title: Association of GM3 with Zap-70 induced by T cell activation in plasma membrane microdomains: GM3 as a marker of microdomains in human lymphocytes.
abstract: Recent evidence demonstrated that T cell activation leads to the redistribution of membrane and intracellular kinase-rich raft microdomains at the site of TCR engagement. In this investigation we demonstrated by high performance thin layer chromatography, gas chromatographic, and mass spectrometric analyses that GM3 is the main ganglioside constituent of these microdomains in human lymphocytes. Then we analyzed GM3 distribution and its interaction with the phosphorylation protein Zap-70. Human T lymphocytes were stimulated with anti-CD3 and anti-CD28. Immunofluorescence microscopy analysis revealed a clustered GM3 distribution over the cell surface and an intracellular localization resembling specific cytoplasmic compartment(s). Scanning confocal microscopy showed that T cell activation induced a significant association between GM3 and Zap-70, as revealed by nearly complete colocalization areas; very few colocalization areas were detected in unstimulated cells. Coimmunoprecipitation experiments revealed that GM3 was immunoprecipitated by anti-Zap-70 only after co-stimulation through CD3 and CD28 as detected by both thin layer chromatography and immunoblotting. Therefore, T cell activation does not promote a redistribution of glycosphingolipid-enriched microdomains but induces Zap-70 translocation in selective membrane domains in which Zap-70 may interact with GM3. These findings suggest that GM3 is a component of a multimolecular signaling complex involved in T cell activation.
authors.affiliation: Dipartimento Medicina Sperimentale e Patologia, Università La Sapienza Roma, Italy.
authors.forename: Tina
authors.initials: T
authors.lastname: Garofalo
authors.forename: Luisa
authors.initials: L
authors.lastname: Lenti
authors.forename: Agostina
authors.initials: A
authors.lastname: Longo
authors.forename: Roberta
authors.initials: R
authors.lastname: Misasi
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Mattei
authors.forename: Giuseppe M
authors.initials: GM
authors.lastname: Pontieri
authors.forename: Antonio
authors.initials: A
authors.lastname: Pavan
authors.forename: Maurizio
authors.initials: M
authors.lastname: Sorice
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005455:Fluorescent Antibody Technique; D005679:G(M3) Ganglioside / Q000378:metabolism*; D006801:Humans; D008213:Lymphocyte Activation; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology*; D051746:ZAP-70 Protein-Tyrosine Kinase
chemical_list: D005679:G(M3) Ganglioside; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human