Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies

Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylatio ns that mediate the assembly of signaling protein complexes. Moreover, chol esterol-sphingolipid raft membrane domains have been implicated to play a r ole in TCR signal transduction. Here, we studied the assembly of TCR with s ignal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate pla sma membrane subfragments that were highly concentrated in activated TCR-CD 3 complexes and associated signaling proteins. We found that the raft trans membrane protein linker for activation of T cells (LAT), but not a palmitoy lation-deficient non-raft LAT mutant, strongly accumulated in TCR-enrichcd immunoisolates in a tyrosine phosphorylation-dependent manner. In contrast, other raft-associated molecules, including protein tyrosine kinases Lck an d Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched p lasma membrane immunoisolates, Many downstream signaling proteins coisolate d with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/ TCR assemblies form a structural scaffold for TCR signal transduction prote ins. Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane pr oteins and lipids, form by a selective protein-mediated anchoring of the ra ft membrane protein LAT in vicinity of TCR.