed Scopus (245) Google Scholar). Agonist-stimulated activation of p70s6k is biphasic, with a rapid transient peak, followed by a sustained plateau (27Kahan C. Seuwen K. Meloche S. Pouysségur J. J. Biol. Chem. 1992; 267: 13369-13375Abstract Full Text PDF PubMed Google Scholar, 32Susa M. Olivier A.R. Fabbro D. Thomas G. Cell. 1989; 57: 817-824Abstract Full Text PDF PubMed Scopus (80) Google Scholar). The relevance of the first peak is not clear, but the sustained phase is thought to represent biological activity (32Susa M. Olivier A.R. Fabbro D. Thomas G. Cell. 1989; 57: 817-824Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Several distinct signal transduction pathways have been implicated in p70s6kactivation. We used transfected L6 myoblasts, expressing either the wild-type FGF receptor-1, or a point-mutated FGF receptor-1, Y766F, to examine signal transduction pathways leading to p70s6kactivation in response to FGF. Fig. 1 A shows that wild-type and Y766F FGF receptor-1-transfected L6 cells expressed similar levels of receptors and that the receptors responded to ligand stimulation with induction of tyrosine kinase activity. In contrast, the parental cells lack detectable expression of FGF receptors, and FGF-2 stimulation of the untransfected parental cells failed to induce p70s6k activation (data not shown). p70s6kactivation was examined by immunoprecipitation of p70s6k, from unstimulated and FGF-2-stimulated cells, followed by incubation in kinase buffer and [γ-32P]ATP, in the presence of 40 S ribosomes, serving as a substrate for the immunoprecipitated p70s6k. After SDS-PAGE of the samples,32P-labeled 40 S ribosomal protein s6 was quantified using a PhosphorImage Analyzer. p70s6k activation was analyzed after 10 and 60 min of FGF-2 treatment, to measure early and sustained phases, respectively. Fig. 1 B shows that p70s6kwas activated in a sustained manner in FGF-2-stimulated L6 cells expressing the wild-type FGF receptor-1. In the mutant Y766F receptor expressing cells (Fig. 1 B), the level of p70s6kactivity was comparatively lower at 10 min treatment. However, at 60 min, similar induction (250–300%) of p70s6k activity was seen in the wild-type and mutant FGF receptor-1 expressing cells. These results indicate that signals for p70s6k activation can be transduced via FGF receptor-1 and that the major autophosphorylation site, Tyr-766, which is required for binding and activation of PLC-γ, is not obligatory for FGF receptor-1-mediated p70s6kactivation. Different signal transduction pathways have been implicated in p70s6k activation, and inhibition of the functions of PI3-kinase and PKC have been used to demonstrate roles for pathways involving these enzymes. We analyzed the effects of rapamycin, wortmannin (PI3-kinase inhibitor), and bisindolylmaleimide (PKC inhibitor) in FGF-2-induced p70s6kactivation. In addition, chronic treatment of cells with PMA was used to down-regulate PKC. Fig. 2 shows that treatment of the wild-type FGFR-1 expressing L6 cells with either of these four different drugs attenuated FGF-2-induced p70s6kactivation, both at 10 and 60 min. Rapamycin failed to bring the activity of p70 down to basal, even at the relatively high dose of 100 ng/ml. Treatment of cells with another PI3-kinase inhibitor, LY294002, brought p70s6k activity down to the basal level when used at a concentration of 30 μm (data not shown). We have previously shown that activation of FGF receptor-1, expressed in porcine aortic endothelial cells, does not lead to activation of PI3-kinase in vitro (33Wennström S. Siegbahn A. Yokote K. Arvidsson A.-K. Heldin C.-H. Mori S. Claesson-Welsh L. Oncogene. 1994; 9: 651-660PubMed Google Scholar). In agreement, we failed to detect activation in vitro of PI3-kinase in L6 myoblasts expressing FGF receptor-1 (Fig. 3). The experiment was performed using L6 myoblasts expressing FGF receptor-1, which were stimulated for 5 or 30 min with FGF-2. As a positive control, PDGF-BB was used to stimulate PDGF receptors, endogenously expressed in the L6 cells. Cells were lysed and immunoprecipitated with phosphotyrosine antibodies, followed by analysis for phosphorylation of phosphatidylinositol by thin layer chromatography. As expected, the PDGF-stimulated cells contained active PI3-kinase, indicated by the 5-fold increase in [32P]PIP relative to basal. In the FGF-2-treated cells, the levels of [32P]PIP did not change as compared with the control, neither at 5 nor 30 min of treatment. Similar results were obtained using antibodies against the p85 regulatory subunit of PI3-kinase for immunoprecipitation (data not shown). Furthermore, analysis of in vivo 32P-labeled L6 cells expressing FGFR-1 showed that FGF-2 also did not stimulate PI3-kinase activity as determined by changes in 3′-phosphorylated lipids (Table I). The table shows that FGF-2 stimulated significant turnover of inositol phospholipids with a greater than 2-fold increase in the radioactivity incorporated into PIP within 5 min and an approximate 50% increase in radioactivity associated with PI(4,5)P2. There was a small increase in radioactivity associated with PIP3; however, the level of PIP3 is extremely low and the apparent change is probably due to an increase in basal PI3-kinase activity acting upon the elevated [32P]PI(4,5)P2; indeed, the radioactivity associated with PIP3 compared with PIP2 remains constant at approximately 0.03%. These data strongly imply that inhibition by wortmannin of p70s6kactivation in the FGF-2-stimulated cells did not involve the classical PI3-kinase.Table IEffect of FGF-2 on inositol lipid turnover in PGF receptor-1 expressing L6 cellsTreatmentcpm 32P detected in inositol phosphate peak corresponding to:PIPPI(4,5)P2PIP3Control437,949814,501263388,387718,245183FGF-2940,1021,250,156431846,1121,030,819259 Open table in a new tab The Y766F FGF receptor-1 mutant lacks the ability to mediate phosphorylation and activation of PLC-γ. Active PLC-γ hydrolyzes PIP2 to inositol 1,4,5-trisphosphate and diacylglycerol (DAG), leading to intracellular Ca2+ fluxes and activation of PKC, respectively. In the Y766F mutant FGF receptor-1 expressing cells, PKC could potentially still be activated in response to FGF-2, via PLC-γ-independent DAG formation. We therefore analyzed formation of DAG, by use of a DAG kinase-linked assay, on FGF-2-stimulated wild-type or Y766F FGFR-1 expressing L6 cells (Table II). In the wild-type receptor expressing L6 cells DAG formation was increased approximately 2-fold, in response to FGF-2, at 10 and 60 min of stimulation. A similar level of DAG formation was induced by vasopressin treatment (data not shown). In the mutant Y776F receptor expressing L6 cell" @default.
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• W1968091333 date "1997-09-01" @default.
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• W1968091333 title "Phosphatidylinositol 3′-Kinase-independent p70 S6 Kinase Activation by Fibroblast Growth Factor Receptor-1 Is Important for Proliferation but Not Differentiation of Endothelial Cells" @default.
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• W1968091333 doi "https://doi.org/10.1074/jbc.272.37.23347" @default.
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