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© The Rockefeller University Press,
0021-9525/2000//199 $5.00
The Journal of Cell Biology, Volume 151, Number 2,
, 2000 199-208
Original Article |
Selective Accumulation of Raft-Associated Membrane Protein Lat in T Cell Receptor Signaling Assemblies
Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR–CD3 complexes and associated signaling proteins. We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation–dependent manner. In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates. Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/TCR assemblies form a structural scaffold for TCR signal transduction proteins. Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.
Key Words: membrane protein assemblies • signal transduction • membrane rafts • palmitoylation • cholesterol
© 2000 The Rockefeller University Press
Abbreviations used in this paper: Ab, antibody; APC, antigen presenting cell; DRMs, detergent resistant membranes; GM1, ganglioside GM1; ITAM, immunoreceptor tyrosine-based activating motif; LAT, linker for activation of T cells; mAb, mouse monoclonal antibody; MβCD, methyl β cyclodextrin; MHC, major histocompatibility complex; PM, plasma membrane; PTK, protein tyrosine kinase; SH2, Src homology 2 domain; TCR, T cell antigen receptor; TfR, transferrin receptor.
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