Lawrence E. Samelson

LAT: The ZAP-70 Tyrosine Kinase Substrate that Links T Cell Receptor to Cellular Activation

Despite extensive study, several of the major components involved in T cell receptor-mediated signaling remain unidentified. Here we report the cloning of the cDNA for a highly tyrosine-phosphorylated 36-38 kDa protein, previously characterized by its association with Grb2, phospholipase C-gamma1, and the p85 subunit of phosphoinositide 3-kinase. Deduced amino acid sequence identifies a novel integral membrane protein containing multiple potential tyrosine phosphorylation sites. We show that this protein is phosphorylated by ZAP-70/Syk protein tyrosine kinases leading to recruitment of multiple signaling molecules. Its function is demonstrated by inhibition of T cell activation following overexpression of a mutant form lacking critical tyrosine residues. Therefore, we propose to name the molecule LAT-linker for activation of T cells.

LAT palmitoylation: its essential role in membrane microdomain targeting and tyrosine phosphorylation during T cell activation.

The linker molecule LAT is a critical substrate of the tyrosine kinases activated upon TCR engagement. Phosphorylated LAT binds Grb2, PLC-gamma1, and other signaling molecules. We demonstrate that human LAT is palmitoylated and that palmitoylated LAT predominantly localizes into glycolipid-enriched microdomains (GEMs). Although the LAT transmembrane domain is sufficient for membrane localization, palmitoylation at C26 and C29 is essential for efficient partitioning into GEMs. LAT palmitoylation is necessary for its tyrosine phosphorylation. After T cell activation, most tyrosine-phosphorylated LAT molecules and a fraction of PLC-gamma1 and other signaling molecules are present in GEMs. LAT is central to T cell activation and is a novel linker molecule shown to require targeting to membrane microdomains for signaling.

Essential Role of LAT in T Cell Development

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement. Phosphorylated LAT binds many critical signaling molecules. The central role of this molecule in TCR-mediated signaling has been demonstrated by experiments in a LAT-deficient cell line. To probe the role of LAT in T cell development, the LAT gene was disrupted by targeting. LAT-deficient mice appeared healthy. Flow cytometric analysis revealed normal B cell populations but the absence of any mature peripheral T cells. Intrathymic development was blocked within the CD4- CD8- stage. No gross abnormality of NK or platelet function was observed. LAT is thus critical to both T cell activation and development.

T cell receptor ligation induces the formation of dynamically regulated signaling assemblies

Tcell antigen receptor (TCR) ligation initiates tyrosine kinase activation, signaling complex assembly, and immune synapse formation. Here, we studied the kinetics and mechanics of signaling complex formation in live Jurkat leukemic T cells using signaling proteins fluorescently tagged with variants of enhanced GFP (EGFP). Within seconds of contacting coverslips coated with stimulatory antibodies, T cells developed small, dynamically regulated clusters which were enriched in the TCR, phosphotyrosine, ZAP-70, LAT, Grb2, Gads, and SLP-76, excluded the lipid raft marker enhanced yellow fluorescent protein–GPI, and were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70–containing clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 continuously dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results emphasize the dynamically changing composition of signaling complexes and indicate that these complexes can form within seconds of TCR engagement, in the absence of either lipid raft aggregation or the formation of a central TCR-rich cluster.

Zeta phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists

Small changes in the peptide-major histocompatibility complex (MHC) molecule ligands recognized by antigen-specific T cell receptors (TCRs) can convert fully activating complexes into partially activating or even inhibitory ones. This study examined early TCR-dependent signals induced by such partial agonists or antagonists. In contrast to typical agonist ligands, both an antagonist and several partial agonists stimulated a distinct pattern of zeta chain phosphorylation and failed to activate associated ZAP-70 kinase. These results identify a specific step in the early tyrosine phosphorylation cascade that is altered after TCR engagement with modified peptide-MHC molecule complexes. This finding may explain the different biological responses to TCR occupancy by these variant ligands.

Complex Complexes: Signaling at the TCR

The focus of this review has been on the complexity of biochemical events at a very proximal position in the TCR signaling cascade. A number of proteins and protein–protein interactions have been described. It should be noted that the function of many of these proteins (e.g., Vav, Cbl, PI3K, and SLP-76) remains to be defined. Likewise, a full accounting of all of the proteins involved in signaling complexes remains to be performed. The reader should know that extensive analysis of the pathways further downstream in the T cell and in other systems suggests that complexity exists at these levels too (Cantrell 1996xT cell antigen receptor signal transduction pathways. Cantrell, D. Annu. Rev. Immunol. 1996; 14: 259–274Crossref | PubMed | Scopus (551)See all ReferencesCantrell 1996). For example, Ras is likely to be only one of the many small G proteins involved in transduction events. Similarly, the well-known ERK kinase, which is most certainly involved in TCR-coupled events, is only one of several related serine/threonine kinases involved in signaling via TCR and other surface receptors. The complexity of the transcriptional machinery assembled at each lymphokine promoter is also well known. We also acknowledge that we have focused almost entirely on TCR-mediated signaling. Biochemical signals arising from engagement of coreceptors, costimulatory receptors, and adhesion molecules are also integrated to give the complex cellular response. Moreover, complex protein–protein interactions driven by these receptors are likely to be found. T cell activation can no longer be described only in terms of simple linear or even branching biochemical steps, but instead must also be thought of as a complex web of associated proteins.Several observations indicate that careful attention to nuances in the T cell signaling pathways is warranted. Activation of T cells with peptides or MHC molecules bearing subtle changes in sequence, so-called altered peptide ligands, results in dramatic differences in TCRζ and ZAP-70 tyrosine phosphorylation and ZAP-70 activity (64xPartial T cell signaling (altered phospho-ζ and lack of ZAP-70 recruitment in APL-induced T cell anergy) . Sloan-Lancaster, J, Shaw, A.S, Rothbard, J.B, and Allen, P.M. Cell. 1995; 79: 913–922Abstract | Full Text PDF | Scopus (541)See all References, 43xζ phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists. Madrenas, J, Wange, R.L, Wang, J.L, Isakov, N, Samelson, L.E, and Germain, R.N. Science. 1995; 267: 515–518Crossref | PubMedSee all References). Similarly, T cell activation schemes that result in anergic T cells fail to induce Ras and ERK activation (40xBlocked signal transduction to the ERK and JNK protein kinases in anergic CD4+ T cells. Li, W, Whaley, C.D, Mondino, A, and Mueller, D.L. Science. 1996; 271: 1272–1276Crossref | PubMedSee all References, 21xBlocked Ras activation in anergic CD4+ T cells. Fields, P.E, Gajewski, T.F, and Fitch, F.W. Science. 1996; 271: 1276–1278Crossref | PubMedSee all References). These examples are likely the first of many instances of variable biochemical responses to receptor engagement. Subtle changes in biochemical signals and in the composition of signaling complexes may have profound effects on cellular physiology. The existence of highly regulated signaling pathways offers the possibility of great flexibility of response to the T cell.

LAT Is Required for TCR-Mediated Activation of PLCγ1 and the Ras Pathway

Abstract In this study, we present the further characterization of a mutant Jurkat T cell line, J.CaM2, that is defective in TCR-mediated signal transduction. Although initial TCR-mediated signaling events such as the inducible tyrosine phosphorylation of the TCR-ζ chain and ZAP-70 are intact in J.CaM2, subsequent events, including increases in intracellular calcium, Ras activation, and IL-2 gene expression are defective. Subsequent analysis of J.CaM2 demonstrated a severe deficiency in pp36/LAT expression, a recently cloned adaptor protein implicated in TCR signaling. Importantly, reexpression of LAT in J.CaM2 restored all aspects of TCR signaling. These results demonstrate a necessary and exclusive role for LAT in T cell activation.

Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation

FcεRI activation of mast cells is thought to involve Lyn and Syk kinases proximal to the receptor and the signaling complex organized by the linker for activation of T cells (LAT). We report here that FcεRI also uses a Fyn kinase–dependent pathway that does not require Lyn kinase or the adapter LAT for its initiation, but is necessary for mast cell degranulation. Lyn-deficiency enhanced Fyn-dependent signals and degranulation, but inhibited the calcium response. Fyn-deficiency impaired degranulation, whereas Lyn-mediated signaling and calcium was normal. Thus, FcεRI-dependent mast cell degranulation involves cross-talk between Fyn and Lyn kinases.

Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT.

T cell activation induces functional changes in cell shape and cytoskeletal architecture. To facilitate the collection of dynamic, high-resolution images of activated T cells, we plated T cells on coverslips coated with antibodies to the T cell receptor (TCR). Using these images, we were able to quantitate the morphological responses of individual cells over time. Here, we show that TCR engagement triggers the formation and expansion of contacts bounded by continuously remodeled actin-rich rings. These processes are associated with the extension of lamellipodia and require actin polymerization, tyrosine kinase activation, cytoplasmic calcium increases, and LAT, an important hematopoietic adaptor. In addition, the maintenance of the resulting contact requires sustained calcium influxes, an intact microtubule cytoskeleton, and functional LAT.

Association of Grb2, Gads, and Phospholipase C-γ1 with Phosphorylated LAT Tyrosine Residues EFFECT OF LAT TYROSINE MUTATIONS ON T CELL ANTIGEN RECEPTOR-MEDIATED SIGNALING

The linker for activation of T cells (LAT) is a critical adaptor molecule required for T cell antigen receptor (TCR)-mediated signaling and thymocyte development. Upon T cell activation, LAT becomes highly phosphorylated on tyrosine residues, and Grb2, Gads, and phospholipase C (PLC)-γ1 bind LAT via Src homology-2 domains. In LAT-deficient mutant Jurkat cells, TCR engagement fails to induce ERK activation, Ca2+ flux, and activation of AP-1 and NF-AT. We mapped the tyrosine residues in LAT responsible for interaction with these specific signaling molecules by expressing LAT mutants with tyrosine to phenylalanine mutations in LAT-deficient cells. Our results showed that three distal tyrosines, Tyr171, Tyr191, and Tyr226, are responsible for Grb2-binding; Tyr171, and Tyr191, but not Tyr226, are necessary for Gads binding. Mutation of Tyr132 alone abolished PLC-γ1 binding. Mutation of all three distal tyrosines also abolished PLC-γ1 binding, suggesting there might be multiple binding sites for PLC-γ1. Mutation of Tyr132 affected calcium flux and blocked Erk and NF-AT activation. Since Grb2 binding is not affected by this mutation, these results strongly suggest that PLC-γ activation regulates Ras activation in these cells. Mutation of individual Grb2 binding sites had no functional effect, but mutation of two or three of these sites, in combination, also affected Erk and NF-AT activation.