Glycosphingolipid-enriched Signaling Domain in Mouse Neuroblastoma Neuro2a Cells
1999; Elsevier BV; Volume: 274; Issue: 30 Linguagem: Inglês
10.1074/jbc.274.30.20916
ISSN1083-351X
AutoresAlessandro Prinetti, Kazuhisa Iwabuchi, Sen‐itiroh Hakomori,
Tópico(s)Signaling Pathways in Disease
ResumoDifferentiation and neuritogenesis of mouse neuroblastoma Neuro2a cells are induced by exogenous ganglioside but are not induced by nerve growth factor because its receptor is absent in these cells. In view of the emerging concept of the "glycosphingolipid-enriched domain" (GEM), we studied the mechanism of the ganglioside effect, focusing on the structure and function of such a domain. GEM in Neuro2a cells, separated as a low density membrane fraction, contains essentially all glycosphingolipids and sphingomyelin, together with five signal transducer molecules (c-Src, Lyn, Csk, Rho A, Ha-Ras). 3H-Labeled Il3NeuAc-LacCer (GM3), Gb4Cer (globoside), and Il3NeuAc-Gg4Cer (GM1) added exogenously to cells were incorporated and concentrated in the low density GEM fraction. In contrast, more than 50% of glycerophospholipids and 30% of cholesterol were found in the high density fraction.3H-Labeled phosphatidylcholine added exogenously to cells was incorporated exclusively in the high density fraction. c-Src, the predominant signal transducer in the microdomain, was coimmunoprecipitated with anti-GM3 antibody DH2 or with anti-Csk; reciprocally, Csk was coimmunoprecipitated with anti-c-Src, indicating a close association of GM3, c-Src, and Csk. Brief stimulation of an isolated GEM fraction by the exogenous addition of GM3, but not lactosylceramide, caused enhanced c-Src phosphorylation with a concomitant decrease of Csk level in GEM. A decreased Csk/c-Src ratio in GEM may cause activation of c-Src because Csk is a negative regulator of c-Src. The effect of exogenous GM3 on c-Src activity was also observed in intact Neuro2a cells. Activation of c-Src was followed by rapid and prolonged (60 min) enhancement of mitogen-activated protein kinase activity leading to neuritogenesis. Thus, the ganglioside induction of neuritogenesis in Neuro2a cells is mediated by GEM structure and function. Differentiation and neuritogenesis of mouse neuroblastoma Neuro2a cells are induced by exogenous ganglioside but are not induced by nerve growth factor because its receptor is absent in these cells. In view of the emerging concept of the "glycosphingolipid-enriched domain" (GEM), we studied the mechanism of the ganglioside effect, focusing on the structure and function of such a domain. GEM in Neuro2a cells, separated as a low density membrane fraction, contains essentially all glycosphingolipids and sphingomyelin, together with five signal transducer molecules (c-Src, Lyn, Csk, Rho A, Ha-Ras). 3H-Labeled Il3NeuAc-LacCer (GM3), Gb4Cer (globoside), and Il3NeuAc-Gg4Cer (GM1) added exogenously to cells were incorporated and concentrated in the low density GEM fraction. In contrast, more than 50% of glycerophospholipids and 30% of cholesterol were found in the high density fraction.3H-Labeled phosphatidylcholine added exogenously to cells was incorporated exclusively in the high density fraction. c-Src, the predominant signal transducer in the microdomain, was coimmunoprecipitated with anti-GM3 antibody DH2 or with anti-Csk; reciprocally, Csk was coimmunoprecipitated with anti-c-Src, indicating a close association of GM3, c-Src, and Csk. Brief stimulation of an isolated GEM fraction by the exogenous addition of GM3, but not lactosylceramide, caused enhanced c-Src phosphorylation with a concomitant decrease of Csk level in GEM. A decreased Csk/c-Src ratio in GEM may cause activation of c-Src because Csk is a negative regulator of c-Src. The effect of exogenous GM3 on c-Src activity was also observed in intact Neuro2a cells. Activation of c-Src was followed by rapid and prolonged (60 min) enhancement of mitogen-activated protein kinase activity leading to neuritogenesis. Thus, the ganglioside induction of neuritogenesis in Neuro2a cells is mediated by GEM structure and function. Glycosphingolipids (GSLs), 1The abbreviations used are: GSL(s), glycosphingolipid(s); NGF, nerve growth factor; GSD, glycosphingolipid signaling domain (this indicates a functional entity); MAPK, mitogen-activated protein kinase; LacCer, lactosylceramide; PC, phosphatidylcholine; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GEM, glycosphingolipid-enriched microdomain (this indicates a physical or chemical entity); MES, 4-morpholineethanesulfonic acid; SM, sphingomyelin; HPTLC, high performance thin layer chromatography; PAGE, polyacrylamide gel electrophoresis1The abbreviations used are: GSL(s), glycosphingolipid(s); NGF, nerve growth factor; GSD, glycosphingolipid signaling domain (this indicates a functional entity); MAPK, mitogen-activated protein kinase; LacCer, lactosylceramide; PC, phosphatidylcholine; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GEM, glycosphingolipid-enriched microdomain (this indicates a physical or chemical entity); MES, 4-morpholineethanesulfonic acid; SM, sphingomyelin; HPTLC, high performance thin layer chromatography; PAGE, polyacrylamide gel electrophoresis particularly gangliosides, have been implicated as mediators of cell adhesion and modulators of signal transduction (1Hakomori S. J. Biol. Chem. 1990; 265: 18713-18716Abstract Full Text PDF PubMed Google Scholar). There has been considerable interest in the functional significance of GSLs in neuronal cells and tissues. Ganglioside patterns in the nervous system display dramatic changes during development, neurite outgrowth, synaptogenesis (2Yavin E. Yavin Z. Dev. Neurosci. 1979; 2: 25-37Crossref Scopus (65) Google Scholar, 3Dreyfus H. Louis J.C. Harth S. Mandel P. Neuroscience. 1980; 5: 1647-1655Crossref PubMed Scopus (120) Google Scholar), and malignant transformation. Sphingolipid biosynthesis is necessary for neuritogenesis in primary cultures of hippocampal neurons (4Harel R. Futerman A.H. J. Biol. Chem. 1993; 268: 14476-14481Abstract Full Text PDF PubMed Google Scholar), and induced expression of GD3 2Glycosphingolipids are abbreviated according to the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature ((1977) Lipids 12, 455–463); however, the suffix -OseCer is omitted. Gangliosides are abbreviated according to Svennerholm ((1964) J. Lipid Res. 5,145–155). 2Glycosphingolipids are abbreviated according to the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature ((1977) Lipids 12, 455–463); however, the suffix -OseCer is omitted. Gangliosides are abbreviated according to Svennerholm ((1964) J. Lipid Res. 5,145–155).synthetase in Neuro2a neuroblastoma cells is followed by neurite outgrowth (5Kojima N. Kurosawa N. Nishi T. Hanai N. Tsuji S. J. Biol. Chem. 1994; 269: 30451-30456Abstract Full Text PDF PubMed Google Scholar). The discovery that the exogenous addition of gangliosides prevents neurodegeneration in vivoand induces neuritogenesis and maintains neurotrophic effects in several cell systems of neural origin (6Tettamanti G. Riboni L. Prog. Brain Res. 1994; 101: 77-100Crossref PubMed Scopus (78) Google Scholar), including neuroblastoma (7Byrne M.C. Ledeen R.W. Roisen F.J. Yorke G. Sclafani J.R. J. Neurochem. 1983; 41: 1214-1222Crossref PubMed Scopus (133) Google Scholar,8Facci L. Leon A. Toffano G. Sonnino S. Ghidoni R. Tettamanti G. J. Neurochem. 1984; 42: 299-305Crossref PubMed Scopus (180) Google Scholar), led to the hypothesis that GSLs and gangliosides play essential roles in the maintenance of the structure and function of neuronal cells. Numerous studies along this line followed (for review, see Refs.9Ledeen R.W. Wu G. Vaswani K.K. Cannella M.S. Horrocks L.A. Neff N.H. Yates A.J. Hadjiconstantinou M. Trophic Factors and the Nervous System. Raven Press, New York1990: 17-34Google Scholar and 10Rösner H. Ledeen R.W. Hakomori S. Yates A.J. Schneider J.S. Yu R.K. Sphingolipids as Signaling Modulators in the Nervous System. New York Academy of Sciences, New York1998: 200-214Google Scholar). A Neuro2a cell model, in contrast to other neuronal cell lines, is unusual in that neuritogenic differentiation is induced readily by various gangliosides (7Byrne M.C. Ledeen R.W. Roisen F.J. Yorke G. Sclafani J.R. J. Neurochem. 1983; 41: 1214-1222Crossref PubMed Scopus (133) Google Scholar), although cells are not susceptible to stimulation by nerve growth factor (NGF) (11Matta S.G. Yorke G. Roisen F.J. Dev. Brain Res. 1986; 27: 243-252Crossref Scopus (56) Google Scholar) and do not contain NGF receptor. The exact mechanism by which gangliosides trigger the molecular events leading to neuronal differentiation remains unexplored.Specific association of c-Src with synaptic vesicles in PC12 cells (12Linstedt A.D. Vetter M.L. Bishop J.M. Kelly R.B. J. Cell Biol. 1992; 117: 1077-1084Crossref PubMed Scopus (65) Google Scholar) and early activation of c-Src kinase in neuroblastoma cells in response to differentiation induction by phorbol esters (13Bjelfman C. Meyerson G. Cartwright C.A. Mellström K. Hammerling U. Påhlman S. Mol. Cell. Biol. 1990; 10: 361-370Crossref PubMed Scopus (77) Google Scholar) or by anti-GM3 antibody (14Chakraborty M. Anderson G.M. Chakraborty A. Chatterjee D. Brain Res. 1993; 625: 197-202Crossref PubMed Scopus (15) Google Scholar) indicate an important role of c-Src or Src family kinases in neural cell differentiation and signal transduction.Recent studies have revealed a novel organization of GSLs and gangliosides in cell membrane, i.e. the majority of them are clustered and associated closely with single or multiple signal transducer molecules. Examples are GM3 organized with c-Src, Rho, FAK, and Ras in B16 melanoma cells (15Yamamura S. Handa K. Hakomori S. Biochem. Biophys. Res. Commun. 1997; 236: 218-222Crossref PubMed Scopus (103) Google Scholar, 16Iwabuchi K. Yamamura S. Prinetti A. Handa K. Hakomori S. J. Biol. Chem. 1998; 273: 9130-9138Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) and GD3 associated with Lyn in rat brain (17Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Such structural units consisting of GM3, c-Src, and Rho can be separated from caveolin-containing units (caveolae) (18Iwabuchi K. Handa K. Hakomori S. J. Biol. Chem. 1998; 273: 33766-33773Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), are involved in signal transduction in response to GSL-mediated stimulation, and are therefore termed the "GSL signaling domain" (GSD) (19Hakomori S. Handa K. Iwabuchi K. Yamamura S. Prinetti A. Glycobiology. 1998; 8: xi-xviiiCrossref PubMed Scopus (186) Google Scholar).Considering the fact that differentiation and neuritogenesis of Neuro2a cells are inducible by gangliosides but not by NGF, we studied the composition and functional organization of GEM at the surface of these cells. Special focus was on the effect of exogenous gangliosides in inducing c-Src activation in GEM, leading to downstream mitogen-activated protein kinase (MAPK) activation resulting in neuritogenesis.EXPERIMENTAL PROCEDURESReagentsGM3 was prepared from dog erythrocytes (20Klenk E. Heuer K. Z. Verdauungs u Stoffwechselkrankheiten. 1960; 20: 180-183PubMed Google Scholar). Gg3 was prepared from guinea pig erythrocytes (21Seyama Y. Yamakawa T. J. Biochem. (Tokyo). 1974; 75: 837-842Crossref PubMed Scopus (56) Google Scholar). GM1 from bovine brain was from Fidia Research Laboratories (Italy). Gb4 and lactosylceramide (LacCer) were from human erythrocytes (22Hakomori S. Kanfer J.N. Hakomori S. Sphingolipid Biochemistry. Plenum Press, New York1983: 1-165Crossref Google Scholar). GM1 and Gb4 were radiolabeled at the terminal sugar residue using the galactose oxidase-[3H4]NaB procedure (23Suzuki Y. Suzuki K. J. Lipid Res. 1972; 13: 687-690Abstract Full Text PDF PubMed Google Scholar). GM3 radiolabeled at C-3 of the long chain base was kindly provided by Prof. S. Sonnino (University of Milan, Italy) (24Sonnino S. Nicolini M. Chigorno V. Glycobiology. 1996; 6: 479-487Crossref PubMed Scopus (44) Google Scholar).l-1-Stearoyl-2-arachidonyl [arachidonyl-5,6,8,9,11,12,14,15-3H]PC (specific activity 175 Ci/mmol) was from NEN Life Science Products.l-1-Stearoyl-2-arachidonyl PC and bovine brain PC were from Sigma. For the specific activity of 3H-labeled GSLs and PC applied for incorporation into cells, see the Fig. 5 legend. Specific anti-GM3 monoclonal antibody DH2 (IgG3) (25Dohi T. Nores G. Hakomori S. Cancer Res. 1988; 48: 5680-5685PubMed Google Scholar) and anti-Gg3 monoclonal antibody 2D4 (IgM) (26Young W.W.J. MacDonald E.M.S. Nowinski R.C. Hakomori S. J. Exp. Med. 1979; 150: 1008-1019Crossref PubMed Scopus (145) Google Scholar) were established as described previously. Specific polyclonal or monoclonal antibodies directed to caveolin, Lyn, Rho A, Ha-Ras, Csk, and other transducer molecules were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat anti-c-Src polyclonal IgG (N-16, Santa Cruz) and rabbit anti-c-Src polyclonal IgG (SRC2), which recognize different epitopes of c-Src protein, were used for combinations of immunoprecipitation and Western blotting experiments. Goat anti-ERK1 polyclonal IgG (Santa Cruz) was used in the MAPK activation assay. Lavendustin C was from Calbiochem. [γ-32P]ATP (3,000 Ci/mmol) and [3H4]NaB (204.1 mCi/mmol) were from NEN Life Science Products.Cell CultureMouse Neuro2a neuroblastoma cells (CCL-131, American Type Culture Collection, Manassas, VA) were cultured in DMEM supplemented with 10% FBS (HyClone, Logan, UT), 4 mml-glutamine, 1 mm pyruvic acid, 4.5 mg/mld-glucose, 100 units/ml potassium penicillin G, and 100 μg/ml streptomycin sulfate in a 5% CO2, 95% air humidified atmosphere.GEM PreparationMembrane fraction presumably corresponding to the GSL-enriched microdomain (GEM) was prepared from Neuro2a cells by ultracentrifugation on a discontinuous sucrose gradient after lysis and homogenization in the presence of 1% Triton X-100 (27Rodgers W. Rose J.K. J. Cell Biol. 1996; 135: 1515-1523Crossref PubMed Scopus (285) Google Scholar) or in hypertonic sodium carbonate medium (28Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (916) Google Scholar) by modification of original procedure as described below. After ultracentrifugation, 1-ml fractions were collected starting from the top of the tube. GEM was also prepared after stimulation of Neuro2a cells with different GSLs as described under "Effect of Gangliosides on c-Src Activation in Intact Cells."Detergent MethodCells were harvested in phosphate-buffered saline containing 0.4 mm Na3VO4, lysed, homogenized, and subjected to sucrose density gradient centrifugation to separate the low density light-scattering membranous fraction (16Iwabuchi K. Yamamura S. Prinetti A. Handa K. Hakomori S. J. Biol. Chem. 1998; 273: 9130-9138Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 27Rodgers W. Rose J.K. J. Cell Biol. 1996; 135: 1515-1523Crossref PubMed Scopus (285) Google Scholar). Briefly, 1–5 × 107 cells were suspended in 1 ml of 10 mm Tris buffer, pH 7.5, 150 mm NaCl, 5 mm EDTA, 1 mmNa3VO4, containing 1% Triton X-100, Dounce homogenized, and the "postnuclear fraction" was subjected to gradient ultracentrifugation (16Iwabuchi K. Yamamura S. Prinetti A. Handa K. Hakomori S. J. Biol. Chem. 1998; 273: 9130-9138Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar), i.e. the fraction was mixed with an equal volume of 85% sucrose (w/v) in the same buffer. The resulting diluent was placed at the bottom of a discontinuous sucrose concentration gradient (30–5%) in the same buffer. Samples were centrifuged for 17 h at 200,000 × g at 4 °C. A white light-scattering band under light illumination located between 5 and 30% sucrose interface was collected and used as the GEM fraction. The entire procedure was performed at 0–4 °C (in ice immersion). The protein content of each fraction was determined using a MicroBCA kit (Pierce Chemical Co.).Hypertonic Sodium Carbonate MethodCells were harvested in 500 mm sodium carbonate, pH 11.0 (2–4 × 107 cells/2 ml) and homogenized using a loose fitting Dounce homogenizer (20 strokes), a Polytron tissue grinder (three 10-s bursts), and a bath sonicator (three 20-s bursts). 1.5 ml of the cell homogenate thus obtained was mixed with an equal volume of 90% sucrose in 25 mm MES, pH 6.5, 150 mm NaCl and overlaid with a discontinuous sucrose gradient (30–5% in the same buffer containing 250 mm sodium carbonate). Samples were submitted to ultracentrifugation, and the light-scattering band just above the 5–30% sucrose interface was collected and designated as the GEM fraction as above. The protein content of each fraction was determined as above.Determination of Distribution Patterns of Glycosphingolipids, Sphingomyelin, Glycerophospholipids, and Cholesterol in Fractions Obtained from Sucrose Gradient CentrifugationGEM and other fractions obtained by sucrose gradient centrifugation as described above were analyzed to determine the lipid content. Each fraction was dialyzed against water to eliminate sucrose and then lyophilized. Residues were extracted with chloroform/methanol (2:1), and the lipid extracts were subjected to repeated Folch-Pi partition (29Folch-Pi J. Arsove S. Meath J.A. J. Biol. Chem. 1951; 191: 819-831Abstract Full Text PDF PubMed Google Scholar). The resulting aqueous phases were purified further using C18 Bond elut packed columns (1 ml, Analytichem International, Harbor, CA) (30Williams M.A. McCluer R.H. J. Neurochem. 1980; 35: 266-269Crossref PubMed Scopus (405) Google Scholar) and subjected to HPTLC. Gangliosides were visualized using orcinol-sulfuric acid staining. GM3 was detected by immunostaining using anti-GM3 monoclonal antibody DH2 and a Vectastain ABC kit (Vector, Burlingame, CA) using biotinylated goat anti-mouse IgG as secondary antibody and diaminobenzidine substrate for the final staining (31Mårtensson S. Brodin T. Carlström A.-S. Dahmen J. Frejd T. Gunnarsson A. Jansson U. Magnusson G. Lundblad A. Glycoconj. J. 1986; 3: 163-174Crossref Scopus (20) Google Scholar). The organic phases from the Folch-Pi partition were subjected to alkaline methanolysis (32Ledeen R.W. Yu R.K. Eng L.F. J. Neurochem. 1973; 21: 829-839Crossref PubMed Scopus (469) Google Scholar) to remove interfering glycerophospholipid, and the content of neutral GSL and SM was analyzed by HPTLC. Glycerophospholipids and cholesterol were separated directly from the lower phase of the Folch-Pi partition without alkaline methanolysis and were subjected to HPTLC. Neutral GSLs and gangliosides were separated by TLC with solvent chloroform/methanol/water 5:4:1 and visualized by spraying with 0.5% orcinol in 10% sulfuric acid. SM and glycerophospholipids were separated by TLC in solvent chloroform/methanol/acetone/acetic acid/water 10:2:4:2:1 and revealed with phosphomolybdate spray (33Vaskovsky V.E. Kostetsky E.Y. J. Lipid Res. 1968; 9: 396Abstract Full Text PDF PubMed Google Scholar). Cholesterol was separated by TLC in solvent hexane/diethyether/acetic acid 80:20:1 and visualized by spraying with 15% solution of concentrated sulfuric acid in 1-butanol. In all cases, the quantity of lipids and their ratio were determined by densitometry in comparison with a known quantity of standard lipid using the Scion Image program (Scion Corporation, Frederick, MD). For determination of 3H-labeled GSLs, TLC autoradiography was performed by exposure to Kodak BioMax MS film at −80 °C with Kodak TranScreen-LE intensifying screen.Distribution of Signal Transducer Molecules in Fractions Obtained from Sucrose Gradient CentrifugationFor analysis of distribution of transducer molecules, GEM and other fractions were subjected to SDS-PAGE followed by Western immunoblotting (34Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44708) Google Scholar) using commercially available specific antibodies as described previously (16Iwabuchi K. Yamamura S. Prinetti A. Handa K. Hakomori S. J. Biol. Chem. 1998; 273: 9130-9138Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). In some experiments, aliquots of GEM (containing ∼30 μg of protein) were diluted 10-fold in immunoprecipitation (IP) buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm NaF, 1 mm EDTA, 1 mm EGTA, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 75 milliunits/ml aprotinin, 1% Triton X-100) and immunoprecipitated by adding 1 μg/ml rabbit anti-Csk polyclonal IgG, 1 μg/ml rabbit anti-c-Src polyclonal IgG, or 1 μg/ml normal rabbit IgG (as negative control). Immunoprecipitates were recovered by adding protein G-Sepharose beads, washed with IP buffer, suspended with 100 μl of SDS-sample buffer, heated to 95 °C for 3 min, subjected to SDS-PAGE, and analyzed by Western blotting.Coimmunoprecipitation of GM3 and c-SrcNeuro2a cells were harvested in phosphate-buffered saline and lysed in lysis buffer (500 μg of protein/ml of buffer) containing 1% Triton X-100, 10 mm Tris-HCl, pH 7.5, 150 mmNaCl, 5 mm EDTA, 1 mmNa3VO4, and 75 milliunits/ml aprotinin and allowed to stand for 20 min. The cell suspension was Dounce homogenized, lysate was centrifuged for 5 min at 1,300 ×g, and 1 ml of supernatant was mixed with protein G-Sepharose beads (50 μl packed) and stirred by rotary shaker for 2 h at 4 °C to preclear nonspecific binding. After centrifugation (500 × g for 1 min), the supernatant was added to 20 μl of DH2 ascites or 20 μl of mouse myeloma SP2 ascites as negative control. The mixtures were placed overnight in a rotary stirrer at 4 °C, added to protein G-Sepharose beads (50 μl packed), and placed again in a rotary mixer for 2 h. Beads were washed three times with IP buffer, recovered by brief weak centrifugation (270 × g, 2 min), suspended in 100 μl of SDS-sample buffer, heated to 95 °C for 3 min, and centrifuged (1,000 × g, 2 min).Immunoprecipitated proteins were analyzed by two-dimensional SDS-PAGE, with the first run performed through 5–15% gradient gel under nonreducing conditions. The second run was performed through 8% gel under reducing conditions. Subsequently, proteins were transferred electrophoretically to polyvinylidene difluoride membranes and immunodetected as described previously (34Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44708) Google Scholar). The purpose of the two-dimensional SDS-PAGE procedure was to improve detection of c-Src, avoiding interference from the presence of mouse IgG (from DH2 antibody).Incorporation of Exogenous Gangliosides, GSLs, and PC in GEM and Other Membrane and Soluble Fractions from Neuro2a CellsPreconfluent Neuro2a cells cultured in 150-mm dishes were washed three times with serum-free DMEM and incubated in the same medium for 10 min or 1 h in the presence of 10 μm[3H-Sph]GM3, [3H-Gal]GM1, [3H-GalNAc]Gb4, or [arachidonyl-3H-]PC (10 ml, 0.25 μCi/ml, specific activity 0.025 Ci/mmol). For specific activity of3H-labeled GSLs and PC applied for incorporation into cells, we followed the protocol described previously (35Riboni L. Prinetti A. Pitto M. Tettamanti G. Neurochem. Res. 1990; 15: 1175-1183Crossref PubMed Scopus (64) Google Scholar). Briefly, [3H]GM3 solution in ethanol (76 μl, containing 73,000 dpm/μl; specific activity 2 Ci/mmol; equivalent to 2.5 μCi and 1.25 nmol) was mixed with 9.87 μl of 10 mm solution of cold GM3 solution in ethanol. Lipid solution was concentrated under a nitrogen stream to near dryness (∼10 μl), and 10 ml of DMEM was added, sonicated, and allowed to stand at 37 °C for 2 h. DMEM solution of 3H-labeled GM1 or Gb4 was prepared in the same way, with approximately the same specific activity. For preparation of DMEM solution of 3H-labeled PC, 25 μl of [3H]PC solution (0.1 mCi/ml; specific activity 175 Ci/mmol) was mixed with 200 μl of 0.5 mm ethanol solution of cold PC prepared from bovine brain and concentrated under a nitrogen stream to near dryness, and 10 ml of DMEM was added, sonicated, and allowed to stand at 37 °C as above. Thus, the DMEM solution of3H-labeled lipids (10 μm) with a specific activity 0.025 Ci/mmol, corresponding to radioactivity of 0.25 μCi/ml, was obtained. After incubation, cells were washed three times with 10% FBS and DMEM and incubated for 30 min in the same medium to remove the loosely bound portion of lipid (38Cannella M.S. Roisen F.J. Ogawa T. Sugimoto M. Ledeen R.W. Dev. Brain Res. 1988; 39: 137-143Crossref Scopus (44) Google Scholar). Cells were rinsed twice with ice-cold phosphate-buffered saline and harvested in the same buffer. Two dishes were pooled for each experimental point and subjected to GEM preparation by the detergent method described above. Radioactivity associated with postnuclear supernatant and sucrose gradient fractions was determined by liquid scintillation with a Beckmann LS6000IC counter.Effect of GM3 on c-Src Activation and Src/Csk Interaction in Isolated GEMGEM was diluted 10× with kinase buffer (30 mmHEPES, pH 7.5, 10 mm MgCl2, 2 mmMnCl2 1 mm CaCl2) (protein content 7–10 μg/ml), and 5-ml aliquots of diluted GEM suspension were used for stimulation by GM3, by the following procedure. The stock solution of GM3 was prepared as a 10 mm solution in absolute ethanol. 5 μl of this solution was added to 5 ml of GEM suspension (final concentration of ethanol in GEM suspension was 0.1%). As a control, GEM suspension was added to an ethanol solution of LacCer having the same concentration as GM3 or ethanol alone (final ethanol concentration in GEM suspension was 0.1%). In some experiments, lavendustin C (36O'Dell T.J. Kandel E.R. Grant S.G. Nature. 1991; 353: 558-560Crossref PubMed Scopus (469) Google Scholar) was added to GEM suspension with GM3. A stock solution of lavendustin C (50 mm) in dimethyl sulfoxide was prepared, and 5, 10, or 15 μl of the stock solution was added to 5 ml of GEM suspension to obtain, respectively, 50, 100, or 150 μm final concentration of lavendustin C. Dimethyl sulfoxide at these concentrations had no effect on c-Src activity. c-Src activity was determined by adding 50 μCi of [γ-32P]ATP solution (370 GBq/mmol, NEN Life Science Products) in 50 μl of kinase buffer and allowed to proceed at 37 °C for 5 min. After incubation, reactions were stopped by placing on ice and adding 5 ml of ice-cold stop buffer (30 mmHEPES, pH 7.5, 300 mm NaCl, 10 mm EDTA, 2 mm Na3VO4, 2% Triton X-100, 2 mm phenylmethylsulfonyl fluoride). Samples were precipitated with 10% trichloroacetic acid. The precipitates were washed twice with acetone and dissolved in 1.0 ml of IP buffer. Samples were added to 20 μl of protein G-Sepharose (Amersham Pharmacia Biotech) and placed on a rotary stirrer for 2 h at 4 °C to preclear nonspecific binding. After centrifugation for 5 min at 270 × g, the supernatants were collected and mixed with 1 μg/ml goat anti-Src IgG. After incubation overnight at 4 °C, 20 μl of protein G-Sepharose was added, and samples were incubated at 4 °C for 2 h. Beads were washed five times with IP buffer containing 0.5 m NaCl and boiled with SDS-sample buffer containing 10% β-mercaptoethanol. The samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. The electroblotted membranes were subjected to autoradiography. Separated proteins were also evaluated by Western blotting. In some experiments, incubation of GEM with GM3 was carried out in the absence of radioactive ATP for different times. After adding stop buffer, samples were immunoprecipitated with anti-c-Src or anti-Csk antibodies as described above, and immunoprecipitates were analyzed by SDS-PAGE followed by Western blotting.Effect of Gangliosides on c-Src Activation in Intact CellsPreconfluent Neuro2a cells cultured in 150-mm dishes were washed extensively with serum-free DMEM and incubated in the presence of 10 μm GM3, GM1, or LacCer (from 10 mm stock solution in ethanol) in serum-free DMEM for 5, 15, or 30 min. Cells were harvested, and GEM was prepared from stimulated cells using the detergent method described above. Aliquots of GEM from different samples containing roughly the same amount of protein (typically 30 μg) were diluted to 500 μl with water and added to the same volume of 2 × IP buffer (20 mm Tris-HCl, pH 7.4, 300 mm NaCl, 2 mm EDTA, 2 mm EGTA, 2 mm Na3VO4, 2 mmphenylmethylsulfonyl fluoride, 0.2% Triton X-100). Mixtures were precleared with protein G-Sepharose. Supernatants were recovered by centrifugation, added to 1 μg/ml goat anti-c-Src IgG, and incubated at 4 °C overnight with rotation. Immunoprecipitates were recovered by centrifugation after adding protein G-Sepharose beads, and the immunocomplex kinase assay was performed as described above.Effect of Lavendustin C on Ganglioside-dependent Neuritogenesis in Neuro2a CellsNeuro2a cells (5,000 cells/cm2) were incubated in 2% FBS and DMEM in the absence or presence of 100 μmlavendustin C. After 1 h, cells were incubated further in the presence of 10 μm GM3 or GM1 in the same medium with or without lavendustin C, and the degree of morphological differentiation was assessed by phase-contrast microscopy. Cell viability was assessed by the trypan blue exclusion test.Measurement of MAPK Activation in Neuro2a CellsNeuro2a cells were plated in 60-mm dishes (15,000 cells/cm2) and cultured 24 h in 10% FBS and DMEM. Cells were washed three times with serum-free DMEM and incubated in the presence of 10 μm GM3, GM1, or LacCer in serum-free DMEM for various times (0–60 min). Cells were rinsed twice with phosphate-buffered saline containing 0.4 mmNa3VO4, scraped in 0.5 ml of lysis buffer (20 mm Tris-HCl, pH 8.0, 20 mmβ-glycerophosphate, 2 mm EGTA, 1 mmNa3VO4, 2 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, 75 units/ml aprotinin), and sonicated for 10 s 10 times. Lysates were centrifuged at 15,000 rpm for 10 min at 4 °C and precleared for nonspecific binding with protein G-Sepharose. Sup
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