Glycosphingolipid-enriched signaling domain in mouse neuroblastoma Neuro2acells - Mechanism of ganglioside-dependent neuritogenesis

Differentiation and neuritogenesis of mouse neuroblastoma Neuro2a cells are induced by exogenous ganglioside but are not induced by nerve growth facto r because its receptor is absent in these cells. In view of the emerging co ncept of the "glycosphingolipid-enriched domain" (GEM), we studied the mech anism of the ganglioside effect, focusing on the structure and function of such a domain. GEM in Neuro2a cells, separated as a low density membrane fr action, contains essentially all glycosphingolipids and sphingomyelin, toge ther with five signal transducer molecules (c-Src, Lyn, Csk, Rho A, Ha-Ras) . H-3-Labeled Il(3)NeuAc-LacCer (GM3), Gb4Cer (globoside), and Il(3)NeuAc-G g4Cer (GM1) added exogenously to cells were incorporated and concentrated i n the low density GEM fk action. In contrast, more than 50% of glycerophosp holipids and 30% of cholesterol were found in the high density fraction. H- 3-Labeled phosphatidylcholine added exogenously to cells was incorporated e xclusively in the high density fraction. c-Src, the predominant signal tran sducer 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 stimula tion 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 a ctivation 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 cel ls. Activation of c-Src was followed by rapid and prolonged (60 min) enhanc ement of mitogen-activated protein kinase activity leading to neuritogenesi s. Thus, the ganglioside induction of neuritogenesis in Neuro2a cells is me diated by GEM structure and function.