LAT (linker for activation of T cells) is a transmembrane adaptor protein that plays an essential role in TCR-mediated signaling and thymocyte development. Because LAT-deficient mice have an early block in thymocyte development, we utilized an inducible system to delete LAT in primary T cells to study LAT function in T cell activation, homeostasis, and survival. Deletion of LAT caused primary T cells to become unresponsive to stimulation from the TCR and impaired T cell homeostatic proliferation and long term survival. Furthermore, deletion of LAT led to reduced expression of Foxp3, CTLA-4, and CD25 in T reg cells and impaired their function. Consequently, mice with LAT deleted developed a lymphoproliferative syndrome similar to that in LATY136F mice, although less severe. Our data implicate that LAT has positive and negative roles in the regulation of mature T cells.Upon engagement of the TCR (T cell receptor), 3 LAT (linker for activation of T cells) is phosphorylated at multiple tyrosine residues by ZAP-70 kinase (1) and functions as a protein scaffold to assemble a large membrane-tethered signalosome (1-3). LAT binding of Gads leads to the membrane recruitment of SLP-76, which in turn interacts with Itk, as well as Vav1 and PLC-␥1. Through its interaction with LAT and SLP-76, PLC-␥1 is also brought to the plasma membrane. Upon activation by ZAP-70 and Itk, PLC-␥1 hydrolyzes phosphatidylinositol 4,5-bisphosphate into the secondary messengers inositol trisphosphate and diacylglycerol (4, 5). Inositol trisphosphate induces Ca 2ϩ mobilization, whereas diacylglycerol binds to RasGRP1 to activate the Ras-MAPK pathway (6, 7). LAT also contributes to Ras-MAPK activation through recruitment of the Grb2-Sos complex to the plasma membrane to activate Ras (1, 8). Initiation of these signaling cascades eventually leads to activation of transcription factors that regulate the genes critical for T cell proliferation and effector functions.The essential role of LAT in T cell activation was initially demonstrated in LAT-deficient Jurkat T cells. Although the proximal signals upstream of LAT phosphorylation remain intact in these cells, TCR-mediated calcium mobilization, Ras-MAPK activation, and NFAT activation are all abolished (9, 10). LAT function during T cell development has also been well characterized. Thymocyte development in LAT Ϫ/Ϫ mice is completely blocked at the CD25 ϩ CD44 Ϫ DN3 stage, indicating an absolute requirement for LAT in pre-TCR signaling (11). Our recent studies using LAT conditional knock-out mice show that LAT is also required during thymocyte development at the DP to SP transition, as positive selection in LAT-deficient DP thymocytes is markedly compromised in these mice. Consequently, the maturation of SP thymocytes is severely blocked (12). In addition, a point mutation at LAT tyrosine 136 (Y136F), which specifically abolishes the LAT-PLC-␥1 interaction, renders both positive and negative thymic selection processes defective in the LATY136F knock-in mice (13), highlighting the importance of LAT-m...

Background: RasGRP4 is one member of the RasGRP family and is highly expressed in mast cells. Results: Fc⑀RI signaling, mast cell function, and thymocyte development are severely impaired by RasGRP1 and RasGRP4 deficiency. Conclusion: This study indicated that the RasGRP family is important in mast cells and T cells.Significance: This work demonstrates immune cells employ multiple members of the RasGRP family to activate the Ras-Erk pathway.

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is essential to bridge T cell receptor (TCR) engagement to downstream signaling events. The indispensable role of LAT in thymocyte development and T cell activation has been well characterized; however, the function of LAT in cytotoxic-T-lymphocyte (CTL) cytotoxicity remains unknown. We show here that LAT-deficient CTLs failed to upregulate FasL and produce gamma interferon after engagement with target cells and had impaired granule-mediated killing. We further dissected the effect of the LAT deletion on each step of granule exocytosis. LAT deficiency led to altered synapse formation, subsequently causing unstable T cell-antigen-presenting cell (APC) conjugates. Microtubule organizing center polarization and granule reorientation were also impaired by LAT deficiency, leading to reduced granule delivery. Despite these defects, granule release was still observed in LAT-deficient CTLs due to residual calcium flux and phospholipase C (PLC) activity. Our data demonstrated that LAT-mediated signaling intricately regulates CTL cytotoxicity at multiple steps.

The Ras-guanyl nucleotide exchange factor RasGRP1 plays a critical role in T cell receptor-mediated Erk activation. Previous studies have emphasized the importance of RasGRP1 in the positive selection of thymocytes, activation of T cells, and control of autoimmunity. RasGRP1 consists of a number of well-characterized domains, which it shares with its other family members; however, RasGRP1 also contains an ∼200 residue-long tail domain, the function of which is unknown. To elucidate the physiological role of this domain, we generated knock-in mice expressing RasGRP1 without the tail domain. Further analysis of these knock-in mice showed that thymocytes lacking the tail domain of RasGRP1 underwent aberrant thymic selection and, following TCR stimulation, were unable to activate Erk. Furthermore, the deletion of the tail domain led to enhanced CD4 + T cell expansion in aged mice, as well as the production of autoantibodies. Mechanistically, the tail-deleted form of RasGRP1 was not able to traffic to the cell membrane following stimulation, indicating a potential reason for its inability to activate Erk. While the DAG-binding C1 domain of RasGRP1 has long been recognized as an important factor mediating Erk activation, we have revealed the physiological relevance of the tail domain in RasGRP1 function and control of Erk signaling.

The LAT Family of Adaptor Proteins Transmembrane adaptor proteins (TRAPs) are critical components of signaling pathways in lymphocytes, linking antigen receptor engagement to downstream cellular processes. While these proteins lack intrinsic enzymatic activity, their phosphorylation following receptor ligation allows them to function as scaffolds for the assembly of multi-molecular signaling complexes. Many TRAPs have recently been discovered and numerous studies demonstrate their roles in the positive and negative regulation of lymphocyte maturation, activation, and differentiation. One such example is the LAT (linker for activation of T cells) family of adaptor proteins. While LAT has been shown to play an indispensable role in T cell and mast cell function, the other family members, LAB (linker for activation of B cells) and LAX (linker for activation of X cells), are necessary to fine-tune immune responses. In addition to its well-established role in the positive regulation of lymphocyte activation, LAT exerts an inhibitory effect on TCR (T cell receptor)-mediated signaling. Furthermore, LAT, along with LAB and LAX, plays a crucial role in establishing and maintaining tolerance. Here, we review recent data concerning the regulation of lymphocyte development and activation by the LAT family of proteins.

The Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76) is a cytosolic adaptor protein essential for thymocyte development and T-cell activation. It contains a sterile-alpha motif (SAM) domain, 3 phosphotyrosine motifs, a proline-rich region, and a Src homology 2 domain. Whereas the other domains have been extensively studied, the role of the SAM domain in SLP-76 function is not known. To understand the function of this domain, we generated SLP-76 knockin mice with the SAM domain deleted. Analysis of these mice showed that thymocyte development was partially blocked at the double-positive to single-positive transition. Positive and negative thymic selection was also impaired. In addition, we analyzed T-cell receptor (TCR)-mediated signaling in T cells from these mutant mice. TCR-mediated inositol 1,4,5-triphosphate production, calcium flux, and extracellular signal-regulated kinase activation were decreased, leading to defective interleukin-2 production and proliferation. Moreover, despite normal association between Gads and SLP-76, TCR-mediated formation of SLP-76 microclusters was impaired by the deletion of the SAM domain. Altogether, our data demonstrated that the SAM domain is indispensable for optimal SLP-76 signaling.

Transmembrane adaptor proteins (TRAPs) link antigen receptor engagement to downstream cellular processes. Although these proteins typically lack intrinsic enzymatic activity, they are phosphorylated on multiple tyrosine residues following lymphocyte activation, allowing them to function as scaffolds for the assembly of multi-molecular signaling complexes. Among the many TRAPs that have been discovered in recent years, the LAT (linker for activation of T cells) family of adaptor proteins plays an important role in the positive and negative regulation of lymphocyte maturation, activation, and differentiation. Of the two members in this family, LAT is an indispensable component controlling T cell and mast cell activation and function; LAB (linker for activation of B cells), also called NTAL, is necessary to fine-tune lymphocyte activation and may be a key regulator of innate immune responses. Here, we review recent advances on the function of LAT and LAB in the regulation of development and activation of immune cells.

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that links T cell receptor (TCR) engagement to downstream signaling events. While it is clear that LAT is essential in thymocyte development and initiation of T cell activation, its function during T cell expansion, contraction, and memory formation remains unknown. To study the role of TCR-mediated signaling in CD8 T cells during the course of pathogen infection, we used an inducible mouse model to delete LAT in antigen-specific CD8 T cells at different stages of Listeria infection and analyzed the effect of deletion on T cell responses. Our data showed that LAT is important for maintaining CD8 T cell expansion during the priming phase; however, it is not required for CD8 T cell contraction and memory maintenance. Moreover, LAT deficiency accelerates memory differentiation during the effector-to-memory transition, leading to a higher frequency of KLRG1lowIL-7RhighCD62Lhigh memory T cells. Nonetheless, these LAT-deficient memory T cells were unable to proliferate or produce cytokines upon secondary infection. Our data demonstrated that, while it is dispensable for contraction and memory maintenance, TCR-mediated signaling regulates CD8 T cell memory differentiation and is essential for the memory response against pathogens.