Engagement of the T cell receptor (TCR) with MHC-peptide complexes leads to the activation of protein tyrosine kinases (PTK), ultimately leading to the activation of downstream signaling events, such as calcium flux and mitogen-activated protein kinase (MAPK) activation (Chan et al. 1994). For many years, it was observed that a 36–38 kDa protein, localized to the plasma membrane, was highly tyrosine phosphorylated upon ligation of the TCR and associated with Grb2, PLC-γ1, and the p85 subunit of PI3K. It was hypothesized that this protein served as a critical link connecting TCR engagement at the membrane to activation of signaling events in the cytosol. In 1998, the gene encoding this protein was identified after microsequencing phosphorylated proteins purified from the membrane fractions of activated Jurkat T cells and was named LAT, linker for activation of T cells. Sequencing of cDNA clones revealed that LAT is a type III transmembrane protein that contains a short extracellular domain, a transmembrane domain, and a long cytoplasmic tail. While LAT has no apparent structural domains, it has multiple tyrosine motifs for binding Grb2. Further biochemical analysis confirmed that it does interact with Grb2, PLC-γ1, and p85 (Zhang et al. 1998a). The indispensible role of LAT in TCR-mediated signaling and thymocyte development was subsequently demonstrated by studies using LAT-deficient Jurkat T cell lines and knockout mice. In 2003, the other member of the LAT protein family, linker for activation of B cells (LAB)/non-T cell activation linker (NTAL), was discovered (Janssen et al. 2003; Brdicka et al. 2002).
Biochemical analyses indicate that LAT is constitutively localized to lipid rafts, similar to Lck and Fyn. In the juxtamembrane region of LAT, there are two conserved cysteine residues, which are covalently modified by palmitate, a 16-carbon fatty acid. Studies using LAT cysteine mutants clearly demonstrate that palmitoylation is required for LAT phosphorylation and function in TCR-mediated signaling (Zhang et al. 1998b). Moreover, LAT palmitoylation is impaired in anergic T cells (Hundt et al. 2006). As of yet, the role of LAT palmitoylation remains unclear. While palmitoylation targets LAT to lipid rafts, raft localization is not essential for LAT function (Zhu et al. 2005). It is possible that palmitoylation is required for trafficking of LAT from the Golgi to the plasma membrane (Hundt et al. 2009) or simply for stably tethering LAT to the plasma membrane.
The Positive Role of LAT in TCR-Mediated Signaling
Upon T cell receptor ligation with an MHC-peptide complex, Src family kinases, specifically Lck and Fyn, are activated and phosphorylate the immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of the TCRζ and CD3 chains. Phosphorylation of ITAMs creates binding sites for ZAP-70, which is subsequently activated by Lck and Fyn. ZAP-70 phosphorylates LAT and other signaling proteins (Samelson 2002). There are nine conserved tyrosine residues between murine and human LAT. Among these tyrosine residues, the four distal tyrosines of LAT mediate the binding of Grb2, Gads, and PLC-γ1 and are vital for T cell activation. Grb2 recruits the Ras guanine nucleotide exchange factor, Son of sevenless (Sos), which activates the Ras-Raf-MAP kinase pathway and results in the activation of the transcription factor AP-1. In addition, Grb2 also stabilizes the interactions between LAT and its other binding partners, Gads and PLCγ1 (Zhu et al. 2003). By binding to LAT, Gads mediates the interaction between LAT and SLP-76, another important adaptor protein. SLP-76 regulates actin polymerization and cytoskeleton rearrangement via recruitment of Vav, the guanine nucleotide exchange factor for Rac/Rho. Additionally, SLP-76 stabilizes PLC-γ1 binding to LAT and recruits the Tec kinase, Itk, which phosphorylates and activates PLC-γ1 (Jordan et al. 2003). PLC-γ1 is responsible for the hydrolysis of phosphatidylinositol bisphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 is important for TCR-mediated calcium mobilization and subsequent activation of the transcription factor NFAT. DAG has dual functions in that it activates protein kinase C (PKC), triggering downstream NF-ĸB nuclear translocation, as well as serving to activate the Ras-Erk pathway through the Ras guanine nucleotide exchange factor, RasGRP1. Since all of these pathways depend on LAT for their activation, the absence of LAT has severe ramifications on TCR signaling. In LAT-deficient Jurkat cell lines, TCR-mediated calcium flux, Erk activation, and IL-2 production are greatly impaired compared to normal Jurkat cells. Phosphorylation of PLC-γ1 and SLP-76 in these cells is also reduced. These signaling defects can be corrected by the reconstitution of LAT expression in these cells (Finco et al. 1998).
In addition to its vital role in the TCR-mediated signaling pathway, LAT is also essential for T cell development in the thymus. LAT-deficient mice show an early block at the DN3 (CD44−CD25+) stage, indicating its importance in pre-TCR signaling. Consequently, LAT-deficient mice lack mature αβ T cells. Moreover, γδ T cells also need LAT signaling during development to enter the periphery (Zhang et al. 1999). Recent data indicate that LAT is necessary to transmit signals through a functional rearranged TCRαβ receptor during the transition from DP (CD4+CD8+) to SP (CD4+ or CD8+) (Shen et al. 2009).
In addition to its well-defined role as a positive regulator of TCR-mediated signaling, LAT is also critical for FcεRI-mediated signaling in mast cells. LAT-deficient mast cells have impaired MAPK activation, cytokine production, and degranulation. LAT−/− mice are resistant to IgE-mediated passive systemic anaphylaxis, indicating the essential role of LAT in FcεRI-mediated signaling in mast cells (Saitoh et al. 2000).
The Negative Role of LAT in TCR-Mediated Signaling
While LAT mainly serves to positively regulate TCR-mediated calcium mobilization and MAPK activation through binding Grb2, Gads, and PLC-γ1, it also negatively impacts TCR signaling by recruiting other proteins, such as Gab2 and SHIP-1. After TCR activation, Grb2 associated binding protein 2 (Gab2), which is constitutively associated with Gads and Grb2, is recruited to LAT and is then phosphorylated by Zap-70. Gab2, in turn, recruits SH2 domain-containing tyrosine phosphatase-2 (SHP2) to dephosphorylate key signaling molecules, such as CD3ζ (Yamasaki et al. 2003).
LAT can also recruit Src homology 2 domain–containing inositol polyphosphate 5-phosphatase-1 (SHIP-1) to the TCR complex through its interaction with Grb2. SHIP-1 is required to anchor downstream of kinase-2 (Dok-2) to the LAT complex as well as for Dok-2 tyrosine phosphorylation. Together with Dok-1, Dok-2 then negatively regulates Zap-70 and Akt kinase activation (Dong et al. 2006). Through its association with negative regulators, LAT plays a critical role in dampening the signals which emanate from the TCR.
LAT in Autoimmunity
Further evidence indicating that LAT plays a negative role in T cell activation comes from studies examining the interaction between LAT with PLC-γ1. LAT-deficient Jurkat T cells reconstituted with the LATY136F mutant, which fails to bind PLC-γ1, have impaired calcium flux and Erk activation (Zhang et al. 2000). To test the role of this interaction in vivo, two groups independently generated LATY136F knock-in mice. Analyses of these mice show that there is a partial block in thymocyte development at the DN3 stage, demonstrating the positive role of the LAT and PLC-γ1 interaction in thymocyte development. However, a small population of thymocytes is able to progress to the DP and SP stages. Mutant T cells that enter the periphery then undergo uncontrolled expansion, revealing the importance of the LAT and PLC-γ1 interaction in the control of T cell homeostasis. LATY136F mice display lymphadenopathy, splenomegaly, as well as lymphocyte infiltration in the lungs, liver, and kidneys. These mice have very few CD8+ T cells in the periphery and the CD4+ T cells are Th2-skewed, producing high levels of IL-4. They have low levels of surface TCR expression and have an activated/effector memory phenotype, characterized by high surface expression of CD44 and low surface expression of CD62L. As a result of the large population of Th2 T cells, B cells are hyperactivated, producing autoantibodies, mainly IgE and IgG1 (Sommers et al. 2002; Aguado et al. 2002).
Further analysis of LATY136F mice demonstrates that they do not contain natural T regulatory cells, which are CD4+CD25+Foxp3+. Adoptive transfer of normal T regulatory cells into neonatal LATY136F mice can prevent development of the autoimmune syndrome (Koonpaew et al. 2006). In addition, transfer experiments show that LATY136F CD4+ T cells can also expand in MHC Class II-deficient mice, indicating that the uncontrolled expansion of these mutant T cells is independent of the TCR-MHC-peptide interaction (Wang et al. 2008). Interestingly, while mice expressing the LATY136F mutant develop a severe lymphoproliferative syndrome, mice with LAT inducibly deleted in mature T cells also develop a similar, albeit less severe, autoimmune syndrome. LAT-deficient CD4+ T cells expand and secrete large amounts of cytokines despite a severe defect in TCR-mediated signaling (Shen et al. 2010).
Additionally, LAT3YF mice, in which LAT fails to bind Gads and Grb2 due to mutations at Y175, Y195, and Y235, exhibit uncontrolled T cell expansion in the periphery. These mice show a block in thymic development at the DN stage, with a complete block in αβ T cell development. Interestingly, γδ T cell development does not seem to be affected, and these cells exit the thymus and populate the periphery. In aged mice, these γδ T cells have undergone significant expansion and, unexpectedly, produce Th2 cytokines. Thus, B cell populations also expand, causing high serum levels of IgG1 and IgE (Nunez-Cruz et al. 2003). These data suggest a role for LAT in the negative regulation of γδ T cell expansion and indicate that αβ and γδ T cells have different requirements for LAT during thymic development. Together, these data clearly indicate the critical role of LAT in T cell homeostasis, but the mechanism by which LAT controls T cell homeostasis remains to be further investigated.
LAT, a transmembrane adaptor protein that is exclusively expressed in hematopoietic cells, is required for thymocyte development and LAT deficiency leads to a complete lack of mature T cells. LAT is also vital for T cell activation downstream of the TCR. LAT is constitutively localized in lipid rafts and contains two cysteine residues that can be palmitoylated. LAT palmitoylation is essential for its function in T cells. Upon T cell activation, LAT is phosphorylated on multiple tyrosine residues by ZAP-70 tyrosine kinase and interacts with Grb2, Gads, and PLC-γ1 directly, as well as Cbl, SLP-76, Vav, and other proteins indirectly. The ability of LAT to interact with these signaling proteins is required for TCR-mediated MAPK activation, calcium mobilization, and cytokine production. Recent studies indicate that LAT also controls T cell homeostasis. The deletion or mutation of LAT causes the development of a lymphoproliferative autoimmune syndrome due to uncontrolled T cell expansion and cytokine production. In summary, LAT is a key signaling molecule that functions in T cell development, activation, and homeostasis. LAT or LAT-mediated signaling pathways could serve as effective targets for the design of improved therapies to enhance or inhibit T cell function during immune responses.
- Wang Y, Kissenpfennig A, Mingueneau M, Richelme S, Perrin P, Chevrier S, et al. Th2 lymphoproliferative disorder of LatY136F mutant mice unfolds independently of TCR-MHC engagement and is insensitive to the action of Foxp3+ regulatory T cells. J Immunol. 2008;180:1565–75.PubMedCrossRefGoogle Scholar