Translocation of tyrosine-phosphorylated TCRζ chain to glycolipid-enriched membrane domains upon T cell activation

Recent studies point to glycolipid-enriched membrane (GEM) microdomains as the critical sites for TCR-mediated signal transduction. However, whether the TCR complex is localized in the GEM domain is not well-defined. In the present study, we analyzed localization of the TCR-CD3 complex in the GEM domain by isolating the GEM fraction with sucrose density gradient centrifugation. Although 10% of TCR zeta chains was localized in the GEM fraction, most of the TCR complexes were excluded from the GEM before and after T cell activation, and the amount of TCR zeta in the GEM was not increased after activation. However, the tyrosine-phosphorylated form of TCR zeta was strongly concentrated in the GEM fraction upon TCR engagement, A kinetic study revealed that tyrosine phosphorylation of TCR zeta occurred initially in the Triton X-100-soluble membrane fraction followed by the accumulation of phosphorylated TCR zeta in the GEM. Thus, these results indicate that phosphorylated TCR zeta migrates into the GEM domains on T cell activation. We speculate that the GEM microdomains may function as a reservoir of activation signals from triggered TCR.