Association of GPI-anchored protein TAG-1 with src-family kinase Lyn in lipid rafts of cerebellar granule cells.
We have demonstrated that antibody-mediated crosslinking of GPI-anchored TAG-1 induced activation of src-family kinase Lyn and rapid tyrosine phosphorylation of an 80-kDa protein (p80), a putative substrate for Lyn, in the lipid raft fraction prepared from primary cerebellar cultures, suggesting the functional association of TAG-1 with Lyn in lipid rafts of the rat cerebellum. In this study, the association was confirmed using a cDNA expression system. TAG-1-expressing CHO transfectants exhibited enhanced self-aggregation and promoted neurite outgrowth of primary cerebellar cultures as a culture substrate. The anti-TAG-1 antibody co-immunoprecipitated Lyn with TAG-1 and induced co-patching of TAG-1 with Lyn in both TAG-1 and Lyn-expressing CHO transfectants. Density gradient analysis revealed that TAG-1 is present in the lipid raft fraction of the CHO transfectants. Furthermore, pretreatment with a sphingolipid biosynthesis inhibitor ISP-1 reduced the extent of tyrosine phosphorylation of p80 by the antibody-mediated crosslinking of TAG-1. Immunocytochemical study showed that both TAG-1 and Lyn are present in cerebellar granule cells. These observations suggest that TAG-1 associates with Lyn in lipid rafts of rat cerebellar granule cells.
Kasahara
Kohji
K
Department of Biochemical Cell Research, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Japan.
Watanabe
Kazutada
K
Kozutsumi
Yasunori
Y
Oohira
Atsuhiko
A
Yamamoto
Tadashi
T
Sanai
Yutaka
Y
D000818:Animals; D016466:CHO Cells; D015816:Cell Adhesion Molecules, Neuronal / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D058969:Contactin 2; D006224:Cricetinae; D003594:Cytoplasmic Granules / Q000201:enzymology / Q000378:metabolism*; D018076:DNA, Complementary; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D050356:Lipid Metabolism; D011233:Precipitin Tests; D012799:Sialyltransferases / Q000235:genetics; D014162:Transfection; D019061:src-Family Kinases / Q000378:metabolism*
D015816:Cell Adhesion Molecules, Neuronal; C548676:Cntn2 protein, rat; D058969:Contactin 2; D018076:DNA, Complementary; D017261:Glycosylphosphatidylinositols; D012799:Sialyltransferases; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases