Cutting Edge: Localization of linker for activation of T cells to lipid rafts is not essential in T cell activation and development

It has been proposed that upon T cell activation, linker for activation of T cells (LAT), a transmembrane adaptor protein localized to lipid rafts, orchestrates formation of multiprotein complexes and activates signaling cascades in lipid rafts. However, whether lipid rafts really exist or function remains controversial. To address the importance of lipid rafts in LAT function, we generated a fusion protein to target LAT to nonraft fractions using the transmembrane domain from a nonraft protein, linker for activation of X cells (LAX). Surprisingly, this fusion protein functioned well in TCR signaling. It restored MAPK activation, calcium flux, and NFAT activation in LAT-deficient cells. To further study the function of this fusion protein in vivo, we generated transgenic mice that express this protein. Analysis of these mice indicated that it was fully capable of replacing LAT in thymocyte development and T cell function. Our results demonstrate that LAT localization to lipid rafts is not essential during normal T cell activation and development.     

Functional complementation of BLNK by SLP-76 and LAT linker proteins

Recent studies have demonstrated a requirement for the SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) and LAT (linker for activation of T cells) adaptor/linker proteins in T cell antigen receptor activation and T cell development as well as the BLNK (B cell linker) linker protein in B cell antigen receptor (BCR) signal transduction and B cell development. Whereas the SLP-76 and LAT adaptor proteins are expressed in T, natural killer, and myeloid cells and platelets, BLNK is preferentially expressed in B cells and monocytes. Although BLNK is structurally homologous to SLP-76, BLNK interacts with a variety of downstream signaling proteins that interact directly with both SLP-76 and LAT. Here, we demonstrate that neither SLP-76 nor LAT alone is sufficient to restore the signaling deficits observed in BLNK-deficient B cells. Conversely, the coexpression of SLP-76 and LAT together restored BCR-inducible calcium responses as well as activation of all three families of mitogen-activated protein kinases. Together, these data suggest functional complementation of SLP-76 and LAT in T cell antigen receptor function with BLNK in BCR function.     

Negative regulation of TCR signaling by linker for activation of X cells via phosphotyrosine-dependent and -independent mechanisms

The activation of T cells and the initiation of an immune response is tightly controlled through the crosstalk of both positive and negative regulators. Two adaptors that function as negative regulators of T cell activation are adaptor in lymphocytes of unknown function X (ALX) and linker for activation of X cell (LAX). Previously, we showed that T cells from mice deficient in ALX and LAX display similar hyperresponsiveness, with increased IL-2 production and proliferation upon TCR/CD28 stimulation, and that these adaptors physically associate. In this study, we analyze the nature of the association between ALX and LAX. We demonstrate that this association occurs in the absence of TCR/CD28 signaling via a mechanism independent of both tyrosine phosphorylation of LAX and the SH2 domain of ALX. Cotransfection of ALX with LAX resulted in LAX tyrosine phosphorylation in the absence of TCR/CD28 stimulation. ALX-mediated LAX phosphorylation depends upon the ALX SH2 domain, which functions to recruit Lck to LAX. We also show that LAX, like ALX, can inhibit RE/AP reporter activation. However, in contrast to its inhibition of NFAT, the inhibition of RE/AP by LAX is independent of its tyrosine phosphorylation. Therefore, it can be concluded that inhibition of signaling events involved in T cell activation by LAX occurs through mechanisms both dependent on and independent of its tyrosine phosphorylation.     

Modeling and simulation of aggregation of membrane protein LAT with molecular variability in the number of binding sites for cytosolic Grb2-SOS1-Grb2

The linker for activation of T cells (LAT), the linker for activation of B cells (LAB), and the linker for activation of X cells (LAX) form a family of transmembrane adaptor proteins widely expressed in lymphocytes. These scaffolding proteins have multiple binding motifs that, when phosphorylated, bind the SH2 domain of the cytosolic adaptor Grb2. Thus, the valence of LAT, LAB and LAX for Grb2 is variable, depending on the strength of receptor activation that initiates phosphorylation. During signaling, the LAT population will exhibit a time-varying distribution of Grb2 valences from zero to three. In the cytosol, Grb2 forms 1:1 and 2:1 complexes with the guanine nucleotide exchange factor SOS1. The 2:1 complex can bridge two LAT molecules when each Grb2, through their SH2 domains, binds to a phosphorylated site on a separate LAT. In T cells and mast cells, after receptor engagement, receptor phosphoyrlation is rapidly followed by LAT phosphorylation and aggregation. In mast cells, aggregates containing more than one hundred LAT molecules have been detected. Previously we considered a homogeneous population of trivalent LAT molecules and showed that for a range of Grb2, SOS1 and LAT concentrations, an equilibrium theory for LAT aggregation predicts the formation of a gel-like phase comprising a very large aggregate (superaggregate). We now extend this theory to investigate the effects of a distribution of Grb2 valence in the LAT population on the formation of LAT aggregates and superaggregate and use stochastic simulations to calculate the fraction of the total LAT population in the superaggregate.     

Minimal requirement of tyrosine residues of linker for activation of T cells in TCR signaling and thymocyte development

Linker for activation of T cells (LAT) is a membrane-associated adaptor protein that is phosphorylated on multiple tyrosines upon TCR cross-linking. Previous studies show that LAT is essential for TCR-mediated signaling and thymocyte development. In this study, we expressed a series of LAT Tyr to Phe mutants in LAT-deficient J.CaM2.5 cells and examined their tyrosine phosphorylation; association with Grb2, Gads, and phospholipase C (PLC)-gamma1; and function in T cell activation. Our results showed that the five membrane-distal tyrosines were phosphorylated upon T cell activation. Grb2, Gads, and PLC-gamma1 associated with LAT preferentially via different sets of tyrosine residues; however, they failed to interact with LAT mutants containing only one tyrosine. We also determined the minimal requirement of LAT tyrosine residues in T cell activation and thymocyte development. Our results showed that a minimum of three tyrosines is required for LAT to function in T cell activation and thymocyte development. LAT mutants that were capable of binding Grb2 and PLC-gamma1 could reconstitute T cell activation in LAT-deficient cells and thymocyte development in LAT-deficient mice.     

Ntal/Lab/Lat2

Non-T cell activation linker (NTAL)/linker for activation of B cells (LAB), now officially termed LAT2 (linker for activation of T cells 2) is a 25-30kDa transmembrane adaptor protein (TRAP) associated with glycolipid-enriched membrane fractions (GEMs; lipid rafts) in specific cell types of hematopoietic lineage. Tyrosine phosphorylation of NTAL/LAB/LAT2 is induced by FcvarepsilonRI aggregation and Kit dimerization in mast cells, FcgammaRI aggregation in monocytes, and BCR aggregation in B cells. NTAL/LAB/LAT2 is also expressed in resting NK cells but, unlike the related TRAP, LAT, not in resting T cells. As demonstrated in monocytes and B cells, phosphorylated NTAL/LAB/LAT2 recruits signaling molecules such as Grb2, Gab1 and c-Cbl into receptor-signaling complexes. Although gene knock out and knock down studies have indicated that NTAL/LAB/LAT2 may function as both a positive and negative regulator of mast cell activation, its precise role in the activation of these and other hematopoietic cells remains enigmatic.     

T cell receptor (TCR)-induced tyrosine phosphorylation dynamics identifies THEMIS as a new TCR signalosome component

Stimulation of the T cell antigen receptor (TCR) induces formation of a phosphorylation-dependent signaling network via multiprotein complexes, whose compositions and dynamics are incompletely understood. Using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we investigated the kinetics of signal propagation after TCR-induced protein tyrosine phosphorylation. We confidently assigned 77 proteins (of 758 identified) as a direct or indirect consequence of tyrosine phosphorylation that proceeds in successive "signaling waves" revealing the temporal pace at which tyrosine kinases activate cellular functions. The first wave includes thymocyte-expressed molecule involved in selection (THEMIS), a protein recently implicated in thymocyte development but whose signaling role is unclear. We found that tyrosine phosphorylation of THEMIS depends on the presence of the scaffold proteins Linker for activation of T cells (LAT) and SH2 domain-containing lymphocyte protein of 76 kDa (SLP-76). THEMIS associates with LAT, presumably via the adapter growth factor receptor-bound protein 2 (Grb2) and with phospholipase Cγ1 (PLC-γ1). RNAi-mediated THEMIS knock-down inhibited TCR-induced IL-2 gene expression due to reduced ERK and nuclear factor of activated T cells (NFAT)/activator protein 1 (AP-1) signaling, whereas JNK, p38, or nuclear factor κB (NF-κB) activation were unaffected. Our study reveals the dynamics of TCR-dependent signaling networks and suggests a specific role for THEMIS in early TCR signalosome function.     

The importance of three membrane-distal tyrosines in the adaptor protein NTAL/LAB

NTAL (non-T cell activation linker)/LAB (linker for activation of B cells) is a LAT (linker for activation of T cells)-like molecule that is expressed in B cells, mast cells, natural killer cells, and monocytes. Upon engagement of the B cell receptor or Fc receptors, it is phosphorylated and interacts with Grb2. LAB is capable of rescuing thymocyte development in LAT(-/-) mice. In this study, we utilized various LAB Tyr to Phe mutants to map the phosphorylation and Grb2-binding sites of LAB. We also examined the function of these mutants by investigating their ability to rescue signaling defects in LAT-deficient Jurkat cells and thymocyte development in LAT(-/-) mice. Our results indicated that human LAB was primarily phosphorylated on three membrane-distal tyrosines, Tyr(136), Tyr(193), and Tyr(233). Mutation of these three tyrosines abolished Grb2 binding and LAB function. Our data suggested that these tyrosines are the most important tyrosines for LAB function.     

MIST functions through distinct domains in immunoreceptor signaling in the presence and absence of LAT.

MIST (also termed Clnk) is an adaptor protein structurally related to SLP-76 and BLNK/BASH/SLP-65 hematopoietic cell-specific adaptor proteins. By using the BLNK-deficient DT40 chicken B cell system, we demonstrated MIST functions through distinct intramolecular domains in immunoreceptor signaling depending on the availability of linker for activation of T cells (LAT). MIST can partially restore the B cell antigen receptor (BCR) signaling in the BLNK-deficient cells, which requires phosphorylation of the two N-terminal tyrosine residues. Co-expression of LAT with MIST fully restored the BCR signaling and dispenses with the requirement of the two tyrosines in MIST for BCR signaling. However, some other tyrosine(s), as well as the Src homology (SH) 2 domain and the two proline-rich regions in MIST, is still required for full reconstitution of the BCR signaling, in cooperation with LAT. The C-terminal proline-rich region of MIST is dispensable for the LAT-aided full restoration of MAP kinase activation, although it is responsible for the interaction with LAT and for the localization in glycolipid-enriched microdomains. On the other hand, the N-terminal proline-rich region, which is a binding site of the SH3 domain of phospholipase Cgamma, is essential for BCR signaling. These results revealed a marked plasticity of MIST function as an adaptor in the cell contexts with or without LAT.     

Role of LAT in the granule-mediated cytotoxicity of CD8 T cells

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 membrane proximal proline-rich region and correct order of C-terminal tyrosines on the adaptor protein LAT are required for TCR-mediated signaling and downstream functions

The primary activating receptor for T cells is the T cell receptor (TCR), which is stimulated upon binding to an antigen/MHC complex. TCR activation results in the induction of regulated signaling pathways vital for T cell differentiation, cellular adhesion and cytokine release. A critical TCR-induced signaling protein is the adaptor protein LAT. Upon TCR stimulation, LAT is phosphorylated on conserved tyrosines, which facilitates the formation of multiprotein complexes needed for propagation of signaling pathways. Although the role of the conserved tyrosines in LAT-mediated signaling has been investigated, few studies have examined the role of larger regions of LAT in TCR-induced pathways. In this study, a sequence alignment of 97 mammalian LAT proteins was used to identify several “functional” domains on LAT. Using LAT mutants expressed in Jurkat E6.1 cells, we observed that the membrane proximal, proline-rich region of LAT and the correct order of domains containing conserved tyrosines are necessary for optimal TCR-mediated early signaling, cytokine production, and cellular adhesion. Together, these data show that LAT contains distinct regions whose presence and correct order are required for the propagation of TCR-mediated signaling pathways.     

Phosphorylation at serine 318 is not required for inhibition of T cell activation by ALX

The activation of T cells and the initiation of an immune response is tightly controlled by both positive and negative regulators. Two adaptors which function as negative regulators of T cell activation are ALX and LAX. ALX constitutively associates with LAX in T cells, and T cells from mice deficient in ALX and LAX display similar hyper-responsiveness upon T cell receptor (TCR)/CD28 stimulation, including increased production of interleukin-2. During T cell activation, ALX is inducibly phosphorylated, however the site of ALX phosphorylation had not been previously identified and the role of phosphorylation in the inhibitory function of ALX was not known. Here, using mass spectrometry, we demonstrate that ALX is phosphorylated on a serine at position 318. Substitution of alanine for serine at this position (ALX S318A) leads to an abrogation of the mobility shift in ALX induced upon TCR/CD28 stimulation. However, ALX S318A retained the ability to bind to and stimulate tyrosine phosphorylation of LAX. In addition, overexpression of ALX S318A inhibited RE/AP activation upon TCR/CD28 stimulation to a similar extent as wild-type ALX. Therefore, although ALX is inducibly phosphorylated upon TCR/CD28 stimulation, this phosphorylation is not required for ALX to inhibit T cell activation.     

Impairment of immunological synapse formation in adaptively tolerant T cells

Adaptive tolerance is a hyporesponsive state in which lymphocyte Ag receptor signaling becomes desensitized after prolonged in vivo encounter with Ag. The molecular mechanisms underlying this hyporesponsive state in T cells are not fully understood, although a major signaling block has been shown to be present at the level of ZAP70 phosphorylation of linker for activation of T cells (LAT). In this study, we investigated the ability of adaptively tolerant mouse T cells to form conjugates with Ag-bearing APCs and to translocate signaling molecules into the interface between the T cells and APCs. Compared with naive or preactivated T cells, adaptively tolerant T cells showed no dramatic impairment in their formation of conjugates with APCs. In contrast, there was a large impairment in immunological synapse formation. Adaptively tolerant T cells were defective in their translocation of signaling molecules, such as ZAP70, LAT, and phospholipase C γ1, into the T cell-APC contact sites. Although Ag-induced activation of VAV1 was normal, VAV's recruitment into the synapse was also impaired. Interestingly, expressions of both IL-2-inducible T cell kinase and growth factor receptor-bound protein 2-related adaptor downstream of SHC were decreased by 60-80% in adaptively tolerant T cells. These decreases, in addition to the impairment in LAT phosphorylation by ZAP70, appear to be the major impediments to the phosphorylation of SLP76 (SRC homology 2 domain-containing leukocyte protein of 76 kDa) and the recruitment of VAV1, which are important for stable immunological synapse formation.     

The missing link(er): a return to symmetry in antigen receptor signaling?

B lymphocytes and T lymphocytes utilize several proteins with common functions to transduce signals from their respective receptors. However, at the hierarchial signalling level of SLP-76 [Src homology 2(SH2) domain-containing leukocyte protein of 76-kDa] and LAT (linker for activation of T cells) in T cells, the only corresponding protein in B cells was known to be BLNK (B cell linker protein). It was thought that perhaps BLNK performed the cognate roles of SLP-76 and LAT in B cells; however, mounting evidence to the contrary revealed that this hypothesis was not robust. Two laboratories have recently described the characterization of a protein expressed in B cells and myeloid cells, alternatively termed NTAL (non-T cell activation linker) or LAB (linker for activation of B cells). NTAL/LAB and LAT may have arisen from a primordial gene-duplicating event, but genes that code for the two proteins do not share a very high degree of sequence identity. Wange discusses the results of the two reports, the evidence for functional homology between LAT and NTAL/LAB, and the possibility that the differences between them might lead to specific clinical therapeutics to manipulate immune cell responses.     

Negative regulation of Fc epsilonRI-mediated signaling and mast cell function by the adaptor protein LAX

LAX is a transmembrane adaptor protein that is expressed in both T and B cells. Upon stimulation via the antigen receptors, it is tyrosine-phosphorylated and binds Grb2 and the p85 subunit of phosphatidylinositol 3-kinase. Disruption of the Lax gene causes hyperresponsiveness in T and B lymphocytes. Here, we showed that LAX was also expressed in mast cells. Upon engagement of the Fc epsilonRI, LAX was also phosphorylated and interacted with Grb2 and p85. LAX-deficient mast cells were hyperresponsive to stimulation via the Fc epsilonRI, as evidenced by enhanced degranulation, p38 MAPK, Akt, and phosphatidylinositol 3-kinase activation. This hyperresponsiveness was likely a consequence of reduced LAB expression after sensitization of mast cells with anti-dinitrophenyl IgE. In addition, Fc epsilonRI-mediated cytokine production and cell survival were also enhanced. These data suggested that LAX negatively regulates mast cell function.     

Synergistic assembly of linker for activation of T cells signaling protein complexes in T cell plasma membrane domains

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.