| 1998 |
LAT (linker for activation of T cells) was cloned as a novel integral membrane protein of 36-38 kDa that is phosphorylated by ZAP-70/Syk protein tyrosine kinases upon TCR engagement, leading to recruitment of multiple signaling molecules including Grb2, PLC-γ1, and the p85 subunit of PI3-kinase. Overexpression of a tyrosine-mutant LAT lacking critical tyrosine residues inhibited T cell activation, demonstrating its essential adapter function. |
cDNA cloning, co-immunoprecipitation, dominant-negative overexpression in T cells |
Cell |
High |
9489702
|
| 1998 |
LAT is palmitoylated at cysteines C26 and C29, and this palmitoylation is essential for its localization into glycolipid-enriched membrane microdomains (GEMs/lipid rafts). Loss of palmitoylation abolishes LAT tyrosine phosphorylation upon TCR activation, demonstrating that GEM targeting is required for LAT signaling function. |
Palmitoylation assay, detergent-resistant membrane fractionation, site-directed mutagenesis of palmitoylation sites, phosphorylation assays |
Immunity |
High |
9729044
|
| 1998 |
LAT is required for TCR-mediated activation of PLC-γ1 and the Ras pathway. The LAT-deficient Jurkat variant J.CaM2 is defective in calcium increases, Ras activation, and IL-2 expression despite intact TCR-ζ chain and ZAP-70 phosphorylation; reconstitution with LAT restored all signaling events. |
Genetic complementation in LAT-deficient Jurkat cell line (J.CaM2), calcium flux, Ras activation assay, IL-2 reporter |
Immunity |
High |
9846483
|
| 1999 |
LAT gene disruption in mice causes a complete block of T cell development at the CD4−CD8− (double-negative) stage in the thymus, with no mature peripheral T cells, while B cell and NK cell populations are grossly normal, establishing LAT as essential for early T cell development. |
Gene targeting (knockout mouse), flow cytometric analysis of lymphocyte populations |
Immunity |
High |
10204488
|
| 2000 |
Mapping of LAT tyrosine residues revealed that Tyr171, Tyr191, and Tyr226 are responsible for Grb2 binding; Tyr171 and Tyr191 (but not Tyr226) are required for Gads binding; and Tyr132 alone is required for PLC-γ1 binding. Mutation of Tyr132 abolished calcium flux and blocked ERK and NFAT activation, while Grb2 binding was unaffected, indicating PLC-γ1 activation regulates Ras activation. |
Tyrosine-to-phenylalanine mutagenesis of LAT, reconstitution in LAT-deficient Jurkat cells, Co-IP, calcium flux, ERK/NFAT assays |
The Journal of biological chemistry |
High |
10811803
|
| 1999 |
Gads constitutively binds SLP-76 via its C-terminal SH3 domain and a proline-rich region of SLP-76, and inducibly associates with tyrosine-phosphorylated LAT via its SH2 domain upon TCR stimulation. Gads thereby bridges LAT and SLP-76 signaling complexes, promoting NFAT activation synergistically. |
Co-immunoprecipitation, domain-deletion mutants, NFAT reporter assay |
Current biology : CB |
High |
10021361
|
| 1999 |
LAT is required for tyrosine phosphorylation of PLC-γ2 and platelet activation downstream of the collagen receptor GPVI. In LAT-deficient platelets, Syk and Btk phosphorylation are maintained but PLC-γ2 phosphorylation, phosphatidic acid formation, PKC substrate phosphorylation, P-selectin expression, and integrin αIIbβ3 activation are markedly reduced in response to CRP. LAT tyrosine phosphorylation is abolished in Syk-deficient platelets, placing LAT downstream of Syk. |
LAT-knockout mouse platelets, phosphorylation assays, aggregation and secretion assays |
Molecular and cellular biology |
High |
10567557
|
| 2000 |
LAT is essential for FcεRI-mediated mast cell activation. LAT-deficient mast cells show intact FcεRI, Syk, and Vav phosphorylation but severely reduced SLP-76, PLC-γ1, and PLC-γ2 phosphorylation, calcium mobilization, MAPK activation, degranulation, and cytokine production after FcεRI cross-linking. LAT-deficient mice are resistant to IgE-mediated passive systemic anaphylaxis. |
LAT-knockout mouse-derived bone marrow mast cells, phosphorylation assays, calcium flux, degranulation assays, anaphylaxis model |
Immunity |
High |
10843385
|
| 1999 |
LAT is required for TCR-mediated Ca2+ mobilization and optimal tyrosine phosphorylation of PLC-γ1, Vav, and SLP-76, as well as ERK activation, CD69 upregulation, and AP-1/NFAT transcription. The LAT transmembrane domain and palmitoylation at Cys26 (but not Cys29) are required for LAT function and TCR signaling. |
LAT-deficient Jurkat cell line generation, reconstitution with LAT mutants, phosphorylation assays, calcium flux, transcription reporter assays |
International immunology |
High |
10360968
|
| 2001 |
TCR engagement triggers actin polymerization-driven T cell spreading requiring LAT. LAT-deficient cells failed to form and maintain the TCR-induced lamellipodia and actin-rich contact rings characteristic of T cell activation, placing LAT upstream of actin cytoskeletal remodeling. |
Live imaging of T cells on antibody-coated coverslips, LAT-deficient cell lines, actin inhibitors, morphometric analysis |
Immunity |
Medium |
11290340
|
| 2002 |
A single tyrosine mutation in LAT (Y136F), disrupting PLC-γ1 binding, causes an early block in T cell maturation but subsequently leads to polyclonal lymphoproliferation and autoimmune disease. TCR-induced PLC-γ1 activation, NFAT/calcium signaling, IL-2 production, and cell death were abolished while Erk activation was intact, demonstrating that LAT-PLC-γ1 interaction is critical for integrated signaling controlling T cell development and homeostasis. |
Knock-in mouse (Y136F point mutation), T cell functional assays, calcium flux, NFAT reporter, IL-2 ELISA, ERK phosphorylation |
Science (New York, N.Y.) |
High |
12065840
|
| 2004 |
Signaling clusters containing LAT are internalized into distinct intracellular compartments after TCR activation. The ubiquitin ligase c-Cbl mediates LAT ubiquitylation, internalization, and downregulation; c-Cbl RING-domain mutants suppress LAT ubiquitylation and increase cellular LAT levels and basal/TCR-induced LAT phosphorylation. |
Live fluorescence imaging of LAT-YFP, c-Cbl RING mutant expression, ubiquitylation assay, c-Cbl knockout T cells |
Molecular and cellular biology |
High |
17938199
|
| 2004 |
LAT exists in two distinct intracellular pools: one at the plasma membrane and one co-distributing with transferrin-labeled intracellular compartments. Distribution between pools is dependent on LAT intracytoplasmic residues. Plasma membrane LAT is recruited to immune synapses within seconds, while the intracellular pool polarizes and recruits over minutes. LAT tyrosines 136, 175, 195, and 235 are required for LAT's own recruitment to the immune synapse. |
Time-lapse video imaging of LAT-GFP in live T lymphocytes conjugated with APCs, transferrin co-localization, LAT mutant analysis |
Journal of cell science |
Medium |
14996932
|
| 2006 |
In anergic T cells, LAT is hypophosphorylated due to impaired palmitoylation, resulting in defective LAT recruitment to the immunological synapse and to detergent-resistant membrane fractions. Upstream signaling (CD3ζ and ZAP-70 phosphorylation) remains intact, identifying defective LAT palmitoylation as the upstream target of anergy induction. This effect was independent of Cbl-b. |
Anergy induction in primary murine CD4+ T cells, palmitoylation assay, DRM fractionation, immunological synapse imaging, phosphorylation assays |
Immunity |
High |
16713970
|
| 2006 |
Palmitoylation of LAT is required not only for lipid raft targeting but also for LAT trafficking to the plasma membrane and protein stability. Non-palmitoylated LAT mutants failed to traffic to the plasma membrane and were unstable, subject to proteasomal degradation. |
LAT palmitoylation-site mutants, subcellular fractionation, proteasome inhibitor treatment, protein stability assays |
Biochemical and biophysical research communications |
Medium |
16460687
|
| 2009 |
TCR and LAT exist in separate protein islands (membrane domains) in resting T cells. Upon T cell activation, these domains concatenate to form signaling microclusters. This was demonstrated using PALM, dual-color fluorescence cross-correlation spectroscopy, and transmission electron microscopy. |
High-speed photoactivated localization microscopy (PALM), dual-color fluorescence cross-correlation spectroscopy, transmission electron microscopy |
Nature immunology |
High |
20010844
|
| 2011 |
LAT is ubiquitylated at specific lysine residues, and ubiquitylation-resistant LAT mutants (lysine-to-arginine substitutions) showed normal internalization rates but defective protein turnover. Cells with ubiquitylation-resistant LAT had elevated T cell signaling, establishing LAT ubiquitylation as a checkpoint for attenuating T cell signaling. |
Site-directed mutagenesis (Lys→Arg), ubiquitylation assay, internalization assay, signaling readouts |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
21282648
|
| 2013 |
VAMP7, a vesicular SNARE, is required for recruitment of LAT-containing vesicles to TCR-activation sites and controls phosphorylation of LAT and formation of the TCR-LAT signaling complex. VAMP7 localizes together with LAT on subsynaptic vesicles, and vesicle docking (not fusion with plasma membrane) regulates TCR-induced signaling. |
siRNA silencing, genetically modified mice (VAMP7-deficient), live imaging, phosphorylation assays, immune synapse analysis |
Nature immunology |
High |
23666293
|
| 2013 |
TRAF6 is recruited to the immune synapse through direct interaction with LAT via its TRAF domain. TRAF6 promotes LAT ubiquitylation at Lys88 via K63-linked chains and LAT tyrosine phosphorylation and association with ZAP-70, thereby positively regulating TCR/CD28-induced NFAT activation. |
Co-immunoprecipitation, immune synapse imaging, TRAF6 knockdown, overexpression of catalytically inactive mutant, site-directed mutagenesis of LAT Lys88 |
Journal of immunology (Baltimore, Md. : 1950) |
Medium |
23514740
|
| 2016 |
SHP-1 directly dephosphorylates LAT (specifically Tyr132) and PLC-γ1/γ2 in NK cells. Dephosphorylation of LAT Tyr132 by SHP-1 abrogates PLC-γ recruitment to the NK-target cell immune synapse, reducing degranulation and target killing. Additionally, LAT is ubiquitylated by c-Cbl and Cbl-b in response to inhibitory receptor engagement, leading to LAT degradation and abrogation of NK cell cytotoxicity. |
SHP-1 phosphatase substrate identification, phospho-specific antibodies, Cbl knockdown, ubiquitylation assays, LAT mutant (ubiquitylation-resistant) expression, NK cytotoxicity assay |
Science signaling |
Medium |
27221712
|
| 2018 |
Lck acts as a molecular bridge to facilitate LAT phosphorylation by ZAP-70. A conserved proline-rich motif in LAT mediates binding to the Lck SH3 domain, while Lck SH2 domain binds phospho-ZAP-70, thereby co-localizing ZAP-70 with LAT. Elimination of this proline-rich motif in LAT compromises TCR signaling and T cell development. |
Identification of LAT proline-rich motif, SH3 binding assay, mutagenesis of LAT proline-rich motif, T cell signaling assays, T cell development analysis |
Nature immunology |
High |
29915297
|
| 2018 |
LAT internalized from the plasma membrane transits through the Golgi/trans-Golgi network (TGN) before being re-delivered to the immune synapse. This retrograde transport depends on the small GTPase Rab6 and the t-SNARE Syntaxin-16. Knockdown or knockout of Rab6 or Syntaxin-16 impairs LAT recruitment to the immune synapse and TCR-stimulated signaling. |
Rab6 knockout mouse, Syntaxin-16 silencing, live-cell imaging of LAT trafficking, Golgi colocalization, TCR signaling assays |
The Journal of experimental medicine |
High |
29440364
|
| 2018 |
Plasma membrane LAT is recruited and phosphorylated at TCR activation sites in the first phase of T cell activation, while vesicular LAT is subsequently recruited dynamically to microclusters via microtubule-directed movement in a second phase. Correlative 3D light and electron microscopy showed absence of vesicles at microclusters at early times but abundance at later times. |
Lattice light sheet microscopy, TIRF-SIM, correlative 3D light and electron microscopy, live-cell imaging |
Nature communications |
High |
29789604
|
| 2019 |
Phosphorylation of LAT at Y132 (the PLC-γ1 recruitment site) by ZAP-70 is rate-limiting for T cell ligand discrimination. The slow phosphorylation kinetics at Y132 are governed by an adjacent glycine residue (G131). Substituting G131 with aspartate or glutamate accelerates Y132 phosphorylation, increases PLC-γ1 activation speed and magnitude, and enhances T cell sensitivity to weak agonists and self-peptides, disrupting ligand discrimination. |
Site-directed mutagenesis (G131D/E), in vitro kinase assay, PLC-γ1 activation assay, T cell ligand discrimination assays |
Nature immunology |
High |
31611699
|
| 2009 |
Cytoskeletal protein 4.1R binds directly to LAT and inhibits its phosphorylation by ZAP-70. 4.1R-deficient CD4+ T cells show enhanced LAT phosphorylation and ERK activation, hyperproliferation, and increased IL-2/IFN-γ production. 4.1R is recruited to the immunological synapse upon TCR stimulation. |
4.1R-knockout mouse, direct binding assay (4.1R–LAT), phosphorylation assays, T cell functional assays |
Blood |
Medium |
19190245
|
| 2019 |
The golgin GMAP210 tethers LAT-containing vesicles to the Golgi and facilitates their specific delivery to the immune synapse. Upon T cell activation, GMAP210 interactions with LAT-containing VAMP7-decorated vesicles increase, and GMAP210 co-migrates with LAT to the immune synapse. Overexpression of GMAP210 domains disrupts LAT delivery, and GMAP210 similarly controls LAT delivery to cilia in a heterologous model. |
Co-immunoprecipitation, vesicle rerouting and capture assay, live-cell imaging, domain overexpression, immune synapse analysis |
Nature communications |
Medium |
31253807
|
| 2015 |
IFT20, an intraflagellar transport protein, controls polarized delivery of intracellular LAT to the immune synapse in primary CD4+ T cells. In IFT20-deficient T cells, centrosome polarization and cell spreading were normal, but LAT recruitment to the immune synapse was reduced, impairing TCR signaling, T cell activation, and proliferation in vitro and antigen-specific responses in vivo. |
Conditional IFT20-knockout mouse in T cells, immune synapse imaging, TCR signaling assays, in vivo T cell response and colitis transfer models |
Proceedings of the National Academy of Sciences of the United States of America |
High |
26715756
|
| 2021 |
Zap70 and LAT form membrane-localized condensates/clusters during T cell activation, and signaling from these clusters requires a positive feedback loop among Zap70, LAT, and Src-family kinases that bind phosphorylated LAT to further activate Zap70. Optogenetically induced LAT clustering (but not one-to-one Zap70:LAT heterodimers) is sufficient to drive downstream signaling and calcium responses. |
Optogenetic clustering system with single-point mutation control, synthetic Zap70:LAT cluster expression in fibroblasts, calcium response assay in T cells |
Cell reports |
High |
34161759
|
| 2022 |
A single pMHC:TCR binding event is sufficient to trigger a LAT condensate. LAT condensate formation is self-limiting and neither size nor lifetime correlates with the originating pMHC:TCR binding duration; only the probability of condensate formation is related to binding dwell time. A LAT mutation facilitating Y132 phosphorylation (G131D) shortens the delay to condensation and alters T cell antigen specificity. |
Single-molecule imaging of pMHC:TCR binding events combined with LAT condensation monitoring, LAT G131D mutant analysis |
Nature communications |
High |
36460640
|
| 2023 |
Accelerating Y136 (mouse; equivalent to human Y132) phosphorylation by the adjacent Gly135Asp substitution in LAT (LATG135D) disrupts ligand discrimination in vivo, causing excessive thymic negative selection, T cell anergy, altered effector/memory balance during infection, and autoimmunity features, demonstrating that the normally slow phosphorylation kinetics of this site constitute a physiological proofreading step. |
LATG135D knock-in mouse, thymic selection assays, infection model (Listeria), autoimmunity phenotyping, T cell anergy assays |
Nature immunology |
High |
36914891
|
| 2017 |
LFA-1 cross-linking activates FAK1/PYK2, which phosphorylates LAT selectively at Y171, promoting formation of a LAT-GRB2-SKAP1 complex distinct from canonical LAT-Gads-SLP-76 complexes. This LFA-1-FAK1-LAT-Y171 pathway decreases T cell–dendritic cell dwell times and reduces T cell conjugation and proliferation. |
Co-immunoprecipitation, site-directed mutagenesis (LAT Y171F), kinase assays, T cell–DC conjugation assay, proliferation assay |
Nature communications |
Medium |
28699640
|
| 2013 |
Absence of LAT results in augmented and persistent tyrosine phosphorylation of CD3ζ and ZAP-70 (revealed by quantitative phosphoproteomics), demonstrating that LAT signaling hub participates in negative feedback to modulate upstream phosphorylation events, in addition to its established role in positive signal propagation. |
MS-based quantitative phosphoproteomics comparing LAT-sufficient and LAT-deficient Jurkat T cells |
PloS one |
Medium |
24204825
|
| 2000 |
Raft-localized LAT, but not palmitoylation-deficient non-raft LAT mutant, selectively accumulates in TCR-enriched plasma membrane immunoisolates in a tyrosine phosphorylation-dependent manner, forming a structural scaffold for TCR signal transduction proteins. Other raft markers (Lck, Fyn, GM1, cholesterol) do not concentrate similarly, indicating protein-mediated selective anchoring. |
Plasma membrane immunoisolation, palmitoylation-deficient LAT mutant, Western blotting for signaling proteins and lipid markers |
The Journal of cell biology |
Medium |
11038169
|
| 2002 |
Redox balance alterations (reduced intracellular glutathione) cause membrane displacement of LAT associated with a conformational change detectable by gel electrophoresis, leading to abrogated TCR-mediated signaling. Mutation of redox-sensitive cysteine residues in LAT produces redox-insensitive mutants that remain membrane-anchored under oxidative stress conditions and restore TCR signaling. |
Glutathione depletion, native vs. non-reducing denaturing PAGE, cysteine mutagenesis, TCR signaling assays |
Molecular and cellular biology |
Medium |
11756537
|
| 2006 |
LAT is a dual substrate for both Lck and Syk/ZAP-70 kinases. LAT phosphorylation is absent in Lck-deficient cells; Lck co-precipitates with LAT; and in vitro kinase assays with purified Lck demonstrate direct phosphorylation of LAT by Lck at ITAM-like motifs Y171/Y191. |
Lck-deficient cell line, co-immunoprecipitation, in vitro kinase assay with purified proteins |
Leukemia research |
Medium |
16938345
|