Synergistic Assembly of Linker for Activation of T Cells Signaling Protein Complexes in T Cell Plasma Membrane Domains

Lorian C Hartgroves,Joseph Lin,Hanno Langen,Tobias Zech,Arthur Weiss,Thomas Harder

doi:10.1074/jbc.m301212200

Abstract

Transmembrane adaptor molecule LAT (linker for activation of T cells) forms a central scaffold for signaling protein complexes that accumulate in the vicinity of activated T cell antigen receptors (TCR). Here we used biochemical analysis of immunoisolated plasma membrane domains and fluorescence imaging of green fluorescence protein-tagged signaling proteins to investigate the contributions of different tyrosine-based signaling protein docking sites of LAT to the formation of LAT signaling protein assemblies in TCR membrane domains. We found that the phospholipase C gamma docking site of LAT and different Grb2/Gads docking sites function in an interdependent fashion and synergize to accumulate LAT, Grb2, and phospholipase C gamma in TCR signaling assemblies. Two-dimensional gels showed that Grb2 is a predominant cytoplasmic adaptor in the isolated LAT signaling complexes, whereas Gads, Crk-1, and Grap are present in lower amounts. Taken together our data suggest a synergistic assembly of multimolecular TCR.LAT signal transduction complexes in T cell plasma membrane domains.

Highlights

Activation of the T cell antigen receptor (TCR)1 by peptide/ major histocompatibility complex ligands triggers tyrosine phosphorylation cascades, which induce downstream signaling events and eventually lead to a physiological T lymphocyte response
LAT Lacking Tyrosine-based Protein Docking Sites Fails to Accumulate at the Site of TCR Engagement—Earlier work of our laboratory showed that TCR1⁄7LAT signaling assemblies (TLAs) form at the contact zone to ␣-CD3 antibody-coated TCR-activating beads [15]
Using imaging and biochemical analysis of TCR signaling membrane domains, we studied the role of the tyrosine-based protein docking sites of LAT in the accumulation of LAT and cytoplasmic signaling proteins in the vicinity of activated TCR

Summary

Introduction

Activation of the T cell antigen receptor (TCR)1 by peptide/ major histocompatibility complex ligands triggers tyrosine phosphorylation cascades, which induce downstream signaling events and eventually lead to a physiological T lymphocyte response. Reconstitution of LAT variants into LAT-deficient JCaM2 cells showed that these three tyrosines (Tyr- 6, -7, and -8) comprise a minimal set of docking sites for transducing Ca2ϩ fluxes and Ras-dependent downstream signaling [11].

Results

Conclusion