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Separation of “Glycosphingolipid Signaling Domain” from Caveolin-containing Membrane Fraction in Mouse Melanoma B16 Cells and Its Role in Cell Adhesion Coupled with Signaling

1998; Elsevier BV; Volume: 273; Issue: 50 Linguagem: Inglês

10.1074/jbc.273.50.33766

ISSN

1083-351X

Autores

Kazuhisa Iwabuchi, Kazuko Handa, S Hakomori,

Tópico(s)

Proteoglycans and glycosaminoglycans research

Resumo

Two membrane subfractions, one enriched in GM3 ganglioside and the other containing caveolin, were separated from low density detergent-insoluble membrane fraction prepared by sucrose density gradient centrifugation of postnuclear fraction of mouse melanoma B16 cells. The GM3-enriched subfraction, separated by anti-GM3 monoclonal antibody DH2, contained sphingomyelin, cholesterol, c-Src, and Rho A but not caveolin. In contrast, the caveolin-containing subfraction, separated by anti-caveolin antibody, contained neither GM3, c-Src, nor Rho A but did contain glucosylceramide, Ras, a very small quantity of sphingomyelin, and a very large quantity of cholesterol. The GM3/c-Src-enriched membrane subfraction was characterized by (i) maintenance of GM3-dependent adhesion and (ii) susceptibility to being activated for signal transduction through GM3. 32P-Phosphorylation of c-Src (M r 60,000) together with two other components (M r 45,000 and 29,000) was enhanced in the fraction bound to dishes coated with asialo-GM2 (Gg3) or with anti-GM3 monoclonal antibody DH2, detected by incubation with [γ-32P]ATP at 37 °C for 5 min. GM3-dependent adhesion of B16 cells to Gg3-coated dishes and associated signaling were not reduced or abolished in the presence of either filipin or nystatin, which are cholesterol-binding reagents known to abolish caveolae structure and function. B16 melanoma cells incubated with filipin (0.16–0.3 μg/ml) or with nystatin (25 μg/ml) for 30 min showed depletion of cholesterol in detergent-insoluble membrane fraction but were still capable of binding to Gg3-coated plate and capable of the associated signaling. Thus, the GM3-enriched subfraction, involved in cell adhesion and capable of sending signals through GM3, represents a membrane domain distinguishable from caveolin-containing subfraction or caveolae. This microdomain is hereby termed the "glycosphingolipid signaling domain" or "glycosignaling domain". Two membrane subfractions, one enriched in GM3 ganglioside and the other containing caveolin, were separated from low density detergent-insoluble membrane fraction prepared by sucrose density gradient centrifugation of postnuclear fraction of mouse melanoma B16 cells. The GM3-enriched subfraction, separated by anti-GM3 monoclonal antibody DH2, contained sphingomyelin, cholesterol, c-Src, and Rho A but not caveolin. In contrast, the caveolin-containing subfraction, separated by anti-caveolin antibody, contained neither GM3, c-Src, nor Rho A but did contain glucosylceramide, Ras, a very small quantity of sphingomyelin, and a very large quantity of cholesterol. The GM3/c-Src-enriched membrane subfraction was characterized by (i) maintenance of GM3-dependent adhesion and (ii) susceptibility to being activated for signal transduction through GM3. 32P-Phosphorylation of c-Src (M r 60,000) together with two other components (M r 45,000 and 29,000) was enhanced in the fraction bound to dishes coated with asialo-GM2 (Gg3) or with anti-GM3 monoclonal antibody DH2, detected by incubation with [γ-32P]ATP at 37 °C for 5 min. GM3-dependent adhesion of B16 cells to Gg3-coated dishes and associated signaling were not reduced or abolished in the presence of either filipin or nystatin, which are cholesterol-binding reagents known to abolish caveolae structure and function. B16 melanoma cells incubated with filipin (0.16–0.3 μg/ml) or with nystatin (25 μg/ml) for 30 min showed depletion of cholesterol in detergent-insoluble membrane fraction but were still capable of binding to Gg3-coated plate and capable of the associated signaling. Thus, the GM3-enriched subfraction, involved in cell adhesion and capable of sending signals through GM3, represents a membrane domain distinguishable from caveolin-containing subfraction or caveolae. This microdomain is hereby termed the "glycosphingolipid signaling domain" or "glycosignaling domain". Glycosphingolipids (GSLs) 1The abbreviations used are: GSL, glycosphingolipid; Cer, ceramide; DIM, detergent-insoluble membrane fraction; Gg3, asialo-GM2 (GalNAcβ4Galβ4Glcβ1Cer); GlcCer, Glcβ1Cer; GM3, NeuAcα3Galβ4Glcβ1Cer; Ley, Fucα2Galβ4(Fucα3)GlcNAcβ; IP, immunoprecipitation; PBS, phosphate-buffered saline; 2-ME, 2-mercaptoethanol; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; SM, sphingomyelin; RIPA, radioimmune precipitation assay; FAK, focal adhesion kinase; PVDF, polyvinylidene difluoride; mAb, monoclonal antibody; GM1, Galβ3GalNAcβ4[NeuAcα3]Galβ4Glcβ1Cer. are known as cell surface antigens (1Hakomori S. Annu. Rev. Biochem. 1981; 50: 733-764Crossref PubMed Scopus (1474) Google Scholar), receptors for bacterial toxins (2Kanfer J.N. Kanfer J.N. Hakomori S. Handbook of Lipid Research 3: Sphingolipid Biochemistry. Plenum Press, New York1983: 437-471Google Scholar), sites for bacterial infection (3Karlsson K.-A. Annu. Rev. Biochem. 1989; 58: 309-350Crossref PubMed Scopus (619) Google Scholar), and modulators of signal transduction through interaction with growth factor receptor kinases (4Hakomori S. Biochem. Soc. Trans. 1993; 21: 583-595Crossref PubMed Scopus (168) Google Scholar, 5Yates A.J. Rampersaud A. Ann. N. Y. Acad. Sci. 1998; 845: 57-71Crossref PubMed Scopus (122) Google Scholar). In their original studies, Tillack, Thompson, Young, and associates (6Tillack T.W. Allietta M. Moran R.E. Young W.W.J. Biochim. Biophys. Acta. 1983; 733: 15-24Crossref PubMed Scopus (96) Google Scholar, 7Rock P. Allietta M. Young W.W.J. Thompson T.E. Tillack T.W. 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Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1868) Google Scholar), and signal transducer molecules such as Src family tyrosine kinases (12Sargiacomo M. Sudol M. Tang Z. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, 13Smart E.J. Ying Y.-S. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (676) Google Scholar, 14Rodgers W. Rose J.K. J. Cell Biol. 1996; 135: 1515-1523Crossref PubMed Scopus (286) Google Scholar, 15Song 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 (920) Google Scholar). Caveolae, morphologically distinct invaginations of plasma membrane known for over 45 years (16Palade G.E. J. Appl. Phys. 1953; 24: 1424Google Scholar, 17Yamada E. J. Biophys. Biochem. Cytol. 1955; 1: 445-457Crossref PubMed Scopus (526) Google Scholar), are characterized by the presence of a distinctive scaffold protein, caveolin (for a review, see Ref. 18Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar). Numerous studies indicate that caveolae play an essential role in endocytosis (e.g. Refs. 19Smart E.J. Mineo C. Anderson R.G. J. Cell Biol. 1996; 134: 1169-1177Crossref PubMed Scopus (79) Google Scholar and 20Vey M. Pilkuhn S. Wille H. Nixon R. DeArmond S.J. Smart E.J. Anderson R.G. Taraboulos A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14945-14949Crossref PubMed Scopus (489) Google Scholar) and signal transduction (e.g. Refs. 21Liu P. Ying Y. Ko Y.-G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar and 22Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). The low density fraction separated from postnuclear membrane fraction of many types of cells by gradient centrifugation in the presence or absence of detergent (10Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2610) Google Scholar, 13Smart E.J. Ying Y.-S. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (676) Google Scholar, 15Song 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 (920) Google Scholar) contains caveolin and is therefore assumed to contain caveolae membrane. The invaginated structure of caveolae and their endocytotic function may depend on cholesterol, since cholesterol-binding reagents such as filipin, nystatin, and methyl-β-cyclodextrin disrupt caveolae structure and function (23Schnitzer J.E. Oh P. Pinney E. Allard J. J. Cell Biol. 1994; 127: 1217-1232Crossref PubMed Scopus (772) Google Scholar, 24Baorto D.M. Gao Z. Malaviya R. Dustin M.L. van der Merwe A. Lublin D.M. Abraham S.N. Nature. 1997; 389: 636-639Crossref PubMed Scopus (244) Google Scholar, 25Orlandi P.A. Fishman P.H. J. Cell Biol. 1998; 141: 905-915Crossref PubMed Scopus (640) Google Scholar). However, little attention has been paid to the distinction between caveolae and GSL-enriched microdomains or to the functional role of GSLs in these microdomains (see "Discussion"). We found previously that a low density membrane fraction from mouse melanoma B16 cells separated by density gradient centrifugation contained all of the GM3 in the cells and enriched levels of c-Src, Rho A, Ras, and FAK (26Yamamura S. Handa K. Hakomori S. Biochem. Biophys. Res. Commun. 1997; 236: 218-222Crossref PubMed Scopus (103) Google Scholar). This pattern was consistent regardless of preparation method, including in the presence versus the absence of detergent. GM3-dependent cell adhesion led to enhanced FAK kinase activity and enhanced GTP binding to Rho A and Ras (27Iwabuchi K. Yamamura S. Prinetti A. Handa K. Hakomori S. J. Biol. Chem. 1998; 273: 9130-9138Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). In this paper, we demonstrate that caveolin-containing fraction can be separated from GM3-enriched fraction and that the former contains a large quantity of cholesterol and small quantities of SM and Ras but does not contain GM3, c-Src, FAK, or Rho A. The latter contains GM3, c-Src, FAK, and Rho A and displays GM3-dependent adhesion coupled with c-Src phosphorylation and signal transduction. Thus, the GM3/c-Src-enriched membrane fraction is functionally distinguishable from caveolae and is hereby termed the "glycosphingolipid signaling domain" or "glycosignaling domain." GM3 (N-acetyl) was prepared from dog erythrocytes (28Klenk E. Heuer K. Z. Verdauungs u Stoffwechselkrankheiten. 1960; 20: 180-183PubMed Google Scholar). Gg3 was prepared from guinea pig blood (29Seyama Y. Yamakawa T. J. Biochem. (Tokyo). 1974; 75: 837-842Crossref PubMed Scopus (56) Google Scholar). GlcCer was purchased from Matreya, Inc. (Pleasant Gap, PA). Anti-GM3 mAb DH2 (mouse IgG3) (30Dohi T. Nores G. Hakomori S. Cancer Res. 1988; 48: 5680-5685PubMed Google Scholar), anti-Gg3 mAb 2D4 (mouse IgM) (31Young W.W.J. MacDonald E.M.S. Nowinski R.C. Hakomori S. J. Exp. Med. 1979; 150: 1008-1019Crossref PubMed Scopus (146) Google Scholar), and anti-Ley mAb AH6 (mouse IgM) (32Abe K. McKibbin J.M. Hakomori S. J. Biol. Chem. 1983; 258: 11793-11797Abstract Full Text PDF PubMed Google Scholar) were established as described previously. Sources and properties of antibodies directed to caveolin, c-Src, and other molecules involved in signal transduction are described under the procedures using these antibodies. Phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, phosphatidylinositol, SM, cholesterol, Cer, PMSF, filipin, and nystatin were purchased from Sigma. Lavendustin C was from Calbiochem. Mouse melanoma B16/F10 cells were obtained from Dr. I. J. Fidler (M.D. Anderson Cancer Center, University of Texas, Houston, TX) and were cultured in high glucose Dulbecco's modified Eagle's medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 2 mml-glutamine, 2 mm pyruvic acid, 100 units/ml penicillin G, and 100 μg/ml streptomycin. For metabolic labeling of [3H]serine, cells were cultured in serine-free RPMI 1640 (Life Technologies, Inc.) containing 10% dialyzed fetal calf serum and 1.5 mCi/ml l-[3H]serine (15–40 Ci/mmol, NEN Life Science Products) in 150-mm tissue culture dishes (Nunc, Naperville, IL) for 20 h. Cells were harvested in 0.02% EDTA-PBS (PBS: 8.1 mm Na2HPO4, 1.5 mmKH2PO4, 2.7 mm KCl, 137 mm NaCl, pH 7.4), lysed, homogenized, and subjected to sucrose density gradient centrifugation to separate low density light-scattering membrane fraction by a modification of the method described previously (14Rodgers W. Rose J.K. J. Cell Biol. 1996; 135: 1515-1523Crossref PubMed Scopus (286) Google Scholar, 27Iwabuchi K. Yamamura S. Prinetti A. Handa K. Hakomori S. J. Biol. Chem. 1998; 273: 9130-9138Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Briefly, 1–5 × 107cells were suspended in 1 ml of lysis buffer (1% Triton X-100, 10 mm Tris-HCl (pH 7.5), 150 mm NaCl, 5 mm EDTA, 1 mm PMSF, and 75 units/ml aprotinin). The postnuclear supernatant was subjected to sucrose density gradient centrifugation. A white light-scattering band located at ∼5–7% sucrose was collected, and termed "DIM." A high density fraction (fraction 12) was collected from the bottom of the gradient. The entire procedure was performed at 0–4 °C. Protein content of each fraction was determined using the Micro-BCA kit (Pierce). GSLs were coated on tissue culture dishes as described previously (27Iwabuchi K. Yamamura S. Prinetti A. Handa K. Hakomori S. J. Biol. Chem. 1998; 273: 9130-9138Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Briefly, 2 ml of Gg3 and GlcCer in ethanol (100 μg/ml) were added to 10-cm dishes (Corning, Cambridge, MA) and dried under gentle shaking on a rocker (20 movements/min) at room temperature. The coated dishes were subsequently treated with 5 ml of 0.1% bovine serum albumin in PBS at room temperature for 1 h with gentle shaking. The dishes were washed five times with ice-cold PBS and used immediately for adhesion experiments. Antibody-coated dishes were prepared by incubation of 5 ml of purified antibody (10 μg/ml in PBS) placed in a 10-cm tissue culture dish at 4 °C overnight. Antibodies used were anti-GM3 mAb DH2 and anti-caveolin rabbit IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Normal mouse IgG and normal rabbit IgG (Santa Cruz Biotechnology) were used as controls. DIM was ∼10× diluted with Tris-NaCl-cation buffer (10 mm Tris-HCl (pH 7.5), 150 mm NaCl, 10 mm MgCl2, 1 mm CaCl2) to obtain a final protein concentration of 2 μg/ml. Aliquots (5 ml) of the diluted fraction were added to dishes coated with Gg3, GM3, or GlcCer, followed by centrifugation at 2000 × g at 4 °C for 30 min. The dishes were left for 15 h on ice to ensure membrane adhesion and then further incubated at 25 °C for 2 h with gentle shaking on rocker (10 movements/min), and washed three times with Tris-NaCl-cation buffer. After washing out of nonadhered material, adherent material on dishes was desorbed by the addition of 5 ml of lysis buffer with sonication for 5 min in a water bath sonicator (Branson, Danbury, CT). The desorbed material was subjected to immunoblot assay to detect GM3 or caveolin using specific antibodies by the method of Towbin et al. (33Towbin H. Gordon J. J. Immunol. Methods. 1984; 72: 313-340Crossref PubMed Scopus (794) Google Scholar) with modification. A PVDF membrane (Immobilon-P, Millipore Corp., Bedford, MA) was soaked in fixation buffer (62.5 mm Tris-HCl (pH 8.9), containing 5% methanol) for at least 2 h before assembly in a Bio-Dot apparatus (Bio-Rad). The collected desorbed fraction was loaded onto PVDF membrane by aspiration. Next, the blotted membranes were washed in fixation buffer and dried. DIM diluted to various concentrations was also blotted and used as standards. After brief soaking in methanol, the membranes were blocked in 5% skim milk in TBS-T (10 mm Tris-HCl (pH 8.0), 150 mm NaCl containing 0.05% Tween 20) at room temperature for 3 h, and then incubated with 1 μg/ml anti-GM3 mAb DH2 or rabbit anti-caveolin IgG in TBS-T containing 1% normal goat serum at room temperature for 2 h. After extensive washing with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) or horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) in TBS-T at room temperature for 1 h. The membrane was washed five times in TBS-T and developed using chemiluminescence method with Super-SignalTM-CL-horseradish peroxidase (Pierce). A 1-ml aliquot of DIM fraction containing 25-30 μg/ml protein was diluted 10 times in IP buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 2 mm NaF, 1 mm EDTA, 1 mm EGTA, 1 mm NaVO4, 1 mm PMSF, 75 units/ml aprotinin, 1% Triton X-100), precleared by incubating with 50 μl of protein G-Sepharose beads (Amersham Pharmacia Biotech), and placed in a rotary mixer at 4 °C for 2 h. After centrifugation at 270 × g for 2 min, the supernatant was separated and added with anti-GM3 mAb DH2 at a final concentration of 1 μg/ml. In addition, 1-ml aliquots of DIM fraction were similarly treated and added with anti-caveolin rabbit IgG (Transduction Laboratories, Lexington, KY), or control normal rabbit IgG, or control mouse IgG (both from Santa Cruz Biotechnology, Santa Cruz, CA), at final concentration of 1 μg/ml. Each mixture was placed overnight in a rotary mixer at 4 °C, then added with 50 μl of protein G-Sepharose beads, and placed again in a rotary mixer for 2 h. The beads were washed three times with IP buffer by centrifugation at 270 × g for 2 min. The immunoprecipitates on beads were treated differently depending on subsequent analysis. (i) For Western blot analysis, the washed beads were suspended with 100 μl of sample buffer with 2-ME, heated to 95 °C for 3 min, and centrifuged at 1000 × g for 2 min, and the supernatant was subjected to SDS-PAGE. (ii) For dot-blot analysis, bound materials on beads were eluted by 1 ml of 0.2m glycine-HCl (pH 2.3) containing 1% Triton X-100 with sonication in ice-cold water for 15 s. After centrifugation at 270 × g for 2 min, resultant supernatants were subjected to the assay. (iii) For the second immunoprecipitation analysis, immunoprecipitates on beads were eluted by mixing with 0.2m glycine-HCl (pH 2.3) containing 1% Triton X-100 as above and centrifugation for 270 × g for 2 min. The supernatants were collected and immediately neutralized with 1m Tris-HCl (pH 10) to pH 7.4 and then diluted 10× with IP buffer and incubated with 1 μg/ml anti-caveolin IgG, anti-GM3 mAb DH2 or normal rabbit IgG in a rotary mixer at 4 °C overnight. The mixtures were added with 50 μl of protein G-Sepharose beads and placed again in a rotary mixer for 2 h. Beads were washed three times with IP buffer, by centrifugation at 270 × g for 2 min, and then suspended with 100 μl of sample buffer with 2-ME, heated to 95 °C for 3 min, and centrifuged at 1000 ×g for 2 min, and the supernatant was subjected to SDS-PAGE. Western blot analysis was performed as described previously (26Yamamura S. Handa K. Hakomori S. Biochem. Biophys. Res. Commun. 1997; 236: 218-222Crossref PubMed Scopus (103) Google Scholar, 27Iwabuchi K. Yamamura S. Prinetti A. Handa K. Hakomori S. J. Biol. Chem. 1998; 273: 9130-9138Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Briefly, proteins separated on 5–15% linear gradient SDS-PAGE under reducing conditions were electroblotted onto PVDF membranes. The membranes were incubated with anti-c-Src rabbit or goat IgG (Santa Cruz Biotechnology), anti-Rho A mouse mAb (Santa Cruz Biotechnology), anti-Ras rat mAb (Santa Cruz Biotechnology), or anti-FAK rabbit IgG (Santa Cruz Biotechnology) for 2 h at room temperature and then incubated with horseradish peroxidase-labeled proper secondary antibody. The reacted proteins were detected by SuperSignal chemiluminescent substrate. Dot blot analysis was performed as described above. DIM was 10× diluted with kinase buffer (30 mm HEPES (pH 7.5), 10 mmMgCl2, 2 mm MnCl2 1 mmCaCl2), with a final protein concentration of 2–3 μg/ml. 5-ml aliquots of this diluted DIM were added to GSL- or antibody-coated 10-cm dishes, followed by centrifugation at 2000 × gat 4 °C for 30 min, and the dishes were kept on ice for 15 h. To determine phosphorylation of control DIM, the same amount of DIM in a polypropylene tube was kept on ice for 15 h (no DIM adhesion occurs under this condition). An aliquot of 50 μCi [γ-32P]ATP (corresponding to 370 GBq/mmol, NEN Life Science Products) was added to each dish and control tube, and the mixture was incubated at 37 °C for 5 min. For some experiments, 100 μm Lavendustin C was added during incubation with [γ-32P]ATP. The reaction was stopped by placing it on ice and adding 5 ml of ice-cold, 2× concentrated stop buffer (15 mm HEPES (pH 7.5), 150 mm NaCl, 5 mm EDTA, 1 mm NaVO4, 1% Triton X-100, 1 mm PMSF). The reaction mixtures in each dish were transferred into another container (part 1), and the remaining adherent material on the dishes was desorbed by the addition of 5 ml of stop buffer followed by sonication in sonicating bath at 0 °C for 5 min (part 2). Parts 1 and 2 were combined and precipitated with trichloroacetic acid at a final concentration of 10%. The precipitates were centrifuged, washed twice with acetone to eliminate trichloroacetic acid, dissolved in 1.0 ml of IP buffer, mixed with 20 μl of protein G-Sepharose, and placed in a rotary mixer at 4 °C for 2 h. After centrifugation at 270 × g for 5 min, the supernatants were collected and added with anti-c-Src goat IgG at final IgG concentration of 1 μg/ml, incubated at 4 °C overnight, added with 20 μl of protein G-Sepharose, and incubated at 4 °C for 2 h. Next, Sepharose beads were washed five times with IP buffer containing 0.5 m NaCl, and boiled with SDS-sample buffer containing 5% 2-ME. In some experiments, RIPA buffer (30 mm HEPES, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mm EDTA, 1 mm NaVO4, 50 mm NaF, 1 mm PMSF, 10 μg/ml pepstatin A, 10 μg/ml leupepsin, 75 units/ml aprotinin) was used for solubilization after acetone wash and for washing of immunoprecipitates instead of IP buffer (34DeSeau V. Rosen N. Bolen J.B. J. Cell. Biochem. 1987; 35: 113-128Crossref PubMed Scopus (31) Google Scholar). The samples were run on 5–15% SDS-PAGE, transferred, and blotted on PVDF membranes. Autoradiography was carried out by exposing the electroblotted membranes to Kodak XAR x-ray film at −80 °C with intensifying screens (DuPont Lightning Plus). After autoradiography, the blotted membranes were probed with anti-c-Src rabbit IgG to determine the amount of protein in each lane. The bound antibodies on PVDF membrane were stripped using 100 mm 2-ME, 2% SDS, 62.5 mm Tris-HCl (pH 6.7), and reprobed with anti-Erk-1 rabbit IgG (Santa Cruz Biotechnology) and anti-myosin light chain mAb (Sigma) to analyze proteins co-immunoprecipitated with c-Src. The membrane subfractions immunoseparated with antibodies were mixed with 1 ml of methanol, sonicated for 15 min, added with 2 ml of chloroform, and further sonicated for 5 min. To extract lipids from liquid samples, 10 volumes of methanol was added, and the mixture was sonicated for 15 min in a water bath sonicator and then added with 20 volumes of chloroform, sonicated for 5 min, and centrifuged at 1500 ×g at 4 °C for 15 min. The supernatants were collected, dried under an N2 stream, sonicated in methanol, added with water, and sonicated again (final solution contained methanol/water, 3:7, v/v). The solutions were applied to Bond Elut packed columns (1 ml, C18, Analytichem International, Harbor, CA) washed with chloroform/methanol (2:1, v/v) and preequilibrated with methanol/water (3:7). The columns were extensively washed with distilled water, and bound materials were eluted with 2 ml of chloroform/methanol (2:1) followed by 1 ml of chloroform. The eluates were dried under N2 and dissolved in ∼20 μl of chloroform/methanol (2:1). Two-dimensional thin layer chromatography was carried out according to the method of Yokoyama et al. (35Yokoyama K. Nojiri H. Suzuki M. Setaka M. Suzuki A. Nojima S. FEBS Lett. 1995; 368: 477-480Crossref PubMed Scopus (22) Google Scholar). In brief, samples were spotted at the lower left-hand corner of a 10 × 10-cm high performance thin layer chromatography plate (Merck). The first chromatographic run was performed with chloroform/methanol/formic acid/water (65:25:8.9:1.1, v/v/v/v). The second run was performed with chloroform/methanol/4.4 n ammonia 50:40:10 (v/v/v) at a rotation of 90° from the first direction. The third run was performed with diethylether in a direction opposite to that of second run. The plate was sprayed by 0.03% primulin (Aldrich) in acetone/water (80:20, v/v) and photographed under UV light. For autoradiography, TLC plates were exposed to Kodak BioMax MS film at −80 °C with Kodak TranScreen-LE intensifying screen. B16 cells were detached with 0.05% trypsin, 0.5 mm EDTA-PBS and suspended in serum-free Dulbecco's modified Eagle's medium at 1.25 × 106 cells/ml. Aliquots of cell suspension were mixed with Me2SO alone (final concentration 0.5%, as control) or Me2SO containing various quantities of filipin (final concentration, 0.02–10 μg/ml) or nystatin (final concentration 12.5–50 μg/ml) in polypropylene tubes (in order to avoid cell adhesion) and incubated for 10 or 30 min at 37 °C. A 500-μl aliquot of the preincubated cell suspension was added to each well of 24-well plates (Corning), which were precoated with Gg3 or GlcCer and blocked with 0.1% bovine serum albumin to observe cell adhesion. In order to observe restoration of caveolae function, filipin-treated cells in polypropylene tubes were centrifuged and suspended in Dulbecco's modified Eagle's medium with 20% fetal calf serum, incubated at 37 °C for 30 min (in CO2incubator), centrifuged, and washed, and aliquots were added to Gg3- or GlcCer-coated plates. After incubation of cells at 37 °C for 30 min, each well was washed with warmed PBS three times to remove nonadherent cells. The quantity of adherent cells was estimated by protein determination using the Micro-BCA kit. In order to observe the effect of filipin on signal transduction induced by GM3-dependent cell adhesion, FAK phosphorylation induced by specific binding to Gg3- as compared with GlcCer-coated plates was measured and quantified as described previously (27Iwabuchi K. Yamamura S. Prinetti A. Handa K. Hakomori S. J. Biol. Chem. 1998; 273: 9130-9138Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). B16 cells were detached with 0.05% trypsin, 0.5 mm EDTA, PBS and suspended in serum-free Dulbecco's modified Eagle's medium at 1.25 × 106 cells/ml. Aliquots of cell suspension were mixed with Me2SO alone (final concentration 0.5%, as control) or Me2SO containing various quantities of filipin (final concentration 0.16–2.5 μg/ml) or 25 μg/ml nystatin in polypropylene tubes and incubated for 30 min at 37 °C. After incubation, the reaction mixtures were centrifuged at 270 × g for 10 min, cell pellets were suspended in 1 ml of lysis buffer, and DIM was prepared as described above. For each DIM sample, cholesterol was isolated as described under "Lipid Component Analysis of DIM and Subfractions" and measured by the method of Gamble et al. (36Gamble W. Vaughan M. Kruth H.S. Avigan J. J. Lipid Res. 1978; 19: 1068-1070Abstract Full Text PDF PubMed Google Scholar). The presence of two clearly distinguishable membrane subfractions in low density DIM prepared from B16 melanoma cells was indicated by two different separation methods,i.e. (i) separation based on antibody binding to the antigen expressed in membrane components (immunoseparation) and (ii) separation based on specific interaction between GM3 (on membrane) and Gg3 (coated on dish) (27Iwabuchi K. Yamamura S. Prinetti A. Handa K. Hakomori S. J. Biol. Chem. 1998; 273: 9130-9138Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), followed by immunoblot analysis of membrane subfractions using anti-GM3 and anti-caveolin antibodies. The DIM subfraction adhered to the Gg3-coated dish, but not the subfraction nonspecifically adhered to the GM3- or GlcCer-coated dish, showed positive blotting with anti-GM3 mAb DH2. Pretreatment of the Gg3-coated dish with anti-Gg3 mAb 2D4, but not with isotype-matched control mAb AH6 (anti-Ley), inhibited binding of the GM3-containing subfraction (Fig. 1 A). The subfraction immunoprecipitated with anti-c-Src showed binding (positive blotting) with anti-GM3, whereas that immunoprecipitated with anti-caveolin IgG did not (negative blotting) (Fig. 1 B). The subfraction bound to anti-caveolin and that unbound to anti-GM3-coated dish were positively immunoblotted with anti-caveolin (Fig.1 C). In Western blotting analysis with anti-caveolin antibody, the IP with anti-caveolin showed a band corresponding to caveolin (M r ∼21,000) (Fig. 1 D,lane 2), but the IP with anti-GM3 or control normal rabbit IgG did not show such a band (lanes 1 and 5). A second IP with anti-GM3, using eluate from IP with anti-ca

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