Association of Src Family Tyrosine Kinase Lyn with Ganglioside GD3 in Rat Brain

Abstract

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 G D3 (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 G M3 , the enzymatic substrate of G D3 synthase. In cotransfection with G D3 synthase and Lyn expression plasmids, R24 immunoprecipitated Lyn and anti-Lyn antibody immunoprecipitated G D3 . 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 G D3 may regulate Lyn in a caveolae-like domain on brain cell membranes.

Gangliosides are functional nerve cell ligands for myelin-associated glycoprotein (MAG), an inhibitor of nerve regeneration

“…It may be significant that GD1a is enriched in these domains (relative to other gangliosides) in CGNs in culture (29). In separate studies, Ab-induced crosslinking of ganglioside GM3 in melanoma cells (43) or GD3 in neurons (44) resulted in activation of raft-resident signaling molecules (Src and Lyn, respectively). These data established a potential functional link between ganglioside crosslinking and intracellular signaling pathways.…”

Src Kinase Mediates the Regulation of Phospholipase C-γ Activity by Glycosphingolipids

“…Signal transduction of non-receptor tyrosine kinases has also been demonstrated in association with several glycosphingolipids. This includes the activation of c-Src by exogenous GM3 (25), the activation of Lyn by anti-ganglioside GD3 antibody (22), activation of Lck with exogenous GM1 (26), the activation of Lyn with anti-␣GalGD1b antibody (27), and the activation of Yes by the globotriaosylceramide binding toxin shiga toxin (23). The mechanism responsible for the regulation of non-receptor tyrosine kinases by glycosphingolipids is not understood.…”

“…Several studies have now implicated the association of most every non-receptor tyrosine kinase with glycosphingolipids. These include c-Src (19), Lyk, Fyn (20), Lyn (21,22), Yes (23), Cbl (24), FAK (19), and Csk (25). These studies have either localized these tyrosine kinases in raft fractions by density gradient separations or have coimmunoprecipitated these kinases with anti-glycosphingolipid antibodies.…”

Involvement of Gangliosides in Glycosylphosphatidylinositol-anchored Neuronal Cell Adhesion Molecule TAG-1 Signaling in Lipid Rafts

“…Previously, we found that R24 treatment of primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of p80 and that Lyn was present in the lipid raft fraction of sucrose density gradient analysis (21). Therefore, we investigated whether tyrosine phosphorylation of p80 was detected in the lipid raft fraction.…”

“…We previously demonstrated that anti-ganglioside GD3 antibody (R24) coimmunoprecipitates the Src family kinase Lyn from the rat cerebellum (21). R24 treatment of primary cerebellar cultures induced Lyn activation and rapid tyrosine phosphorylation of an 80-kDa protein (p80).…”