Association of GM3 with Zap-70 Induced by T Cell Activation in Plasma Membrane Microdomains

Recent evidence demonstrated that T cell activationleads to the redistribution of membrane and intracellu-lar kinase-rich raft microdomains at the site of TCRengagement. In this investigation we demonstrated byhigh performance thin layer chromatography, gas chro-matographic, and mass spectrometric analyses thatGM3 is the main ganglioside constituent of these mi-crodomains in human lymphocytes. Then we analyzedGM3 distribution and its interaction with the phospho-rylation protein Zap-70. Human T lymphocytes werestimulated with anti-CD3 and anti-CD28. Immunofluo-rescence microscopy analysis revealed a clustered GM3distribution over the cell surface and an intracellularlocalization resembling specific cytoplasmic compart-ment(s). Scanning confocal microscopy showed that Tcell activation induced a significant association be-tween GM3 and Zap-70, as revealed by nearly completecolocalization areas; very few colocalization areas weredetected in unstimulated cells. Coimmunoprecipitationexperiments revealed that GM3 was immunoprecipi-tated by anti-Zap-70 only after co-stimulation throughCD3 and CD28 as detected by both thin layer chromatog-raphy and immunoblotting. Therefore, T cell activationdoes not promote a redistribution of glycosphingolipid-enriched microdomains but induces Zap-70 translocationin selective membrane domains in which Zap-70 mayinteract with GM3. These findings suggest that GM3 is acomponent of a multimolecular signaling complex in-volved in T cell activation.