FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1996532068> ?p ?o ?g. }

• W1996532068 endingPage "11488" @default.
• W1996532068 startingPage "11484" @default.
• W1996532068 abstract "Addition of sphingosine 1-phosphate induces proliferation of quiescent Swiss 3T3 fibroblasts by unknown mechanisms. To identify the pathways involved, the ability of sphingosine 1-phosphate to activate mitogen-activated protein (MAP) kinase was studied. Sphingosine 1-phosphate rapidly activated the Raf/MAP kinase kinase (MKK)/MAP kinase pathway, and the concentration dependence for MAP kinase activation correlated with that for induction of DNA synthesis. Both MKK1 and MKK2 were activated by sphingosine 1-phosphate, assessed by specific immune complex kinase assays. Prior treatment of the Swiss 3T3 cells with pertussis toxin inhibited 70–80% of the sphingosine 1-phosphate-stimulated MAP kinase activity. Thus, one of the direct or indirect targets of exogenous sphingosine 1-phosphate appears to be a Gi/Go protein. Addition of sphingosine 1-phosphate induces proliferation of quiescent Swiss 3T3 fibroblasts by unknown mechanisms. To identify the pathways involved, the ability of sphingosine 1-phosphate to activate mitogen-activated protein (MAP) kinase was studied. Sphingosine 1-phosphate rapidly activated the Raf/MAP kinase kinase (MKK)/MAP kinase pathway, and the concentration dependence for MAP kinase activation correlated with that for induction of DNA synthesis. Both MKK1 and MKK2 were activated by sphingosine 1-phosphate, assessed by specific immune complex kinase assays. Prior treatment of the Swiss 3T3 cells with pertussis toxin inhibited 70–80% of the sphingosine 1-phosphate-stimulated MAP kinase activity. Thus, one of the direct or indirect targets of exogenous sphingosine 1-phosphate appears to be a Gi/Go protein. Recently, many metabolites of complex lipids have been shown to function as agonists or second messengers in signal transduction (1Hannun Y.A. Bell R.M. Science. 1989; 243: 500-507Crossref PubMed Scopus (1103) Google Scholar, 2Liscovitch M. Cantley L.C. Cell. 1994; 77: 329-334Abstract Full Text PDF PubMed Scopus (311) Google Scholar, 3Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Abstract Full Text PDF PubMed Google Scholar). Sphingosine 1-phosphate, a breakdown product of sphingolipids, has been implicated in diverse cellular functions (4Spiegel S. Olivera A. Carlson R.O. Adv. Lipid Res. 1993; 25: 105-129PubMed Google Scholar). In Swiss 3T3 fibroblasts, sphingosine 1-phosphate stimulates DNA synthesis and cell division (5Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (810) Google Scholar, 6Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Crossref PubMed Scopus (560) Google Scholar). Several reported biological effects of sphingosine 1-phosphate may contribute to its mitogenic effect, including mobilization of intracellular calcium (6Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Crossref PubMed Scopus (560) Google Scholar, 7Mattie M. Brooker G. Spiegel S. J. Biol. Chem. 1994; 269: 3181-3188Abstract Full Text PDF PubMed Google Scholar, 8Ghosh T.K. Bian J. Gill D.L. J. Biol. Chem. 1994; 269: 22628-22635Abstract Full Text PDF PubMed Google Scholar), activation of phospholipase D (9Zhang H. Desai N.N. Murphey J.M. Spiegel S. J. Biol. Chem. 1990; 265: 21309-21316Abstract Full Text PDF PubMed Google Scholar), and enhancement of the DNA binding activity of transcriptional factor AP-1 (10Su Y. Rosenthal D. Smulson M. Spiegel S. J. Biol. Chem. 1994; 269: 16512-16517Abstract Full Text PDF PubMed Google Scholar).MAP 1The abbreviations used are: MAP kinase, mitogen-activated protein kinase; MKK, MAP kinase kinase; MEK, MAP kinase/extracellular signal-regulated kinase kinase; DMEM, Dulbecco's modified Eagle's medium; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; BSA, bovine serum albumin; KR, recombinant kinase-defective p42mapk; LPA, lysophosphatidic acid; pNPP, p-nitrophenyl phosphate; TNFα, tumor necrosis factor α.1The abbreviations used are: MAP kinase, mitogen-activated protein kinase; MKK, MAP kinase kinase; MEK, MAP kinase/extracellular signal-regulated kinase kinase; DMEM, Dulbecco's modified Eagle's medium; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; BSA, bovine serum albumin; KR, recombinant kinase-defective p42mapk; LPA, lysophosphatidic acid; pNPP, p-nitrophenyl phosphate; TNFα, tumor necrosis factor α. kinases are serine/threonine protein kinases that play important roles in mitogenic signaling of a variety of ligands for many receptors, including receptor tyrosine kinases and G protein-coupled receptors (11Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1152) Google Scholar, 12Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 13Blumer K.J. Johnson G.L. Trends Biochem. Sri. 1994; 19: 236-240Abstract Full Text PDF PubMed Scopus (420) Google Scholar). MAP kinases are activated by dual tyrosine and threonine phosphorylations occurring in a TXY motif near the conserved A(S)PE and phosphorylate (S/T)P motifs in their target proteins. A 42-kDa MAP kinase (p42mapk/ERK2) and a 44-kDa MAP kinase (p44mapk/ERK2) are the two best characterized MAP kinases, collectively referred to here as MAP kinase. Both enzymes are activated by two remarkably specific dual specificity MAP kinase kinases (MKKs or MEKs) (14Ahn N.G. Seger R. Krebs E.G. Curr. Opin. Cell Biol. 1992; 4: 992-999Crossref PubMed Scopus (228) Google Scholar, 15Crews C.M. Alessandrini A. Erikson R.L. Science. 1992; 258: 478-480Crossref PubMed Scopus (736) Google Scholar, 16Wu J. Harrison J.K. Vincent L.A. Haystead C. Haystead T. Michel H. Hunt D. Lynch K.R. Sturgill T.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 173-177Crossref PubMed Scopus (117) Google Scholar, 17Wu J. Harrison J.K. Dent P. Lynch K.R. Weber M.J. Sturgill T.W. Mol. Cell. Biol. 1993; 13: 4539-4548Crossref PubMed Scopus (122) Google Scholar). MKKs are in turn regulated by serine phosphorylation by several MKK kinases, including MEK kinase and Raf-1 (18Dent P. Haser W. Haystead T.A. Vincent L.A. Robert T.M. Sturgill T.W. Science. 1992; 257: 1404-1407Crossref PubMed Scopus (496) Google Scholar, 19Kyriakis J.M. App H. Zhang X.F. Banerjee P. Brautigan D.L. Rapp U.R. Avruch J. Nature. 1992; 358: 417-421Crossref PubMed Scopus (966) Google Scholar, 20Lange-Carter C.A. Pieiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (869) Google Scholar). The diversity in MAPKKKs is regarded as a major factor accounting for the multiple pathways linked to MAP kinase. However, very recently MEK kinase has been argued to be a physiologic activator of Jun kinase and not MAP kinase (21Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1010) Google Scholar).Recent evidence has suggested that branching pathways of sphingolipid metabolism may mediate either mitogenic or apoptotic effects (1Hannun Y.A. Bell R.M. Science. 1989; 243: 500-507Crossref PubMed Scopus (1103) Google Scholar, 3Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Abstract Full Text PDF PubMed Google Scholar, 4Spiegel S. Olivera A. Carlson R.O. Adv. Lipid Res. 1993; 25: 105-129PubMed Google Scholar, 22Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1598) Google Scholar, 23Jarvis W.D. Fornari Jr., F.A. Browning J.L. Gewirtz D.A. Kolesnick R.N. Grant S. J. Biol. Chem. 1994; 269: 31685-31692Abstract Full Text PDF PubMed Google Scholar). Ceramide has been reported to induce apoptosis in several mammalian cell lines, whereas sphingosine and sphingosine 1-phosphate are mitogenic. It is critically important to identify the downstream components involved. To this end, the potential involvement of MAP kinase in signaling for sphingosine 1-phosphate in Swiss 3T3 fibroblasts was studied. We report that exogenous sphingosine 1-phosphate rapidly stimulates the activation of the MAP kinase pathway by a pertussis toxin-sensitive mechanism.EXPERIMENTAL PROCEDURESMaterialsPDGF-BB was from Upstate Biotechnology Inc., EGF was from Collaborative Research. Sphingosine 1-phosphate was prepared by enzymatic digestion of sphingosylphosphorylcholine with phospholipase D as described, and its purity was confirmed by thin layer chromatography in five different solvent systems (6Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Crossref PubMed Scopus (560) Google Scholar). Rabbit polyclonal anti-raf-1 antibody and the Raf-1 C-terminal peptide were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against p42mapk, MKK1, and MKK2 were kindly provided by Michael Weber (Dept. of Microbiology, University of Virginia); pertussis toxin was a generous gift of Erik Hewlett (Dept. of Medicine, University of Virginia). Recombinant MKK1 was expressed in Escherichia coli and purified as described (22Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1598) Google Scholar).Cell Culture and MAP Kinase AssaySwiss 3T3 fibroblasts were grown in DMEM, 10% fetal calf serum at 37 °C to confluence. The confluent cells were washed two times with DMEM without serum and then incubated overnight (approximately 20 h) with DMEM/Way-mouth's medium (1:1) containing 0.1% dialyzed calf serum. The serum-starved cells were washed two times with Krebs-Ringer bicarbonate/Hepes (25Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (817) Google Scholar) and incubated in Krebs-Ringer bicarbonate/Hepes for 1.5 h before use. MAP kinase activity was determined by an immune complex kinase assay using a p42mapk-specific antibody TR12 as previously described (25Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (817) Google Scholar). The data shown are the means and standard deviations of triplicate determinations expressed as pmol of phosphate incorporated into myelin basic protein per min. Data shown are representative of multiple (two to five) experiments.MAP Kinase Kinase AssaysTotal MKK activity was determined by KR phosphorylation as described (25Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (817) Google Scholar). To determine MKK1 and MKK2 activities separately, cell lysate supernatants were prepared from Swiss 3T3 cells using the buffer and methodologies described for rat1 cells (25Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (817) Google Scholar). Lysate supernatant protein (75 µg total protein each) were immunoprecipitated with saturating amounts (15 µl) of anti-MKK1 or anti-MKK2 antibodies (precoupled to protein A-agarose) for 2 h at 4 °C. The immune complexes were washed three times with lysis buffer (25Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (817) Google Scholar) and once in lysis buffer without detergent. Washed complexes were incubated (30 °C, 15 min) with 40 µl of MKK reaction mix (1 µg of KR, 25 mm Hepes (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol, 40 mn pNPP, 50 µm [γ-32P]ATP (5,000 cpm/pmol)), and processed for SDS-polyacrylamide gel electrophoresis. The relative activities of MKK1 and MKK2 were compared by quantitating the phosphorylation of KR with a PhosphorImager (Molecular Dynamics) following SDS-polyacrylamide gel electrophoresis.Raf-1 Activity DeterminationCell lysate supernatants (100 µg total protein each) were precleared with protein A-agarose (Pierce). Cleared supernatants were incubated (4 °C, 30 min) with 0.4 µg of affinity-purified anti-Raf-1 or with antibody preincubated with 4 µg of Raf-1 C-terminal peptide (approximately 1000-fold molar excess). Immune complexes were adsorbed (4 °C, 2 h) to protein A-agarose (20 µl of 50% slurry). Beads were washed three times with lysis buffer and once omitting Triton X-100. Assays were initiated by addition of 30 µl of reaction mixture containing 33 mM Hepes (pH 7.5), 14 mM MgCl2, 1.3 mM dithiothreitol, 53 mM pNPP, 30 µm [γ-32P]ATP (10,000 cpm/pmol), and 0.5 µg of recombinant MKK1 expressed in E. coli. After incubation (6 min, 30 °C), 1 µg of KR protein (in 10 µl of 25 mM Tris/Cl, pH 7.5 (4 °C), 40 mM pNPP, 2 mM EGTA, 1 mM dithiothreitol, 37% (v/v) ethylene glycol) was added and incubated (30 °C, 6 min) before quenching with SDS sample buffer for SDS-polyacrylamide gel electrophoresis.DNA SynthesisDNA synthesis was determined by [3H]thymidine incorporation as described (6Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Crossref PubMed Scopus (560) Google Scholar).RESULTSSphingosine 1-Phosphate Activates MAP Kinase in Swiss 3T3 CellsTo determine whether exogenous sphingosine 1-phosphate can stimulate MAP kinase activity in Swiss 3T3 fibroblasts, quiescent Swiss 3T3 fibroblasts were treated with a mitogenic concentration of sphingosine 1-phosphate (5 µm) or PDGF (65 ng/ml) for 1–15 min, and p42mapk activities were assayed by a specific immune complex assay (25Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (817) Google Scholar). MAP kinase was rapidly activated in cells treated with sphingosine 1-phosphate or PDGF. BSA vehicle added alone had no discernible effect on MAP kinase (Fig. 1).Activation of MAP kinase by sphingosine 1-phosphate was time and concentration dependent. Sphingosine 1-phosphate rapidly stimulated MAP kinase activity, reaching a maximal level within 2.5 min and declining thereafter (Fig. LA). The response to PDGF was similar, but MAP kinase activity remained elevated for at least 15 min (Fig. 1A). Half-maximal activation of MAP kinase was observed at approximately 1 µm sphingosine 1-phosphate (Fig. LB). This concentration dependence correlates closely with the concentration dependence for induction of DNA synthesis as measured by [3H]thymidine uptake (Fig. 1B).Signaling Pathway for Activation of MAP Kinase by Sphingosine 1-PhosphateMAP kinases are activated by phosphorylation on tyrosine and threonine residues by MAP kinase kinases (MKKs or MEKs), which in turn are regulated by one of several potential upstream serine/threonine kinases including Raf-1 (18Dent P. Haser W. Haystead T.A. Vincent L.A. Robert T.M. Sturgill T.W. Science. 1992; 257: 1404-1407Crossref PubMed Scopus (496) Google Scholar, 19Kyriakis J.M. App H. Zhang X.F. Banerjee P. Brautigan D.L. Rapp U.R. Avruch J. Nature. 1992; 358: 417-421Crossref PubMed Scopus (966) Google Scholar) and MEKK (20Lange-Carter C.A. Pieiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (869) Google Scholar). To investigate the pathway(s) by which sphingosine 1-phosphate activates MAP kinase in Swiss 3T3 fibroblasts, we measured MKK and Raf-1 activities in cells treated with sphingosine 1-phosphate. Both sphingosine 1-phosphate and PDGF significantly stimulated MKK activity in 3T3 fibroblasts (Fig. 2). Similarly, sphingosine 1-phosphate and PDGF activated Raf-1 kinase activity in 3T3 fibroblasts approximately 2- and 3-fold, respectively (Fig. 2). Thus, Raf-1 appears to be an important MKK activator for the response to sphingosine 1-phosphate. Preliminary experiments have not revealed any direct effects of sphingosine 1-phosphate on Raf, MKK, or MAP kinase studied in vitro (data not shown), implying action(s) upstream of Raf-1.FIG. 2Activation of MEK and Raf-1 by sphingosine 1-phosphate and PDGF. Quiescent Swiss 3T3 fibroblasts were treated with 7.5 µm sphingosine 1-phosphate or 65 ng/ml PDGF and MKK (A), or Raf-1 kinase activities (B and C) were determined. A, phosphorylation of KR substrate incubated with buffer alone (lane 1) (autophosphorylation control) or with lysate protein from cells treated with BSA vehicle (lane 2), 7.5 sphingosine 1-phosphate for 1 min (lane 3), 7.5 µm sphingosine 1-phosphate for 2 min (lane 4), or 65 ng/ml PDGF for 3 min (lane 5). Arrow, KR band. B, Raf-1 activity from cells treated (as indicated) with BSA vehicle, sphingosine 1-phosphate (7.5 µm) for 1 min (SPP-1′) or 2 min (SPP-2′), or PDGF (65 ng/ml, 3 min) and assayed by activation of wild type MKK1 enzymatically coupled to phosphorylation of KR. Phosphorylation of KR was incubated with MKK1 alone (lane 1). Lanes 2, 5, 8, and 11, assays with immune complexes in the absence of MKK1; lanes 3. 6, 9. and 12. assays with immune complexes plus MKK1; lanes 4, 7, 10, 13, immune complexes isolated in the presence of peptide antigen and assayed in the presence of MKK1. C, quantitation of raf-1 activities (B) by analysis with a Phosphorlmager (Molecular Dynamics). The signal of the KR band in lane 1, representing the basal activity of MKK1, was subtracted.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Both MKK1 and MKK2 Are Activated by Sphingosine 1-Phos-phate and Growth FactorsTwo MAP kinase kinases, MKK1 and MKK2, have been identified by molecular cloning, which share 81% identity (15Crews C.M. Alessandrini A. Erikson R.L. Science. 1992; 258: 478-480Crossref PubMed Scopus (736) Google Scholar, 16Wu J. Harrison J.K. Vincent L.A. Haystead C. Haystead T. Michel H. Hunt D. Lynch K.R. Sturgill T.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 173-177Crossref PubMed Scopus (117) Google Scholar, 17Wu J. Harrison J.K. Dent P. Lynch K.R. Weber M.J. Sturgill T.W. Mol. Cell. Biol. 1993; 13: 4539-4548Crossref PubMed Scopus (122) Google Scholar). To determine whether one or both MKKs are stimulated by sphingosine 1-phosphate in comparison with PDGF and EGF, specific antibodies 2M. Weber, unpublished data. to MKK1 and MKK2 were used to assay specifically each enzyme in immune complexes by phosphorylation of added KR substrate. MKK1 activity was found to be approximately six times higher than that of MKK2 in quiescent Swiss 3T3 cells by Phosphorlmager analysis (data not shown). Both MKK1 and MKK2 were markedly activated by sphingosine 1-phosphate, PDGF, and EGF (Fig. 3). With each stimulus, MKK1 activity was about 6-fold greater than MKK2 activity.FIG. 3Both MKK1 and MKK2 are activated by sphingosine 1-phosphate and growth factors. MKK1 and MKK2 activities in Swiss 3T3 cells were determined by phosphorylating KR after inimunoprecipitation with anti-MKK1-(lanes 1, 3, 5, 7) or anti-MKK2 (lanes 2, 4, 6, 8)-specific antibodies. Cells were treated with BSA (lanes 1 and 2), 10 µm sphingosine 1-phosphate for 2 min (lanes 3 and 4), 65 ng/ml PDGF for 3 min (lanes 5 and 6), or 100 ng/ml EGF for 3 min (lanes 7 and 8). Lane 0, lysis buffer control. Arrow, KR band.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Exogenous Sphingosine 1-Phosphate-induced MAP Kinase Activation Is Sensitive to Pertussis Toxin InhibitionThe rapid action of sphingosine 1-phosphate is consistent with stimulation of a plasma membrane receptor but could also result from rapid uptake (4Spiegel S. Olivera A. Carlson R.O. Adv. Lipid Res. 1993; 25: 105-129PubMed Google Scholar) and action at intracellular target(s). LPA is a mitogenic phospholipid structurally similar to sphingosine 1-phosphate (26Durieux M.E. Lynch K.R. Trends Pharmacol. Sci. 1993; 14: 249-254Abstract Full Text PDF PubMed Scopus (108) Google Scholar). LPA is thought to activate MAP kinase in Rat1 cells via a putative Grcoupled receptor inferred primarily from inhibition of the actions of LPA by pertussis toxin (27van Corven W.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). Pertussis toxin catalyzes the ADP-ribosylation of Gi/Go subunits of the heterotrimeric G proteins and has been used extensively to investigate the role of these G proteins in signal transduction. The modified serine residue is located in a domain that interacts with G protein-coupled receptors, uncoupling the activated G protein-coupled receptor to Gi/Go. To examine whether a pertussis toxin-sensitive G protein is involved in the pathway utilized by sphingosine 1-phosphate to stimulate MAP kinase, Swiss 3T3 cells were treated with 100 ng/ml pertussis toxin for 20 h before stimulation with sphingosine 1-phosphate, LPA, PDGF, or EGF. As shown in Fig. 4A, pertussis toxin inhibited the activation of MAP kinase by sphingosine 1-phosphate and lysophosphatidic acid. In contrast, pertussis toxin had no apparent effect on the PDGF- or EGF-stimulated MAP kinase activation.FIG. 4Activation of MAP kinase by exogenous sphingosine 1-phosphate is sensitive to pertussis toxin inhibition. A, MAP kinase activities in Swiss 3T3 cells treated without or with 100 ng/ml pertussis toxin for 20 h and stimulated with BSA carrier (“None”), 5 µm sphingosine 1-phosphate for 2.5 min (SPP), 20 µm lysophosphatidic acid for 3 min (LPA), 65 ng/ml PDGF for 5 min, or 100 ng/ml EGF for 5 min. B, MAP kinase activities in Swiss 3T3 cells pretreated with indicated concentrations of pertussis toxin for 20 h and stimulated with 5 µm sphingosine 1-phosphate for 2.5 min or 65 ng/ml PDGF for 5 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT)About 20% of the MAP kinase activity induced by sphingosine 1-phosphate appeared to be insensitive to inhibition by pertussis toxin (Fig. 4A). To ensure that the pertussis toxin effect was a maximal one, its concentration dependence for inhibition of stimulation of MAP kinase by sphingosine 1-phosphate was studied (Fig. 4B). 70% inhibition of the sphingosine 1-phosphate-stimulated MAP kinase activation was observed at 10 ng/ml pertussis toxin. Higher concentrations (up to 400 ng/ml) of pertussis toxin had little effect in further increasing the extent of inhibition; these higher concentrations still had no significant effect on the response to PDGF. Thus, about 20% of the sphingosine 1-phosphate-stimulated MAP kinase activity is insensitive to pertussis toxin, an activity still representing a significant activation of MAP kinase over the basal activity. Therefore, sphingosine 1-phosphate can activate the MAP kinase pathway by both pertussis toxin-sensitive and -insensitive pathways.DISCUSSIONOur results showed that sphingosine 1-phosphate added to Swiss 3T3 cells rapidly activates the protein kinase cascade sequentially involving Raf-1, MAP kinase kinase, and MAP kinase. The ability of sphingosine 1-phosphate to stimulate MAP kinase activity has important implications for its mitogenic action. A requirement for MAP kinase for cell cycle progression in fibroblasts is supported by several lines of evidence. Expression of MAP kinase proteins rendered kinase-defective, by mutation of either the essential lysine or the sites of activating phosphorylation, blocks cell growth of Chinese hamster fibroblasts (28Pages G. Lenormand P. L'Allemain G. Chambard J.C. Meleche S. Pouyssegur J. Proc. Natl. Acad. Sci. V. S. A. 1993; 90: 8319-8323Crossref PubMed Scopus (923) Google Scholar), as does expression of activation site mutants of its specific activator, MKK, in NIH 3T3 cells (29Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar). Similarly, expression of kinase-defective MAP kinase inhibits the mitogenic effect of small t antigen in CV-1 cells (30Sontag E. Fedorov S. Kamibayashi C. Robbins D. Cobb M. Mumby M. Cell. 1993; 75: 887-897Abstract Full Text PDF PubMed Scopus (459) Google Scholar). Secondly, apparently specific pharmacologic MKK inhibitors have recently been identified that block the mitogenic but not the metabolic effects of insulin. 3A. Saltiel, personal communication. Finally, expression of MKP1, a highly specific MAP kinase phosphatase, blocks DNA synthesis induced by v-Ras (31Sun H. Tonks N.K. Barsagi D. Science. 1994; 266: 285-288Crossref PubMed Scopus (206) Google Scholar). Activation of MAP kinase by various mitogens in fibroblasts appears to be sufficient for stimulating cell growth of fibroblasts since expression of activated mutants of MKK causes proliferation of NIH 3T3 cells (29Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar, 32Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1254) Google Scholar).Recently, sphingosine 1-phosphate was shown to stimulate DNA binding of activator protein 1 (AP-1) transcription factor complexes (10Su Y. Rosenthal D. Smulson M. Spiegel S. J. Biol. Chem. 1994; 269: 16512-16517Abstract Full Text PDF PubMed Google Scholar) that modulate expression of genes involved in growth regulation and neoplastic transformation. AP-1 complexes are composed of either homodimers of c-Jun or heterodimers of c-Jun and c-Fos, both of which are regulated at transcription and by phosphorylation (33Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3248) Google Scholar, 34Treisman R. Curr. Opin. Genet. Dev. 1994; 4: 96-10135Crossref PubMed Scopus (618) Google Scholar). MAP kinase phosphorylates ternary complex factor(s), which, in complex with serum response factor, enhance Fos transcription (35Hipskind R.A. Baccarini M. Nordheim A. Mol. Cell. Biol. 1994; 14: 6219-6231Crossref PubMed Scopus (137) Google Scholar). Thus, the increase in AP-1 activity by sphingosine 1-phosphate previously observed may be explained by the data herein that establish sphingosine 1-phosphate as a potent activator of the MAP kinase pathway.The concentration dependence for activation of MAP kinase by sphingosine 1-phosphate correlated precisely with that for stimulation of DNA synthesis. Furthermore, sphingosine 1-phosphate-stimulated MAP kinase activation was largely sensitive to pertussis toxin inhibition. Additional data obtained 4K. A. Goodmote, M. E. Mattie, A. Berger, and S. Spiegel, manuscript in preparation. has demonstrated that sphingosine 1-phosphate-stimulated DNA synthesis is also sensitive to pertussis toxin inhibition. Thus, all of the available data suggest that the MAP kinase cascade mediates the mitogenic effect of sphingosine 1-phosphate.It was previously thought that sphingosine 1-phosphate was taken up by cells to exert all of its actions. Several pieces of evidence lend support to this notion. Sphingosine 1-phosphate can be formed intracellularly in response to sphingosine, PDGF, and serum, and inhibition of sphingosine 1-phosphate formation with a sphingosine kinase inhibitor inhibited cellular proliferation induced by these mitogens (5Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (810) Google Scholar). Moreover, the amount of sphingosine 1-phosphate formed intracellularly in response to these mitogens was nearly the same as that taken up by the cells after treatment with mitogenic concentrations of sphingosine 1-phosphate. However, data presented in this study demonstrated that a large proportion of the sphingosine 1-phosphate-stimulated MAP kinase activity was sensitive to pertussis toxin inhibition. This implies that the primary signaling mechanism of exogenous sphingosine 1-phosphate for activation of MAP kinase involves one or more pertussis toxin-sensitive G proteins, members of the Gi/Go families. This activation mechanism is similar in this regard to that demonstrated for LPA. Exogenous sphingosine 1-phosphate might activate a receptor coupled to Gi/Go or alternatively activate Gi/Go proteins in a receptor-independent fashion. Further studies of the cellular target(s) for sphingosine 1-phosphate are clearly warranted.Differences in physiologic responses to sphingosine 1-phosphate and LPA in Swiss 3T3 cells make it unlikely that these two agonists are simply redundant for the same receptor or G protein target. First, sphingosine 1-phosphate, in contrast to LPA, can release calcium from internal sources independent of an increase in intracellular concentrations of inositol triphosphate (7Mattie M. Brooker G. Spiegel S. J. Biol. Chem. 1994; 269: 3181-3188Abstract Full Text PDF PubMed Google Scholar). Although sphingosine 1-phosphate increased inositol triphosphate, complete inhibition of formation of inositol phosphates by a brief treatment with 12-O-tetradecanoylphorbol 13-acetate did not inhibit sphingosine 1-phosphate-mediated calcium responses, indicating that formation of inositol 1,4,5-trisphosphate is not required for release of calcium by sphingosine 1-phosphate. In contrast and in agreement with previous studies (36van Corven E.J. Groenink A. Jalink K. Eichholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (677) Google Scholar), 12-O-tetradecanoylphorbol 13-acetate pretreatment of Swiss 3T3 cells suppressed not only inositol 1,4,5-trisphosphate formation but also calcium responses elicited by LPA (7Mattie M. Brooker G. Spiegel S. J. Biol. Chem. 1994; 269: 3181-3188Abstract Full Text PDF PubMed Google Scholar). Second, sphingosine 1-phosphate had no significant effect on the release of arachidonic acid in Swiss 3T3 fibroblasts cells (7Mattie M. Brooker G. Spiegel S. J. Biol. Chem. 1994; 269: 3181-3188Abstract Full Text PDF PubMed Google Scholar), whereas LPA stimulated arachidonic acid release in these cells (7Mattie M." @default.
• W1996532068 created "2016-06-24" @default.
• W1996532068 creator A5014072958 @default.
• W1996532068 creator A5014705936 @default.
• W1996532068 creator A5037738083 @default.
• W1996532068 date "1995-05-01" @default.
• W1996532068 modified "2023-09-26" @default.
• W1996532068 title "Sphingosine 1-Phosphate Rapidly Activates the Mitogen-activated Protein Kinase Pathway by a G Protein-dependent Mechanism" @default.
• W1996532068 cites W1493153573 @default.
• W1996532068 cites W1501369263 @default.
• W1996532068 cites W1501693497 @default.
• W1996532068 cites W1520730446 @default.
• W1996532068 cites W1543411735 @default.
• W1996532068 cites W1555579413 @default.
• W1996532068 cites W1566441785 @default.
• W1996532068 cites W1573821584 @default.
• W1996532068 cites W1583840873 @default.
• W1996532068 cites W1601124709 @default.
• W1996532068 cites W1607687470 @default.
• W1996532068 cites W1613312511 @default.
• W1996532068 cites W1935609469 @default.
• W1996532068 cites W1963852241 @default.
• W1996532068 cites W1971863225 @default.
• W1996532068 cites W1983475354 @default.
• W1996532068 cites W1989480480 @default.
• W1996532068 cites W1992494265 @default.
• W1996532068 cites W1993657069 @default.
• W1996532068 cites W1995560813 @default.
• W1996532068 cites W1996068610 @default.
• W1996532068 cites W1996192399 @default.
• W1996532068 cites W2001521752 @default.
• W1996532068 cites W2005900169 @default.
• W1996532068 cites W2007476270 @default.
• W1996532068 cites W2009338295 @default.
• W1996532068 cites W2009993180 @default.
• W1996532068 cites W2017729786 @default.
• W1996532068 cites W2033421786 @default.
• W1996532068 cites W2045876058 @default.
• W1996532068 cites W2046216255 @default.
• W1996532068 cites W2050944466 @default.
• W1996532068 cites W2051538397 @default.
• W1996532068 cites W2057126981 @default.
• W1996532068 cites W2061574494 @default.
• W1996532068 cites W2064129448 @default.
• W1996532068 cites W2082190322 @default.
• W1996532068 cites W2091536410 @default.
• W1996532068 cites W2116716030 @default.
• W1996532068 cites W2149439788 @default.
• W1996532068 cites W2244032147 @default.
• W1996532068 cites W2330457575 @default.
• W1996532068 cites W75708282 @default.
• W1996532068 doi "https://doi.org/10.1074/jbc.270.19.11484" @default.
• W1996532068 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7744787" @default.
• W1996532068 hasPublicationYear "1995" @default.
• W1996532068 type Work @default.
• W1996532068 sameAs 1996532068 @default.
• W1996532068 citedByCount "174" @default.
• W1996532068 countsByYear W19965320682012 @default.
• W1996532068 countsByYear W19965320682015 @default.
• W1996532068 countsByYear W19965320682017 @default.
• W1996532068 countsByYear W19965320682018 @default.
• W1996532068 countsByYear W19965320682019 @default.
• W1996532068 countsByYear W19965320682021 @default.
• W1996532068 crossrefType "journal-article" @default.
• W1996532068 hasAuthorship W1996532068A5014072958 @default.
• W1996532068 hasAuthorship W1996532068A5014705936 @default.
• W1996532068 hasAuthorship W1996532068A5037738083 @default.
• W1996532068 hasConcept C111472728 @default.
• W1996532068 hasConcept C124160383 @default.
• W1996532068 hasConcept C132149769 @default.
• W1996532068 hasConcept C137361374 @default.
• W1996532068 hasConcept C138885662 @default.
• W1996532068 hasConcept C170493617 @default.
• W1996532068 hasConcept C184235292 @default.
• W1996532068 hasConcept C185592680 @default.
• W1996532068 hasConcept C2776596873 @default.
• W1996532068 hasConcept C2778703144 @default.
• W1996532068 hasConcept C55493867 @default.
• W1996532068 hasConcept C86803240 @default.
• W1996532068 hasConcept C89611455 @default.
• W1996532068 hasConcept C90934575 @default.
• W1996532068 hasConcept C95444343 @default.
• W1996532068 hasConcept C97029542 @default.
• W1996532068 hasConceptScore W1996532068C111472728 @default.
• W1996532068 hasConceptScore W1996532068C124160383 @default.
• W1996532068 hasConceptScore W1996532068C132149769 @default.
• W1996532068 hasConceptScore W1996532068C137361374 @default.
• W1996532068 hasConceptScore W1996532068C138885662 @default.
• W1996532068 hasConceptScore W1996532068C170493617 @default.
• W1996532068 hasConceptScore W1996532068C184235292 @default.
• W1996532068 hasConceptScore W1996532068C185592680 @default.
• W1996532068 hasConceptScore W1996532068C2776596873 @default.
• W1996532068 hasConceptScore W1996532068C2778703144 @default.
• W1996532068 hasConceptScore W1996532068C55493867 @default.
• W1996532068 hasConceptScore W1996532068C86803240 @default.
• W1996532068 hasConceptScore W1996532068C89611455 @default.
• W1996532068 hasConceptScore W1996532068C90934575 @default.
• W1996532068 hasConceptScore W1996532068C95444343 @default.

• next