Association of Src Family Tyrosine Kinase Lyn with Ganglioside GD3 in Rat Brain
1997; Elsevier BV; Volume: 272; Issue: 47 Linguagem: Inglês
10.1074/jbc.272.47.29947
ISSN1083-351X
AutoresKohji Kasahara, Yumiko Watanabe, Tadashi Yamamoto, Yutaka Sanai,
Tópico(s)Galectins and Cancer Biology
ResumoAssociation of gangliosides with specific proteins in the central nervous system was examined by co-immunoprecipitation with anti-ganglioside antibody. Protein kinase activity was detected in precipitates with monoclonal antibody to ganglioside GD3 (R24) from membranal fraction of rat brain. Using in vitro kinase assay, several phosphorylated proteins of 40, 53, 56, and 80 kDa were isolated by gel electrophoresis. Of these proteins, the proteins of 53 and 56 kDa (p53/56) were identified as two isoforms of Src family tyrosine kinase Lyn, based on co-migration during gel electrophoresis, comparative peptide mapping, and sequential immunoprecipitation with anti-Lyn antibody. The identification was confirmed using a cDNA expression system in Chinese hamster ovary (CHO) cells, which express solely ganglioside GM3, the enzymatic substrate of GD3synthase. In co-transfection with GD3 synthase and Lyn expression plasmids, R24 immunoprecipitated Lyn and anti-Lyn antibody immunoprecipitated GD3. R24 treatment of rat primary cerebellar cultures induced Lyn activation and rapid tyrosine phosphorylation of several substrates including mitogen-activated protein kinases. Furthermore, sucrose density gradient analysis showed that Lyn of cerebellum and CHO transfectants were detected in a low density light-scattering band, i.e. the caveolae membrane fraction. R24 immunoprecipitated caveolin from Triton X-100 extract of CHO transfectants. These observations suggest that GD3 may regulate Lyn in a caveolae-like domain on brain cell membranes. Association of gangliosides with specific proteins in the central nervous system was examined by co-immunoprecipitation with anti-ganglioside antibody. Protein kinase activity was detected in precipitates with monoclonal antibody to ganglioside GD3 (R24) from membranal fraction of rat brain. Using in vitro kinase assay, several phosphorylated proteins of 40, 53, 56, and 80 kDa were isolated by gel electrophoresis. Of these proteins, the proteins of 53 and 56 kDa (p53/56) were identified as two isoforms of Src family tyrosine kinase Lyn, based on co-migration during gel electrophoresis, comparative peptide mapping, and sequential immunoprecipitation with anti-Lyn antibody. The identification was confirmed using a cDNA expression system in Chinese hamster ovary (CHO) cells, which express solely ganglioside GM3, the enzymatic substrate of GD3synthase. In co-transfection with GD3 synthase and Lyn expression plasmids, R24 immunoprecipitated Lyn and anti-Lyn antibody immunoprecipitated GD3. R24 treatment of rat primary cerebellar cultures induced Lyn activation and rapid tyrosine phosphorylation of several substrates including mitogen-activated protein kinases. Furthermore, sucrose density gradient analysis showed that Lyn of cerebellum and CHO transfectants were detected in a low density light-scattering band, i.e. the caveolae membrane fraction. R24 immunoprecipitated caveolin from Triton X-100 extract of CHO transfectants. These observations suggest that GD3 may regulate Lyn in a caveolae-like domain on brain cell membranes. Gangliosides, sialic acid-containing glycosphingolipids, are found in the outer leaflet of the plasma membrane of all vertebrate cells and are thought to play functional roles in cellular interactions and control of cell proliferation (1Yamakawa T. Nagai Y. Trends Biochem. Sci. 1978; 3: 128-131Abstract Full Text PDF Scopus (260) Google Scholar, 2Hakomori S. Annu. Rev. Biochem. 1981; 50: 733-764Crossref PubMed Scopus (1480) Google Scholar, 3Hakomori S. J. Biol. Chem. 1990; 265: 18713-18716Abstract Full Text PDF PubMed Google Scholar, 4Hakomori S. Igarashi Y. J. Biochem. 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The addition of exogenous gangliosides or anti-ganglioside antibody to the primary neurons and neuroblastomasin vitro has been shown to stimulate the differentiation with concomitant neurite sprouting and extension (8Roisen F.J. Bartfeld H. Nagele R. Yorke G. Science. 1981; 214: 577-578Crossref PubMed Scopus (311) Google Scholar, 9Ledeen R.W. J. Neurosci. Res. 1984; 12: 147-159Crossref PubMed Scopus (288) Google Scholar, 10Chatterjee D. Chakraborty M. Anderson G.M. Brain Res. 1992; 583: 31-44Crossref PubMed Scopus (24) Google Scholar). Transfection of ganglioside GD3(NeuAcα2,8NeuAcα2,3Galβ1,4Glcβ1,1-ceramide) 1The nomenclature for gangliosides follows the system of Svennerholm (79Svennerholm L. J. Neurochem. 1963; 10: 613-623Crossref PubMed Scopus (1313) Google Scholar). synthase cDNA into neuroblastoma induced cholinergic differentiation with neurite sprouting (11Kojima N. Kurosawa N. Nishi T. Hanai N. Tsuji S. J. Biol. 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Ganglioside biosynthesis takes place in the Golgi apparatus, where glucosylceramide is glycosylated by sequential addition of galactose, sialic acid, andN-acetylgalactosamine. Ganglioside GD3 is important as a precursor of the b and c series ganglioside. Recently we isolated GD3 synthase (α2,8-sialyltransferase) cDNA and found that the GD3 synthase expression was regulated in stage- and spatio-restricted manners in the rat central nervous system (17Nara K. Watanabe Y. Maruyama K. Kasahara K. Nagai Y. Sanai Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7952-7956Crossref PubMed Scopus (123) Google Scholar, 19Watanabe Y. Nara K. Takahashi H. Nagai Y. Sanai Y. J. Biochem. (Tokyo). 1996; 120: 1020-1027Crossref PubMed Scopus (23) Google Scholar). GD3 is the predominant ganglioside of the early, immature nervous system of birds and mammals, but its amount decreases in contrast with the accumulation of higher sialylated gangliosides during maturation (21Yu R.K. Macala L.J. Taki T. Weinfield H.M. Yu F.S. J. Neurochem. 1988; 50: 1825-1829Crossref PubMed Scopus (250) Google Scholar). GD3 is implicated in cell attachment (22Cheresh D.A. Pierschbacher M.D. Herzig M.A. Mujoo K. J. Cell Biol. 1986; 102: 688-696Crossref PubMed Scopus (265) Google Scholar) and cell-to-cell interactions during embryogenesis (23Sariola H. Aufderheide E. Bernhard H. Henke F.S. Dippold W. Ekblom P. Cell. 1988; 54: 235-245Abstract Full Text PDF PubMed Scopus (106) Google Scholar). Non-receptor protein-tyrosine kinases of the Src subfamily are implicated in signal transduction systems that control cell proliferation and differentiation (24Cooper J.A. Kemp B. Peptides and Protein Phosphorylation. CRC Press, Boca Raton, FL1990: 85-113Google Scholar). In the present study, we isolated ganglioside GD3 binding proteins from rat brain to clarify the ganglioside-mediated signal transduction and identified two of them as Src family tyrosine kinase Lyn. The mouse IgG3 anti-ganglioside GD3monoclonal antibody R24 was obtained from hybridoma R24(American Type Culture Collection no. HB8445). Anti-Lyn (Lyn8), anti-Fyn (Fyn301), and anti-Yes (3H9) monoclonal antibodies were purchased from Wako Chemicals (Osaka, Japan). Anti-Src monoclonal antibody (GD11) was a gift from Dr. Y. Fukui (Faculty of Life Science and Agriculture, University of Tokyo). Anti-Lyn rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-caveolin polyclonal antibody and horseradish peroxidase-conjugated anti-phosphotyrosine antibody (PY20) were purchased from Transduction Laboratories (Lexington, KY). Fluorescein isothiocyanate (FITC) 2The abbreviations used are: FITC, fluorescein isothiocyanate; MAPK, mitogen-activated protein kinase; PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; DIG, detergent-insoluble glycosphingolipid-enriched complex. -conjugated goat anti-mouse IgG antibody was purchased from Zymed (San Francisco, CA). Phosphospecific mitogen-activated protein kinase (MAPK) antibody was purchased from New England BioLabs (Beverly, MA). Triton X-100 and EGTA were purchased from Sigma. Cultures were prepared from cerebella of 7-day-old rats as described by Yuzaki (25Yuzaki M. Mikoshiba K. J. Neurosci. 1992; 12: 4253-4263Crossref PubMed Google Scholar). In brief, cells were isolated by trypsinization, followed by trituration in DNase solution; they were then suspended in Fischer's serum-free medium. Membrane fraction was prepared from adult Wistar rat brain. Brains were homogenized in ice-cold buffer A (0.32 m sucrose 1 mm Tris-HCl, pH 7.4, 0.1 mm EDTA) using a Teflon motor-driven glass homogenizer. The homogenate was centrifugated at 900 × g for 10 min. The supernatant was centrifugated at 11,500 × g for 20 min. The resulting pellet of the rat brain or centrifugated cultured cells was solubilized in lysis buffer (1% Triton X-100, 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mmNa3VO4, 1 mm EGTA, 1 mmphenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml pepstatin A) at 4 °C for 20 min. The postnuclear supernatants were collected after centrifugation at 14,000 rpm for 3 min. Aliquots (0.5 ml, 750 μg of protein) of the supernatants were precleared with protein G-Sepharose (7.5 μl), and then incubated with anti-GD3antibody R24 (2.5 μg) for 1 h and precipitated with protein G-Sepharose (7.5 μl). Following immunoprecipitation, the beads were washed three times with lysis buffer, washed once with kinase buffer (30 mm HEPES, pH 7.5, 10 mm MgCl2, 2 mm MnCl2), and resuspended in 20 μl of kinase buffer. The reaction was started by addition of 5 μCi of [γ-32P]ATP (3,000 Ci/mmol, NEN Life Science Products) and incubated for 10 min at room temperature. Phosphorylation was stopped by the addition of Laemmli sample buffer, and samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by autoradiography. In a re-immunoprecipitation experiment, after the kinase reaction the samples were boiled for 5 min in lysis buffer with 1% SDS, diluted 10-fold with lysis buffer, and then re-immunoprecipitated with anti-Src family kinase antibodies or R24. Phosphoamino acid analysis was performed as described (26Mori A. Aizawa H. Saido T.C. Kawasaki H. Mizuno K. Murofushi H. Suzuki K. Sakai H. Biochemistry. 1991; 30: 9341-9346Crossref PubMed Scopus (38) Google Scholar). Phosphoprotein radiolabeled with32Pi (p53/56) was eluted from a polyacrylamide gel and hydrolyzed in 6 m hydrochloric acid at 105 °C for 2 h. The hydrolysate was evaporated and resuspended in 50 μl of carrier phosphoamino acid solution containing 1 mmphosphotyrosine, phosphothreonine, and phosphoserine. The solution was then subjected to cellulose thin layer chromatography (TLC) with a developing solution consisting of 1-butanol, isopropyl alcohol, formic acid, and water (3:1:1:1). The plate was dried, sprayed with ninhydrin to determine the positions of phosphoamino acids, and then subjected to autoradiography. Rat primary cerebellar cultures were harvested 24 h after seeding by pipetting. The cells were treated first with R24 and then with FITC-conjugated anti-mouse IgG antibody and were analyzed on a FACScan (Becton-Dickinson). Chinese hamster ovary (CHO) cells were transfected transiently (48 h) with the pME18S expression plasmid (27Takebe Y. Seiki M. Fujisawa J.-I. Hoy P. Yokota K. Arai K.-I. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar) containing Lyn (28Takeuchi M. Kuramochi S. Fusaki N. Nada S. Kawamura-Tsuzuku J. Matsuda S. Semba K. Toyoshima K. Okada M. Yamamoto T. J. Biol. Chem. 1993; 268: 27413-27419Abstract Full Text PDF PubMed Google Scholar) or c-Src (29Umemori H. Sato S. Yagi T. Aizawa S. Yamamoto T. Nature. 1994; 367: 572-576Crossref PubMed Scopus (352) Google Scholar) cDNA using LipofectAMINE™ (Life Technologies, Inc.) according to the manufacturer's instructions. Expression of transgenes was confirmed by immunoblotting. Lyn was not detected endogenously in CHO cells. Although Src was endogenously detected in CHO cells, transient expression greatly enhanced Src. CHO cells express solely the ganglioside GM3, an enzymatic substrate of GD3 synthase. We previously established a CHO cell line, CST, expressing GD3 synthase and synthesizing GD3 constitutively by stable transfection of full-length human GD3 synthase cDNA (20Ogura K. Nara K. Watanabe Y. Kono K. Tai T. Sanai Y. Biochem. Biophys. Res. Commun. 1996; 225: 932-938Crossref PubMed Scopus (67) Google Scholar). In this experiment, we used CST cells as GD3-positive cells and parental CHOP cells (CHO expressing polyoma large T) as GD3-negative cells (17Nara K. Watanabe Y. Maruyama K. Kasahara K. Nagai Y. Sanai Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7952-7956Crossref PubMed Scopus (123) Google Scholar). Metabolic labeling of glycosphingolipids was performed using 2 μCi/ml [14C] galactose (300 mCi/mmol, NEN Life Science Products) for 24 h. Lipids in immunoprecipitates were extracted with chloroform/methanol (1/1, v/v) by sonication and separated on a silica gel TLC in a solvent system of chloroform, methanol, and 0.5% CaCl2 (55/45/10, v/v/v). Cells (5 × 106) were incubated with 20 μg/ml R24 at 37 °C for the indicated times on dish. After washing with ice-cold PBS, lysates were prepared in 1% Triton X-100, 50 mm Tris-HCl, pH 7.4, 150 mmNaCl, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, and 5 μg/ml pepstatin A at 4 °C. After centrifugation at 14,000 rpm for 3 min, the supernatants were incubated with anti-Lyn antibody and precipitated with protein G-Sepharose. The immunoprecipitates were incubated with kinase buffer containing 10 μm ATP and 5 μCi of [γ-32P]ATP (3,000 Ci/mmol). Kinase activity was measured by in vitro autophosphorylation. Sucrose density gradient analysis was performed according to the method described (30Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2618) Google Scholar). Rat cerebellum or CHO transfectants were homogenized using a Teflon glass homogenizer in TNE/Triton X-100 buffer (1% Triton X-100, 25 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EGTA). The lysate was brought to 40% sucrose. A linear sucrose gradient (5–30%) in TNE without Triton X-100 was layered over the lysate. Gradients were centrifuged for 16–20 h at 39,000 rpm at 4 °C in a Hitachi RPS40T rotor. Nine fractions were harvested from 5% to 30% sucrose by bottom puncture. Fraction 10 was collected from 40% sucrose. Immunoprecipitates with anti-GD3 antibody (R24) from Triton X-100 extract of adult rat brain membrane were analyzed for the presence of protein kinase activity by an in vitro kinase assay. In vitrokinase reaction resulted in phosphorylation of several proteins of 40, 53, 56, and 80 kDa, as judged by SDS-PAGE (Fig.1). No kinase activity was detected in R24 precipitates performed in the presence of 30 μmGD3 or in immunoprecipitates with control mouse IgG3. The present study dealt with identification of 53- and 56-kDa proteins (p53/56). The 32P-labeled p53/56 were eluted from SDS-PAGE and hydrolyzed with 6 m hydrochloric acid. The hydrolysate was separated by thin layer chromatography. Radioactivity was detected only in the position of phosphotyrosine (Fig.2).Figure 2Phosphoamino acid analysis of p53/56.The phosphoamino acid of the p53/56 band in in vitro kinase assay was examined. The markers used were phosphotyrosine (P-Tyr), phosphothreonine (P-Thr), and phosphoserine (P-Ser).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The molecular weight and tyrosine phosphorylation of p53/56 suggested that it could be a Src family tyrosine kinase. To investigate this possibility, we have compared the SDS-PAGE patterns of in vitro phosphorylated proteins that were immunoprecipitated from membrane fraction of rat brain by antibodies specific to several members of the Src tyrosine kinase family, i.e. Src, Fyn, Yes, and Lyn, to that of GD3 specific antibody R24. The pattern of phosphorylated proteins obtained with anti-Lyn antibody resembled most closely the protein pattern resulted from R24 immunoprecipitation, with p53/56 migrating exactly as two autophosphorylated splice isoforms, p53 lyn and p56 lyn , of Lyn (Fig. 3 A). Phosphorylation of 40- and 80-kDa proteins were also observed in immunoprecipitates by anti-Lyn antibody. The bands of p53/56 and p53/56 lyn were excised and subjected to comparative peptide mapping after digestion with V8 protease. As shown in Fig. 3 B, the maps of p53/56 and p53/56 lyn appeared indistinguishable. The maps differed from those of Src, Fyn, and Yes (data not shown). The identification was confirmed by sequential immunoprecipitation with R24 and anti-Lyn antibody. The in vitro kinase assay was performed with R24 immunoprecipitates, after which the immune complexes were disrupted by boiling in SDS-containing buffer and subjected to a second immunoprecipitation with antibodies specific to Src, Fyn, Yes, and Lyn. Anti-Lyn antibody, but not anti-Src, -Fyn, or -Yes antibodies, precipitated specifically the p53/56 in re-immunoprecipitation experiments (Fig. 3 C). R24 did not precipitate p53/56 after SDS boiling, indicating that R24 does not bind p53/56 directly. The association of Lyn with GD3 was confirmed using a cDNA expression system in CHO cells. GM3(NeuAcα2,3Galβ1, 4Glcβ1,1-ceramide) is the only ganglioside synthesized in CHO cells and is an enzymatic substrate of GD3 synthase. We have previously established an CHO cell line, CST, constitutively expressing GD3 synthase (20Ogura K. Nara K. Watanabe Y. Kono K. Tai T. Sanai Y. Biochem. Biophys. Res. Commun. 1996; 225: 932-938Crossref PubMed Scopus (67) Google Scholar). Both CHO and CST cells were transfected transiently with an expression plasmid carrying cDNA for a p56 splice isoform of Lyn. Lyn activity was co-precipitated by R24 from CST cells that constitutively synthesized GD3, but not from CHO cells (Fig.4 A). In a control transfection with a Src expression plasmid, R24 did not precipitated Src activity in CST cells expressing Src (Fig. 4 B). This finding suggests that R24 does not precipitate the whole membrane protein. To substantiate further the association of Lyn with GD3, CST cells overexpressing p56 lyn were metabolically labeled with [14C]galactose. After immunoprecipitation with anti-Lyn antibody, lipids were extracted from the co-immunoprecipitates and subjected to TLC and autoradiography. 14C-Labeled GD3 was detected in the immunoprecipitate with anti-Lyn antibody, but not with control mouse IgG. 3K. Kasahara, Y. Watanabe, T. Yamamoto, and Y. Sanai, unpublished observation. Therefore, we conclude that ganglioside GD3 associates with Src family tyrosine kinase Lyn in rat brain. Lyn is known to be localized in cerebellar granule cells (31Umemori H. Wanaka A. Kato H. Takeuchi M. Tohyama M. Yamamoto T. Mol. Brain Res. 1992; 16: 303-310Crossref PubMed Scopus (117) Google Scholar). We examined the possibility of GD3-Lyn association in rat primary cerebellar cultures, of which more than 90% are granule cells. Flow cytometric analysis using R24 revealed that GD3was expressed on the cell surface (Fig.5 A). As expected, R24 also co-precipitated Lyn from Triton X-100 extract of the cells (Fig.5 B). To clarify the possibility of GD3-mediated signal transduction via Lyn, we measured Lyn activity in primary cerebellar cultures after treatment with R24. For this, Triton X-100 extracts were prepared from primary cerebellar cultures, which were treated with R24 for 0.5–30 min. Immunoprecipitation was carried out with anti-Lyn antibody. Kinase activity was measured by in vitro autophosphorylation. R24 treatment resulted in a rapid (within 1 min) and significant (3-fold) increase of Lyn activity, with no change in the amount of Lyn protein (Fig. 6). A possible increase in tyrosine phosphorylation of cellular proteins as a result of treatment of rat primary cerebellar cultures with R24 was investigated. After incubation with R24, cells were extracted with 1% Triton X-100 and cell extracts were fractionated into supernatant and particulate fraction. After SDS-PAGE, phosphotyrosines were detected by anti-phosphotyrosine immunoblotting. Treatment with R24 induced tyrosine phosphorylation of several proteins in the supernatants, including a prominent phosphorylation of a protein of about 80 kDa (Fig. 7). The phosphorylation peaked at 1 min and returned to the control level at 30 min. One of the detected proteins was identified as MAPK, using phosphospecific MAPK antibody. The antibody detects p42 and p44 MAPK only when it is catalytically activated by phosphorylation at Tyr-204. The phosphorylation peaked at 5 min and returned to the control level at 30 min. This observation suggests that R24 stimulates the MAPK cascade via a Lyn signaling pathway. Ganglioside is known to form clusters in the outer leaflet of the lipid bilayer (32Thompson T.E. Tillack T.W. Annu. Rev. Biophys. Chem. 1985; 14: 361-386Crossref PubMed Scopus (331) Google Scholar). Lyn anchors on the inner leaflet via N-terminal lipid modification, palmitoylation, and myristoylation (33Resh M.D. Cell. 1994; 76: 411-413Abstract Full Text PDF PubMed Scopus (593) Google Scholar). How does GD3associate with Lyn? The association is probably due to the presence of detergent-insoluble glycosphingolipid-enriched complex (DIG) or caveolae-like domains on the cell surface of the brain (34Parton R.G. Simons K. Science. 1995; 269: 1398-1399Crossref PubMed Scopus (296) Google Scholar). These complexes are known to be enriched in Src family tyrosine kinase, and can be isolated as a low density light-scattering band by sucrose density gradient analysis. Therefore, we investigated the distribution of Lyn in a sucrose density gradient. Subcellular fractionation of extracts from either rat cerebellum or CST cells transfected with p56 lyn was performed in parallel. Most of Lyn from a rat cerebellum and about 50% of Lyn from CST cells in a 150-mm dish was present in the light scattering band at fractions 3–5 (Fig.8, B and E, respectively). Src from rat cerebellum was detected in the same fractions as Lyn; however, most of the Src from CST cells overexpressing Src in a 150-mm dish was found at the bottom of the gradient (Fig. 8, C and F, respectively). This discrepancy probably derives from differences in the protein/detergent ratio during the initial homogenization, as has recently been pointed out (35Millán J. Puertollano R. Fan L. Alonso M.A. Biochem. Biophys. Res. Commun. 1997; 233: 707-712Crossref PubMed Scopus (38) Google Scholar), because most of Lyn and about 70% of Src from the transfectants in 10 150-mm dishes were present in the light scattering band (data not shown). In the CST cells, caveolin (a marker protein of caveolae) was also present in the low density fraction (Fig.8 G). Caveolin was not detected in homogenate of cerebellum (data not shown). We investigated whether R24 immunoprecipitated caveolae of CHO cells. In CHO cells transfected with GD3 synthase, R24 co-precipitated caveolin (Fig. 9). In the present study, we demonstrated that a monoclonal antibody to ganglioside GD3, R24, co-immunoprecipitates Src family tyrosine kinase Lyn from Triton X-100 extracts of rat brain and primary cerebellar cell cultures. This suggests that there is a specific association of Lyn with GD3 on rat brain cell membrane. The main region of the GD3-Lyn association in brain may be cerebellar granule cells. Indeed, both Lyn mRNA and protein are predominantly expressed in the cerebellar granule cells, as has been shown by in situ hybridization and immunohistochemistry (31Umemori H. Wanaka A. Kato H. Takeuchi M. Tohyama M. Yamamoto T. Mol. Brain Res. 1992; 16: 303-310Crossref PubMed Scopus (117) Google Scholar,36Chen S. Bing R. Rosenblum N. Hillman D.E. Neuroscience. 1996; 71: 89-100Crossref PubMed Scopus (17) Google Scholar). The same area expresses GD3 synthase and synthesizes GD3 (19Watanabe Y. Nara K. Takahashi H. Nagai Y. Sanai Y. J. Biochem. (Tokyo). 1996; 120: 1020-1027Crossref PubMed Scopus (23) Google Scholar, 37Graus F. Cordon-Cardo C. Houghton A.N. Melamed M.R. Old L.J. Brain Res. 1984; 324: 190-194Crossref PubMed Scopus (38) Google Scholar, 38Kotani M. Kawashima I. Ozawa H. Ogura K. Ishizuka I. Terashima T. Tai T. Glycobiology. 1994; 4: 855-865Crossref PubMed Scopus (63) Google Scholar, 39Kotani M. Terashima T. Tai T. 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The phosphatidylinositol turnover and cell proliferation can be blocked by a tyrosine kinase inhibitor (41Welte K. Miller G. Chapman P.B. Yuasa H. Natoli E. Kunicka J.E. Cordon-Cardo C. Buhrer C. Old L.J. Houghton A.N. J. Immunol. 1987; 139: 1763-1771PubMed Google Scholar, 42Norihisa Y. McVicar D.W. Ghosh P. Houghton A.N. Longo D.L. Creekmore S.P. Blake T. Ortaldo J.R. Young H.A. J. Immunol. 1994; 152: 485-495PubMed Google Scholar, 43Ortaldo J.R. Mason A.T. Longo D.L. Beckwith M. Creekmore S.P. McVicar D.W. J. Leukocyte Biol. 1996; 60: 533-539Crossref PubMed Scopus (36) Google Scholar), suggesting that GD3-mediated tyrosine kinase activation has an important role in the activation of T cells. The association of Src family kinase Lyn with gangliosides has also been reported for rat basophilic leukemia RBL-2H3 cells (44Oliver C. Sahara N. Kitani S. Robbins A.R. Mertz L.M. Siraganian R.P. J. Cell Biol. 1992; 116: 635-646Crossref PubMed Scopus (46) Google Scholar, 45Minoguchi K. 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These data suggest that the interaction of Src family kinase with ganglioside could play a role in receptor-mediated signal transduction. Binding of R24 to GD3 in rat primary cerebellar cultures induced phosphorylation of MAPK at Tyr-204, which was shown to be critical
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