Glycosphingolipid-enriched Signaling Domain in Mouse Neuroblastoma Neuro2a Cells

Abstract

Differentiation and neuritogenesis of mouse neuro- blastoma Neuro2a cells are induced by exogenous gan- glioside 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-en- riched domain” (GEM), we studied the mechanism of the ganglioside effect, focusing on the structure and func- tion 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 exog- enously 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 in- corporated exclusively in the high density fraction. c- Src, the predominant signal transducer in the microdo- main, 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 addi- tion 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. Acti- vation 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.