Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor

The role of lipid rafts in T cell antigen receptor (TCR) signaling was inve stigated using fluorescence microscopy. Lipid rafts labeled with cholera to xin B subunit (CT-B) and cross-linked into patches displayed characteristic s of rafts isolated biochemically, including detergent resistance and coloc alization with raft-associated proteins. LCK, LAT, and the TCR all colocali zed with lipid patches, although TCR association was sensitive to nonionic detergent. Aggregation of the TCR by anti-CD3 mAb cross-linking also caused coaggregation of raft-associated proteins. However, the protein tyrosine p hosphatase CD45 did not colocalize to either CT-B or CD3 patches. Cross-lin king of either CD3 or CT-B strongly induced tyrosine phosphorylation and re cruitment of a ZAP-70(SH2)(2)-green fluorescent protein (GFP) fusion protei n to the lipid patches. Also, CT-B patching induced signaling events analag ous to TCR stimulation, with the same dependence on expression of key TCR s ignaling molecules. Targeting of LCK to rafts was necessary for these event s, as a nonraft-associated transmembrane LCK chimera, which did not colocal ize with TCR patches, could not reconstitute CT-B-induced signaling. Thus, our results indicate a mechanism whereby TCR engagement promotes aggregatio n of lipid rafts, which facilitates colocalization of LCK, LAT, and the TCR whilst excluding CD45, thereby triggering protein tyrosine phosphorylation .