title: Involvement of gangliosides in the process of Cbp/PAG phosphorylation by Lyn in developing cerebellar growth cones.
abstract: The association of gangliosides with specific proteins in the central nervous system was examined by coimmunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 (R24) immunoprecipitated the Csk (C-terminal src kinase)-binding protein (Cbp). Sucrose density gradient analysis showed that Cbp of rat cerebellum was detected in detergent-resistant membrane (DRM) raft fractions. R24 treatment of the rat primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of Cbp. Treatment with anti-ganglioside GD1b antibody also induced tyrosine phosphorylation. Furthermore, over-expressions of Lyn and Cbp in Chinese hamster ovary (CHO) cells resulted in tyrosine 314 phosphorylation of Cbp, which indicates that Cbp is a substrate for Lyn. Immunoblotting analysis showed that the active form of Lyn and the Tyr314-phosphorylated form of Cbp were highly accumulated in the DRM raft fraction prepared from the developing cerebellum compared with the DRM raft fraction of the adult one. In addition, Lyn and the Tyr314-phosphorylated Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. Immunoelectron microscopy showed that Cbp and GAP-43, a growth cone marker, are localized in the same vesicles of the growth cone fraction. These results suggest that Cbp functionally associates with gangliosides on growth cone rafts in developing cerebella.
authors.affiliation: Laboratory of Biomembrane, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Sekino-Suzuki
authors.forename: Kohei
authors.initials: K
authors.lastname: Yuyama
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Miki
authors.forename: Mizuho
authors.initials: M
authors.lastname: Kaneda
authors.forename: Hidenori
authors.initials: H
authors.lastname: Suzuki
authors.forename: Naomasa
authors.initials: N
authors.lastname: Yamamoto
authors.forename: Tadashi
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Chitose
authors.initials: C
authors.lastname: Oneyama
authors.forename: Masato
authors.initials: M
authors.lastname: Okada
authors.forename: Kohji
authors.initials: K
authors.lastname: Kasahara
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies / Q000494:pharmacology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology* / Q000254:growth & development*; D006224:Cricetinae; D005732:Gangliosides / Q000276:immunology / Q000378:metabolism*; D020439:Growth Cones / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D000906:Antibodies; D005732:Gangliosides; D008565:Membrane Proteins; C408142:Pag1 protein, rat; D010750:Phosphoproteins; C033851:ganglioside, GD1b; D014443:Tyrosine; C026226:ganglioside, GD3; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases