GM3-enriched Microdomain Involved in Cell Adhesion and Signal Transduction through Carbohydrate-Carbohydrate Interaction in Mouse Melanoma B16 Cells

Abstract

Mouse melanoma B16 cells are characterized by the predominant presence of ganglioside GM3 and adhere to lactosylceramide- or Gg3-coated plates through interaction of GM3 with lactosylceramide or Gg3, whereby not only adhesion but also spreading and enhancement of cell motility occur (Kojima, N., Hakomori, S. (1991) J. Biol. Chem. 266, 17552–17558). We now report that the adhesion process is based essentially on a glycosphingolipid-enriched microdomain (GEM) at the B16 cell surface, since >90% of GM3 present in the original cells is found in GEM, and GEM is also enriched in several signal transducer molecules, e.g. c-Src, Ras, Rho, and focal adhesion kinase (FAK). GEM was isolated as a low density membranous fraction by homogenization of B16 cells in lysis buffer under two different conditions (i.e. buffer containing 1% Triton X-100, or hypertonic sodium carbonate without detergent), followed by sucrose density gradient centrifugation. A close association of GM3 with c-Src, Rho, and FAK was indicated by co-immunoprecipitation of GM3 present in GEM by anti-GM3 monoclonal antibody DH2, followed by Western blotting with antibodies directed to these transducer molecules. The following data indicate that GEM is a structural and functional unit for initiation of GM3-dependent cell adhesion coupled with signal transduction. 1) Tyrosine phosphorylation in FAK was greatly enhanced in B16 cells adhered to Gg3-coated plates but was minimal in cells adhered to GM3-coated, GlcCer-coated, or noncoated plates. 2) GTP loading on Ras and Rho increased significantly when cells were adhered to Gg3-coated plates, compared with GM3-coated, GlcCer-coated, or noncoated plates. Since Ras and Rho are closely associated with GM3 in GEM, cell adhesion/stimulation through GM3 in GEM may induce activation of Ras and Rho through enhanced GTP binding. Mouse melanoma B16 cells are characterized by the predominant presence of ganglioside GM3 and adhere to lactosylceramide- or Gg3-coated plates through interaction of GM3 with lactosylceramide or Gg3, whereby not only adhesion but also spreading and enhancement of cell motility occur (Kojima, N., Hakomori, S. (1991) J. Biol. Chem. 266, 17552–17558). We now report that the adhesion process is based essentially on a glycosphingolipid-enriched microdomain (GEM) at the B16 cell surface, since >90% of GM3 present in the original cells is found in GEM, and GEM is also enriched in several signal transducer molecules, e.g. c-Src, Ras, Rho, and focal adhesion kinase (FAK). GEM was isolated as a low density membranous fraction by homogenization of B16 cells in lysis buffer under two different conditions (i.e. buffer containing 1% Triton X-100, or hypertonic sodium carbonate without detergent), followed by sucrose density gradient centrifugation. A close association of GM3 with c-Src, Rho, and FAK was indicated by co-immunoprecipitation of GM3 present in GEM by anti-GM3 monoclonal antibody DH2, followed by Western blotting with antibodies directed to these transducer molecules. The following data indicate that GEM is a structural and functional unit for initiation of GM3-dependent cell adhesion coupled with signal transduction. 1) Tyrosine phosphorylation in FAK was greatly enhanced in B16 cells adhered to Gg3-coated plates but was minimal in cells adhered to GM3-coated, GlcCer-coated, or noncoated plates. 2) GTP loading on Ras and Rho increased significantly when cells were adhered to Gg3-coated plates, compared with GM3-coated, GlcCer-coated, or noncoated plates. Since Ras and Rho are closely associated with GM3 in GEM, cell adhesion/stimulation through GM3 in GEM may induce activation of Ras and Rho through enhanced GTP binding. Glycosphingolipids (GSLs) 1The abbreviations used are: GSL, glycosphingolipid; DIM, detergent-insoluble material; DISAM, detergent-insoluble substrate attachment matrix; GEM, glycosphingolipid-enriched microdomain(s); PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; FAK, focal adhesion kinase; mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium; GPI, glycosylphosphatidylinositol; MES, 4-morpholineethanesulfonic acid. have been implicated as modulators of signal transduction through their effect on protein kinase activity (for a review, see Ref. 1Hakomori S. J. Biol. Chem. 1990; 265: 18713-18716Abstract Full Text PDF PubMed Google Scholar), particularly that associated with growth factor receptors (1Hakomori S. J. Biol. Chem. 1990; 265: 18713-18716Abstract Full Text PDF PubMed Google Scholar, 2Bremer E.G. Hakomori S. Biochem. Biophys. Res. 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Cancer Res. 1988; Google Scholar). or monoclonal antibodies for Western and directed to transducer such as c-Src, FAK, and were directed to c-Src, FAK, and were antibody directed to was monoclonal and antibody directed to was monoclonal were with lysis buffer containing 1% Triton X-100, the other with hypertonic sodium carbonate were in and to sucrose density gradient to low density membranous fraction to a of the J.K. J. Cell Biol. 1996; PubMed Scopus Google Scholar). were in of lysis buffer containing 1% Triton X-100, and of and to for a phosphorylation was was to The cell was with and the was for at to and The fraction was to sucrose density gradient fraction was with of sucrose containing buffer with or without The was at the of a sucrose gradient with were for at at A under to the of the membranous at sucrose was other at different sucrose The was at The protein of fraction was a The original S. T. M. J. Biol. 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J. 1986; Scopus Google Scholar), as was with substrate The GM3 on was also by GEM and other protein were to with Western protein on were to in and at for The were at in containing in containing and for with antibodies directed to with antibody with the was in containing and with or with for for of or The was in containing and the with a substrate of GEM of was with protein and by a for at to binding. for the was with of anti-GM3 or with of or as The were in a at with protein and in a for were with containing by for and with of buffer with to for and for The were to to and with antibodies directed to transducer molecules, under the conditions as under of were with antibody by the as B16 melanoma characterized by of and enhanced motility on or is based essentially on interaction of GM3 on B16 cells with or on the plates (17Kojima N. Hakomori S. J. Biol. Chem. 1989; 264: 20159-20162Abstract Full Text PDF PubMed Google Scholar, 18Kojima N. Hakomori S. J. Biol. Chem. 1991; 266: 17552-17558Abstract Full Text PDF PubMed Google Scholar) and is to as of signal transduction by GM3-dependent the following was of Gg3, or was to plates and a the of was to plates and were with Gg3, or and was with a of B16 cell in plates were in a to a at for at and at in a for or as of cell were on by and Cell were for the of FAK and associated with FAK and for the associated with Rho and Ras the effect of interaction of on with GM3 on B16 Gg3-coated plates were with of Hakomori S. J. Exp. PubMed Scopus Google Scholar) or directed to K. J.M. Hakomori S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) for at were with PBS, and B16 cells were by and in at for were and the of FAK and phosphorylation were as Since of B16 cells on Gg3-coated was by anti-GM3 N. Hakomori S. J. Biol. Chem. 1991; 266: 17552-17558Abstract Full Text PDF PubMed Google Scholar), the effect of anti-GM3 on of FAK and were The cells were in at and for at in the presence of or the was in and of containing was were and followed the to FAK and phosphorylation as through adhesion to Gg3-coated plates or cells on or plates as were in of lysis buffer 1% Triton by for of containing the of protein were for in buffer containing and on and proteins were to The were with in containing for at and with for at The were with containing with for at with and by the with a substrate the of associated with FAK, FAK was isolated the cell by of cell of was with protein and for at for the was with of at and with protein for at The was at for and were with and in buffer containing The FAK of was by Western blotting containing of FAK were on and to The were with and the followed by The original for of GTP and to Ras J. S. D.A. 1990; PubMed Scopus Google Scholar) was modified at as of gel since to Ras and Rho in B16 cells not of cells in with was with for was to the under and was for cells were with TBS, with and in of cells were by low for on plates with Gg3, or The plates were for at and the was by on and the of of were by in of lysis buffer 1% Triton X-100, and at for the was with for at and were by with protein for at at for at and with or monoclonal or antibodies or with as at The were with protein and for at The were with buffer A and buffer in buffer at for and The was with and at for at was and was with of and to and was The were and were by The was in of and on plates, which were with and GTP by under and by were and was by of B16 melanoma cells in lysis buffer containing 1% Triton or hypertonic sodium carbonate followed by sucrose density gradient a in the low density The of the was for under or under the two conditions the presence of membranous are in GEM fraction by sodium carbonate without are in GEM fraction by the 1% Triton The of of GM3 in fraction sucrose density gradient in 1% Triton is in the was sodium carbonate not of GM3 in the original cells was found in the low density membranous under or the only a of GM3 was only in other fraction which of protein of and other transducer present in GEM and in the fraction are in of and transducer in low density membranous fraction and in density protein in containing 1% Triton in hypertonic sodium density membranous fraction density protein density membranous fraction density protein of proteins and GM3 present in fraction to present in of GM3 was found in of in Western blotting by the of protein present in The of protein used in a was not of proteins and GM3 present in fraction to present in of GM3 was found in of in Western blotting by the of protein present in The of protein used in a was not in a and Ras of Rho, and of FAK present in B16 cells were found in GEM fraction under Triton conditions The of c-Src, Rho, and FAK was but different for GEM in hypertonic sodium carbonate a of FAK was present in GEM fraction in sodium Ras was in fraction by sodium carbonate since Ras under of c-Src, Rho, and FAK in low density GEM by two different is in of the that fraction only of and receptors such as growth factor and were in low density GEM fraction but present in density which of of GEM were with anti-GM3 and protein GM3 and with transducer present in GEM were the and to Western blotting with as under and and were on Western blotting A and the of or to GEM not in of or The to FAK was by Western blotting with antibodies of the of containing of FAK were B16 cells adhered to GM3-coated, or noncoated plates for FAK was by by Western phosphorylation of the was compared by Western blotting with A enhancement of was in cells adhered to Gg3-coated plates. activity was for cells on or noncoated plates The FAK in these was of cells were adhered to GM3-coated, or noncoated plates of in cells adhered to Gg3-coated plates as compared with or noncoated plates are in of phosphorylation in FAK of cell adhesion based on interaction and the of enhancement of phosphorylation in FAK, FAK activity by was compared B16 cells adhered to Gg3-coated plates with different 1) 2) and 3) Tyrosine phosphorylation of cells adhered to plates with was greatly 1) to cells on plates with or antibodies and FAK of and were or the was and indicate that enhancement of phosphorylation in FAK GM3-dependent B16 cell adhesion to Gg3-coated plates is by or not Since B16 cell adhesion on a Gg3-coated coupled with FAK was when the Gg3-coated was with as and the adhesion process was in the presence of anti-GM3 N. Hakomori S. J. Biol. Chem. 1991; 266: 17552-17558Abstract Full Text PDF PubMed Google Scholar), the effect of anti-GM3 on phosphorylation in B16 cells was greatly phosphorylation in and D. The the that GM3 is the signal transduction to of Ras and Rho, the of signal transduction through these was in B16 cells adhered to Gg3-coated plates in with GM3-coated, GlcCer-coated, and noncoated plates, since Ras and Rho are found in GEM and associated closely with GTP to Ras and Rho was increased greatly in cells adhered to Gg3-coated plates and was minimal in cells to or noncoated plates The of to Ras and Rho, by a was when cells were adhered to a Gg3-coated but was when cells were to plates. The associated with Rho for cells adhered to plates and a that for cells adhered to in the associated with Ras was for plates for B16 cells adhered to plates, the Rho was different the Ras of function in cell have been 1) GSLs function of adhesion receptors integrin or are involved in cell adhesion as for carbohydrate-binding receptors or for by 2) GSLs involved in signal such as growth factor and protein kinase (for a review, see Ref. 1Hakomori S. J. Biol. Chem. 1990; 265: 18713-18716Abstract Full Text PDF PubMed Google Scholar). two occur and in close association with of GSLs with other is a for the and is by the of GSLs in There is increasing evidence that GSLs are not at the cell but are to or with other these have been GEM A of as 1) The of of GSLs at the cell surface as by with the are M. R.E. Biophys. PubMed Scopus Google Scholar) and are also in the surface of M. Biochemistry. 1990; PubMed Scopus Google Scholar, M. 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Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The present that GM3-dependent adhesion of B16 cells to Gg3-coated plates two in signal 1) FAK activation increased of and 2) enhanced GTP to Rho and FAK activation was B16 cells were adhered to Gg3-coated plates but was not the of cells to GM3-coated, GlcCer-coated, or noncoated plates. GTP to Rho was significantly enhanced when cells were to plates, to a when to Gg3-coated plates. that GM3 in GEM, interaction with Gg3, may induce signal the and motility and to The transducer associated with GM3 in GEM of B16 cells is of GM3-dependent cell adhesion were not of in of is that the of associated with GEM is in since the kinase for M. S. Y. T. H. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar), is in GEM and in fraction is also that may interaction of GM3 with Gg3, in to activation of The by which cell adhesion through GM3 in GEM in GEM is to may to the by and Schlessinger A. 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Glycobiology. 1991; 1: 623-630Crossref PubMed Scopus (57) Google Scholar). is that or receptors may close to GEM when GSLs in GEM are involved in cell such a may not occur in A for function of GEM in with and in with other receptors is in GEM as a structural unit of adhesion coupled with signal transduction is with growth factor growth factor is of only to a and a signal based on activation of a GEM may of to and We for and of the