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.
Harder
T
T
Basel Institute for Immunology, CH-4005, Basel, Switzerland.
Kuhn
M
M
D048868:Adaptor Proteins, Signal Transducing; D017252:CD3 Complex / Q000276:immunology / Q000302:isolation & purification; D002352:Carrier Proteins / Q000302:isolation & purification*; D002458:Cell Fractionation / Q000379:methods; D002784:Cholesterol / Q000302:isolation & purification; D006801:Humans; D018189:Immunomagnetic Separation; D019169:Jurkat Cells; D021962:Membrane Microdomains; D008565:Membrane Proteins / Q000302:isolation & purification*; D019308:Palmitic Acid / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000302:isolation & purification*; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D017252:CD3 Complex; D002352:Carrier Proteins; C110810:LAT protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; D019308:Palmitic Acid; D002784:Cholesterol