THE SIR HANS KREBS LECTURE LAT – an important raft-associated transmembrane adaptor protein Delivered on 6 July 2009 at the 34th FEBS Congress in Prague, Czech Republic Va ´ clav Hor ˇ ejs ˇ ı ´ , Pavel Ota ´ hal and Toma ´ s ˇ Brdic ˇ ka Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Introduction A number of immunologically important receptors, e.g. T cell and B cell antigen receptors (TCR, BCR), Fc-receptors, natural killer (NK) ⁄ myeloid cell activat- ing receptors, collagen receptor on platelets, some cytokine receptors, employ common functional princi- ples for signal transduction. These multichain receptor complexes consist of a ligand-recognition module and noncovalently associated signalling subunits. The sig- nalling subunits are transmembrane proteins contain- ing in their intracellular domains tyrosine residues that can be phosphorylated by kinases associated constitu- tively or, more often just very transiently, with the receptor. Extracellular domains of these signalling subunits are in some cases large, sometimes contributing to ligand binding (many cytokine receptors). In other cases the extracellular domains are relatively small and participate rather in interactions with the ligand-bind- ing chains of the receptor complexes, such as the CD3c, d, e subunits of the TCR complex [1] or CD79a, b components of the BCR complex [2]. Some of the receptor-associated signalling chains have only very short extracellular segments (f chain of the TCR complex [3], c chain of several Fc receptors [4], DAP12 and DAP10 chains of several NK ⁄ myeloid cell activating receptors [5]). Keywords immunoreceptor signalling; LAT; raft; transmembrane adaptor protein; tyrosine phosphorylation Correspondence V. Hor ˇ ejs ˇ ı ´ , Institute of Molecular Genetics, AS CR, Vı ´ den ˇ ska ´ 1083, 142 20 Prague 4, Czech Republic Fax: 420 244472282 Tel: 420 241729908 E-mail: horejsi@biomed.cas.cz (Received 8 July 2010, revised 12 August 2010, accepted 24 August 2010) doi:10.1111/j.1742-4658.2010.07831.x Membrane rafts are microdomains involved in a number of biologically important processes, including immunoreceptor signalling. Among the functionally important protein components of these microdomains are transmembrane adaptor proteins, containing in their intracellular domains tyrosine residues that can be phosphorylated and bind other cytoplasmic signalling proteins. The most important leukocyte transmembrane adaptor protein is LAT (linker for activation of T cells), which is critically involved in T cell receptor signalling, but also plays important roles in signal initia- tion by several other immunologically important receptors. Here we review recent progress in the elucidation of several aspects of this protein, e.g. the controversy concerning the importance of LAT being present in membrane rafts, the involvement in signalling through a number of receptors other than the T cell receptor and the puzzling phenotype of some LAT mutants. Abbreviations BCR, B cell receptor; cSMAC, central supramolecular activation cluster; DRM, detergent-resistant membrane complex; GPVI, glycoprotein VI; LAT, linker for activation of T cells; NK, natural killer; PI3K, phosphatidylinositol 3-kinase; TCR, T cell receptor; TRAP, transmembrane adaptor protein. FEBS Journal 277 (2010) 4383–4397 ª 2010 The Authors Journal compilation ª 2010 FEBS 4383 Several other proteins structurally similar to the last group (f chain-like) exist that are not directly associated with any receptor, but also play more or less important roles in the regulation of receptor sig- nalling. Some of these transmembrane adaptor pro- teins (TRAPs) are palmitoylated and targeted to membrane rafts (LAT, NTAL, LIME, PAG), others are found in nonraft membrane (SIT, TRIM, LAX, GAPT) [6,7]. In our opinion, the term TRAP can also be used for the abovementioned proteins closely asso- ciated with receptors, i.e. f, c chains, DAP12, DAP10. Common features of TRAPs thus include: short extra- cellular domain, single transmembrane domain, intra- cellular domain containing signalling-relevant motifs, such as potentially phosphorylated tyrosine motifs, polyproline sequences, PDZ-binding motifs, etc. This review deals mainly with the functionally most important TRAP, linker for activation of T cells (LAT). We will concentrate mainly on the latest devel- opments in the field, but will also review the literature on rather neglected roles of LAT in non-T cells. Several relatively recent reviews exist, dealing with TRAPs in general or specifically with some of them [4,5,8–16]. Membrane rafts Membrane rafts are membrane microdomains enriched in cholesterol, sphingolipids and glycerolipids contain- ing mainly saturated fatty acid residues. These lipids have a tendency to form a specific ‘ordered liquid phase’ distinguished from the less ordered rest of the membrane composed mainly of lipids possessing mostly polyunsaturated fatty acids. The term ‘lipid raft’ has been used more frequently in the literature, but because not only lipids, but also proteins, are essential for the formation of this type of membrane microdomain, the term ‘membrane rafts’ has been recommended [17] and therefore will be used through- out this review. Most transmembrane proteins are excluded from the rafts, exceptions being mostly palmitoylated molecules, such as several members of the tumour necrosis factor (TNF) receptor family, TRAPs LAT, NTAL, PAG, LIME or the coreceptors CD4 and CD8. Typical com- ponents of membrane rafts are extracellularly oriented proteins anchored in the membrane through a glyco- lipid moiety (glycosylphosphatidylinositol) [18,19] such as CD14, CD16b, CD24, CD48, CD52, CD55, CD58, CD59, CD73, CD87, CD90 (Thy-1), CD108, CD109, CD157, CD160, CD177, CD228, CD230 (prion pro- tein), Ly-6 family. Importantly, several lipid-modified cytoplasmic molecules are present in the rafts, e.g. Src family kinases [20] heterotrimeric and small G-proteins [21]. Because of the presence of important signalling mole- cules, membrane rafts have been implicated in signal- ling through a wide range of receptors, including immunoreceptors, and also in many other biologically important processes, such as antigen presentation, cell interactions with pathogens and bacterial toxins, bud- ding of viruses from a host cell membrane, pathogene- sis of prion and other neurodegenerative diseases, specific forms of endocytosis, vesicle trafficking and establishing cell polarity [22–28]. Although native rafts are, due to their small size and dynamic nature, difficult to observe directly, they can be visualized using, for example, specific lipid probes [29] or electron microscopy [30,31]. A special type of raft microdomain, caveolae, can be readily observed by electron microscopy [32]. ‘Elementary rafts’ are probably quite small (diameter < 20 nm) and dynamic and contain very few (perhaps even single and some none at all) protein molecules surrounded by a ‘shell’ of several hundreds of the specific lipid molecules. These ‘elementary rafts’ may easily coalesce into larger patches, especially after membrane exposure to certain types of detergent or after cross-linking of their protein or glycolipid com- ponents by antibodies or natural multivalent ligands [25,27,33,34]. Because of their specific lipid composition, mem- brane rafts are, especially at low temperatures, rela- tively resistant to solubilization by some detergents commonly used for membrane solubilization, such as polyoxyethylene type (Brij-series, Triton X-100), but are readily solubilized in other detergents, such as octyl- glucoside or SDS. The detergent-resistant membrane complexes (DRMs) derived from the rafts can be easily purified by density gradient ultracentrifugation or size- exclusion chromatography [35]. There are probably several types of membrane raft in the plasma membrane of a cell type, differing in their lipid and protein composition. Recently we described a novel type of raft (‘heavy rafts’) producing upon detergent solubilization complexes that do not flotate in a density gradient [36]. It is not clear to what extent the DRM preparations obtained from detergent-solubilized cells correspond to the native rafts. The detergent usually used as a stan- dard in the raft studies, Triton X-100, is probably a bad choice, as it may dissolve the raft membrane essentially completely at increased temperature or after prolonged exposure. Brij-98 appears to be a much bet- ter alternative, as it produces much more stable and reproducible DRMs, presumably corresponding much LAT, a key membrane raft-associated protein V. Hor ˇ ejs ˇ ı ´ et al. 4384 FEBS Journal 277 (2010) 4383–4397 ª 2010 The Authors Journal compilation ª 2010 FEBS better to raft microdomains present in the membrane before detergent exposure [26,37,38]. In the following text, ‘rafts’ usually refers to ‘DRMs’ derived from the native rafts. We are fully aware of the fact that the DRMs are not identical to native rafts, but we believe that this simplification is useful. LAT – basic properties, roles in TCR signalling One of the functionally most important leukocyte raft molecules is the TRAP LAT. LAT was originally called pp36-38 and was of great interest as it was the most rapidly tyrosine-phosphorylated protein upon TCR engagement, associated with several signalling molecules (see [39] and references therein). Cloning of the LAT cDNA [40,41] revealed it as a type III (leaderless) transmembrane protein of 262 amino acids (human) or 242 amino acids (mouse). A shorter human isoform exists (233 amino acids), which arises by alternative splicing and lacks residues 114– 142 of the long form. So far nothing is known about the possible functional importance of this difference between the two LAT forms. This prototypic TRAP (Fig. 1) is expressed in thymo- cytes and T cells, NK cells and mast cells; later it was also found in pre-B cells (but not in mature B cells) [42,43], myeloid cells [44], megakaryocytes and platelets [45,46]. The LAT polypeptide chain contains in its mem- brane-proximal part two cysteine residues (C26, C29 in humans, C27, C30 in mouse), which can be palmitoy- lated by a so far unidentified palmitoyl transferase(s). This post-translational modification is essential for LAT membrane and raft association (see below); recent data demonstrate that monopalmitoylation of LAT on C26 is sufficient for its association with the plasma membrane and function (however, it was not reported whether the monopalmitoylated mutant is present in membrane rafts to the same extent as the double-palmitoylated wild-type protein) [47]. Upon immunoreceptor engagement of several recep- tors [most notably TCR, FccR, FceRI, collagen recep- tor glycoprotein VI (GPVI), but see below for more examples] at least five of its nine tyrosine motifs can be phosphorylated by ZAP-70 or Syk kinases [40], but also by Itk [48] and possibly Lck [49]. Phosphorylated LAT associates with several SH2-containing molecules [Grb2, Gads, Grap, PLCc1, p85, phosphatidylinositol 3-kinase (PI3K), Vav], thereby organizing signalosomes needed for the initiation of several intracellular signal- ling pathways [40,50–52]. A key cytoplasmic adaptor, SLP-76, is recruited to phospho-LAT via its constitutive association with Gads [53]. It is not known how many different phospho-LAT containing complexes exist, differing in their composition. The formation of phosphotyrosine-dependent multiprotein signalling complexes organized around phospho-LAT was also examined more rigorously in an in vitro system based on recombinant LAT incorporated in liposomes and recruitment of signalling protein complexes from Jurkat cell cytosol [54]. Little is known about the structural details of differ- ential recognition of tyrosine-phosphorylated sites in LAT by SH2-containing ligands, an exception being the adaptor Gads; high-resolution structures of Gads–SH2 complexed with phosphopeptides corresponding to sites 171, 191 and 226 revealed the structural basis for prefer- ential recognition of specific phospho-LAT sites by Gads, as well as for the related adaptor Grb2 [55]. LAT – negative regulation in TCR signalling LAT was reported to interact with the active (open) form of Lck in rafts and possibly induce its transition into the inactive (closed) conformation [56]. The inter- action of LAT with a negative regulator of the Ras–MAPK pathway of receptor tyrosine kinases, Sprouty1, negatively regulates LAT phosphorylation. A C-terminal deletion mutant of Sprouty1 is unable to translocate to the immune synapse and interact with LAT [57]. Cytoskeletal protein 4.1R negatively regu- lates T cell activation by directly binding to LAT, and thereby inhibiting its phosphorylation by ZAP-70 [58]. Tyrosine phosphatase SHP-2 is recruited to the LAT– Gads–SLP-76 complex and regulates the phosphoryla- tion of signalling proteins Vav1 and ADAP. This enzyme is transiently inactivated by reactive oxygen species produced after TCR stimulation [59]. The inhibitory Fc receptor FccRIIB (present also on acti- vated T cells) associates with phosphatases SHP-1, SHP-2 and SHIP-1 inhibit TCR-mediated Ca 2+ mobi- lization, in part through the inhibition of LAT phos- phorylation followed by the inhibition of PI3K activation [60]. Cellular localization and functionality of LAT was reported to be sensitive to intracellular redox status. Oxidative stress results in conformational changes (the formation of intramolecular dislufidic bridges) causing membrane displacement of LAT and consequent hyporesponsiveness of T lymphocytes [61]. LAT, as a key component of the TCR activation pathways, may be expected to be a target of pathogens trying to eliminate T cell-based immune responses. Indeed, Yersinia suppresses T lymphocyte activation through the virulence factor YopH, a tyrosine V. Hor ˇ ejs ˇ ı ´ et al. LAT, a key membrane raft-associated protein FEBS Journal 277 (2010) 4383–4397 ª 2010 The Authors Journal compilation ª 2010 FEBS 4385 phosphatase that dephosphorylates LAT and SLP-76 in activated T cells [62]. LAT – signalling clusters, membrane rafts and interactions with TCR complexes One of the current models postulates that TCR molecules or their clusters in the plasma membrane of resting T cells are physically separated from raft micr- odomains containing several important signalling mol- ecules, e.g. Lck, Fyn, LAT, PAG, PIP2 (it is not clear whether individual rafts contain several of the proteins or rather there are separate LAT-containing, Lck- containing, etc. rafts) [23]. A variant of the model assumes that TCR clusters and a subset of rafts are preassembled even in resting T cells and TCR ligation just reorients them such that the raft signalling pro- teins start to interact functionally with the TCR [37]. After TCR ligation, the TCR clusters are either mixed or concatenated with the rafts, which may be simulta- neously fused to form larger patches. Such processes also apparently accompany the formation of physio- logical immunological synapses or ‘patches’ or ‘caps’ induced by artificial cross-linking of TCR [63]. The understanding of the involvement of rafts in this process is complicated by unresolved problems, such as the heterogeneity of raft microdomains and techni- cal problems in studies on the nature of apparently highly dynamic raft assemblies. An illustration of the raft heterogeneity is provided by the observations that cholesterol extraction destabilizes the membrane micr- odomains containing Lck, whereas those containing LAT remain almost intact. As shown by electron microscopy, following T cell activation, both LAT and Lck colocalize in 50–100 nm microdomains, which cor- relates with the initiation of T cell signal transduction [64]. The involvement of LAT-containing rafts in TCR-ini- tiated activation was demonstrated using transfectants expressing LAT-GFP [65]. After stimulation with anti- CD3-coated beads, LAT-GFP translocated to the area of T cell contact with the beads. The LAT-GFP present in the contact area was markedly immobilized compared with the membrane outside the contact. The mobility increased after raft disruption by cholesterol depletion, and was also dependent on the integrity of critical bind- ing sites (PLCc) in the cytoplasmic domain of LAT. At present it is not entirely clear why the presence of LAT in membrane rafts is functionally important and how these LAT-containing rafts are related to ‘signalling clusters’ described in several papers. Transmembrane glycoprotein CD2 involved in T cell costimulation, LAT, and tyrosine kinase Lck were reported to be coclustered in discrete T cell plasma membrane microdomains. The integrity of these micr- odomains was dependent on protein–protein interac- tions based on phosphorylated LAT, but apparently independent of interactions with rafts or actin [66]. In quiescent T cells, LAT and TCR were observed in sep- arate ‘protein islands’, which became concatenated upon T cell activation [67]. The signalling complexes organized around phospho-LAT and apparently vital for intracellular signalling appear to be oligomerized by multipoint co-operative binding of several cytoplas- mic SH2 and SH3 domain-containing signalling pro- teins to LAT [68–70]. The involvement of LAT in T cell activation is also regulated by another type of membrane microdomain heterogeneity. LAT molecules are preferentially located in the uropod of migrating T cells. In activated T cells forming stable immunological synapses with antigen- loaded B cells, LAT accumulates at the contact between the two cells (immunological synapse) [71]. LAT was reported to exist in two distinct cellular pools, one at the plasma membrane and the other in endocytic vesicles also containing a transferrin receptor and the TCR f chain [72]. The plasma membrane-asso- ciated LAT is rapidly recruited to the immune synapse, whereas the intracellular pool is first polarized and Y37 Y46 Y67 Y113 Y132 Y175 (Gads, Grb2) Y195 (Gads, Grb2) Y235 (Grb2) Y136 (PLCγ) Plasma membrane Membrane raft Fig. 1. A model of the LAT molecule. A schematic representation of mouse LAT with a palmitoylation site (orange) and the positions of all tyrosines (yellow). The binding partners for the key phosp- hotyrosine residues are indicated. LAT, a key membrane raft-associated protein V. Hor ˇ ejs ˇ ı ´ et al. 4386 FEBS Journal 277 (2010) 4383–4397 ª 2010 The Authors Journal compilation ª 2010 FEBS recruited to the immunological synapse with a delay. Critical tyrosine residues of LAT are necessary for recruitment to the immunological synapse and a juxta- membrane region of LAT is involved in the intracellu- lar pool localization of LAT and T cell signalling. This aspect was recently examined in more detail by Purbhoo et al. [73]. The study found that the kinase ZAP-70 and the adaptor proteins LAT and SLP-76 accumulate in separate clusters at the immunological synapse. Importantly, a sizeable fraction of LAT was found in vesicles that migrated to surface microclusters containing SLP-76 and the adaptor protein GADS, where they became temporarily immobilized. The results suggest a surprising additional mechanism of LAT participation in the TCR signalling process. The involvement of LAT-containing membrane rafts in the formation and signalling of TCR microclusters and central supramolecular activation clusters (cSMACs) at the immunological synapse remains con- troversial. A recent study [74] did not find accumula- tion of raft probes at TCR microclusters or cSMACs. Raft association of LAT mutants was dispensable for TCR microcluster formation. Observable accumulation of raft probes in the cell interface actually occurred after cSMAC formation and could rather be due to membrane ruffling or endocytosis. The results of this study suggest that membrane rafts may actually not serve as a platform for T cell activation. Is the presence of LAT in rafts necessary for its function in TCR signalling? Proper functioning of LAT appeared to be dependent on its targeting to membrane rafts [75–77]. This target- ing was thought to be due to palmitoylation of its juxta- membrane cysteine motif (CxxC) because the cysteine mutants were not able to reconstitute TCR signalling in LAT-negative T cell lines [76,77]. Furthermore, target- ing of SLP-76 constitutively to plasma membrane rafts in LAT-deficient Jurkat T cells largely restores the sig- nalling defects, indicating that recruitment of SLP-76 to the membrane raft environment via phospho-LAT is the crucial LAT-dependent signalling event [78]. Also, the displacement of LAT from membrane rafts was demonstrated as a molecular mechanism responsible for the inhibition of T cell signalling by polyunsaturated fatty acids [79]. Furthermore, palmitoylation of LAT was shown to be defective in anergic T cells [80]. Although f chain or ZAP-70 phosphorylation were normal in these cells, LAT tyrosine phosphorylation and PLCc1 activation were markedly decreased. Inhibi- tion of T cell activation by a cytoplasmic LAT mutant lacking the transmembrane domain is accompanied by reduced recruitment of signalling molecules to glyco- lipid-enriched microdomains [81]. However, the importance of LAT localization in membrane rafts became recently doubtful as a result of several studies. First, the nonpalmitoylated LAT cyste- ine mutants were shown to be not only absent from membrane rafts, but not even properly transported to the plasma membrane and remained retained in the endoplasmic reticulum [47,82,83]. It was suggested that in addition to proper acylation, homotypic or hetero- typic protein–protein interactions may also contribute to LAT targeting to rafts [83]. Second, it was demon- strated that a LAT construct composed of the cyto- plasmic region of LAT fused with the extracellular and transmembrane regions of the nonraft transmembrane adaptor, LAX, restored TCR signalling in LAT-defi- cient cell line and normal development of T cells from LAT ) ⁄ ) haematopoietic precursors [84]. A similar con- clusion was reached using another LAT construct (the cytoplasmic part of LAT equipped with a membrane- anchoring motif of Src) not targeted to membrane rafts but yet fully functional [47]. These results, which might demolish the generally accepted concept of the membrane raft’s importance in immunoreceptor signalling, were recently explained by results from our laboratory [36]. We demonstrated the existence of a novel type of membrane raft-like micr- odomain (‘heavy rafts’) containing a number of mem- brane molecules, including, for example, the LAX and the LAX-LAT chimaeric construct. The LAT con- structs targeted to the newly identified ‘heavy rafts’ are also able to support TCR signalling, albeit less effi- ciently than the wild-type LAT present in ‘classical rafts’; the least efficient are constructs targeted to non- raft membrane. This difference may be minimized by increased levels of LAT-construct expression in the heavy rafts or nonraft membrane. Therefore, different types of membrane microdomain appear to provide environment regulating functional efficiency of signal- ling molecules present therein. Role of LAT in anergy induction LAT was reported to be hypophosphorylated and mis-localized in anergic T cells, apparently as a conse- quence of a selective palmitoylation defect; it was largely absent from DRM fractions corresponding to rafts and was not normally recruited to the immuno- logical synapse. The defects were selective for LAT, because DRM localization and palmitoylation of Fyn were intact. These defects were not due to enhanced LAT degradation [80]. It should be noted that induction V. Hor ˇ ejs ˇ ı ´ et al. LAT, a key membrane raft-associated protein FEBS Journal 277 (2010) 4383–4397 ª 2010 The Authors Journal compilation ª 2010 FEBS 4387 of T cell anergy is accompanied by defective palmitoy- lation and displacement from rafts of yet another TRAP, PAG [85]. Regulation of palmitoylation of LAT (and of other palmitoylated membrane proteins) and its functional consequences remain a rather unknown and potentially very important area. The use of LAT by T lymphocyte receptors other than TCR LAT is an important component of signalling path- ways initiated not only by TCR, but also by other T cell surface receptors. LAT was reported to associate with CD4 and CD8 coreceptors via the cysteine motifs in these coreceptors that mediate Lck binding [86]. However, this poten- tially important result has not been confirmed by other studies and it is perhaps only because of the concomi- tant presence of these molecules in membrane rafts. TCR-independent ligation of the T cell surface glyco- protein ⁄ coreceptor CD2 induces LAT tyrosine phos- phorylation and association with other tyrosine phosphorylated proteins, including PLCc-1, Grb-2 and SLP-76 [87,88]. LAT is associated (evidently via lipid- based raft microdomains) with a glycosylphosphat- idylinositol-anchored glycoprotein, CD48; functional association of the CD48 ⁄ LAT raft complex with TCR was dependent on CD2 [89]. Stimulation of Eph- rinB1receptor (a receptor tyrosine kinase interacting with the transmembrane ligand EphrinB1) led to increased LAT phosphorylation and p44 ⁄ 42 and p38 MAPK activation [90]. This signalling pathway may be essential in T cell–T cell costimulation and in the regulation of a T cell response threshold in response to antigen stimulation. LAT is involved in apoptosis induced in double positive thymocytes by ligation of CD8 in the absence of TCR engagement (a mechanism that may remove thymocytes that have failed positive selection) [91]. LAT (as well as several other signalling proteins) becomes tyrosine phosphorylated during CXCR3-mediated T cell chemotaxis [92]. On the other hand, the treatment of T cells with the chemokine SDF-1 (ligand of the CXCR4 receptor) caused a reduction in tyrosine phosphorylation of the TCR downstream effectors, ZAP-70, SLP-76 and LAT (and also of ZAP-70 and SLP-76), indicating that this chemokine may negatively regulate the threshold of T cell activation [93]. Regulation of LAT expression LAT expression is markedly increased upon TCR-med- iated activation; this is inhibited by rapamycin at the translational level. In contrast, cyclosporin A and FK506 strongly enhance TCR-induced LAT expression in T cells [94]. LAT protein levels are regulated by ubiquitination, recycling through trans-Golgi ⁄ endo- some compartments and clathrin-dependent internali- zation and proteasome-dependent degradation [95]. Interestingly, the amount of LAT (and its phosphory- lation) in membrane rafts is increased in cells lacking the structurally related adaptor NTAL (LAB) [96]. This is probably due to the competition between NTAL and LAT for the limited raft space available. Signalling clusters containing LAT are internalized and dissociate rapidly upon TCR activation. This process is linked to the ubiquitin ligase c-Cbl [97]. Sustained tyrosine phosphorylation of LAT and SLP-76 is observed in thymocytes deficient in c-Cbl [98]. Functional defects of LAT tyrosine mutants in vivo – possible negative regulatory roles of LAT The essential importance of LAT for T cell develop- ment (and therefore functioning of pre-TCR) is evi- denced by the fact that LAT ) ⁄ ) mice have thymocyte development completely blocked at the double-negative stage [99]. LAT-negative Jurkat T cell line mutants have severely impaired TCR signalling [100,101]. Interestingly, the development of other cells naturally expressing LAT is not impaired in LAT ) ⁄ ) mice [99]. Thorough studies on genetically engineered mice using the mutant gene-knock-in approach revealed the relative importance of LAT individual tyrosine resi- dues. Mutants lacking all four distal tyrosine residues (Y136, Y175, Y195 and Y235 in mice, i.e. those bind- ing after phosphorylation PLCg, PI3K, Gads, Grb2) had identical severe defects in thymocyte development as the LAT knock-outs [102]; these tyrosine residues were also essential for LAT-dependent signalling in FceRI-mediated mast cell activation [103]. On the other hand, mice with only mutated LAT Y136, which binds (after phosphorylation) PLCc1, or only the other three critical tyrosines (Y175, Y195, and Y235) exhib- ited an incomplete block of thymocyte differentiation accompanied by the development of striking autoim- mune phenotypes [104–107], which may be partially related to a defect in Treg development [108–110]; for a detailed review see [15,111,112]. These results suggest that LAT may also play a negative regulatory role(s) in TCR signalling, in addition to its well-established crucial positive regulatory role. Actually, LAT associ- ates with an inhibitory complex containing cytoplasmic adaptors Dok-2 and Grb2 and SHIP-1 phosphatase [113]. LAT, a key membrane raft-associated protein V. Hor ˇ ejs ˇ ı ´ et al. 4388 FEBS Journal 277 (2010) 4383–4397 ª 2010 The Authors Journal compilation ª 2010 FEBS Moreover, recruitment of inhibitory SHP-1 phospha- tase to rafts and its association with LAT were mark- edly increased after TCR engagement [114]. Another inhibitory mechanism may be based on threonine 155 phosphorylation of LAT by Erk and JNK following TCR engagement, which leads to defective recruitment of PLC-c1 and SLP-76 [115]. Furthermore, Gab2 (con- stitutively associating with Gads ⁄ Grb2) is recruited to membrane rafts by LAT upon TCR ligation. Gab2 may inhibit signalling by competing with SLP-76 for Gads ⁄ Grb2 binding and by binding SHP-2 phospha- tase, which inhibits TCR signalling by dephosphoryla- tion of the f chain and other important signalling molecules [116,117]. Participation of LAT in signalling by platelet receptors As stated above, LAT is also of essential importance in signalling pathways initiated by the ligation of GPVI (platelet collagen receptor). This receptor resem- bles TCR and some activating Fc-receptors in some respects – it uses the associated FcRc chain as a sig- nalling subunit (similar to the f chain in TCR com- plex), the signalling is initiated by Src family kinases and involves Syk and further downstream molecules participating in the TCR signalling, including LAT. After stimulation of the receptor by collagen or con- vulxin, LAT is phosphorylated and associates with multiple cytoplasmic signalling proteins [45,118–121]. However, the GPVI signalling pathway is less depen- dent on LAT as compared with TCR; platelet activa- tion downstream of GPVI shows a much greater dependency on SLP-76 than on LAT [122], yet the absence of LAT leads to decreased platelet activation, degranulation and aggregation [123]. Studies on mice carrying LAT with mutated tyrosine residues demon- strated a crucial role of tyrosine residues 175, 195 and 235 in the phosphorylation of LAT induced via GPVI. These tyrosine residues appear to be important in the recruitment of the tyrosine kinase Fyn, which may be, in addition to Syk, involved in LAT phosphorylation. The binding of PLCc2 via GPVI is dependent on an interaction with phospho-tyrosine 136 of LAT [124]. Similar to other immunoreceptors, GPVI is down- regulated following activation. This occurs either by ectodomain proteolytic shedding or internalization. In mice lacking LAT, GPVI shedding (but not internali- zation) is inhibited, indicating that a LAT-dependent signalling pathway is involved in the activation of the process [125]. LAT also participates in platelet activation via cross-linking of FccRIIa, which relocates in membrane rafts where the kinase Lyn and LAT are among the major phosphotyrosyl proteins [126,127]. LAT is also involved in platelet activation through the C-type lectin receptor CLEC-2 after binding the snake venom rhodocytin [123,128] and in platelet acti- vation by the peptide LSARLAF mediated by an unidentified receptor(s) [129]. LAT is also tyrosine phosphorylated in response to stimulation (followed by aggregation) of platelets by ADP and thrombin, impli- cating this adaptor in signalling pathways of the rele- vant G-protein coupled receptors [130]. Platelet aggregation induced by the C-terminal peptide of thrombospondin-1 (RFYVVMWK) also requires LAT [131]. The tyrosine phosphatase 1B [132] and the 18 kDa low molecular mass phosphotyrosine phospha- tase [133] were identified as the enzymes dephosphoryl- ating LAT (and activated FccRIIA) in platelets activated via FccR. Participation of LAT in signalling by Fc receptors on mast cells, monocytes and macrophages LAT was identified as an important component involved in macrophage activation via FccRIII and FccRIV (linked to anaphylatoxin receptor activation and generation of inflammation) [134]. Cross-linking of high-affinity FccRI CD64 on THP-1 monocytic cells induces tyrosine phosphorylation of multiple proteins, including Lck, Syk and LAT, which is inhibited by coligation of an ITIM-bearing immuno- globulin-like receptor LILRB4 [135]. LAT associates with both FccRI and FccRII and enhances signal transduction elicited by these receptors in myeloid cells [44]. LAT is an important component in the activation of mast cells, where it plays similar roles as in activated T cells. Following the ligation of FceRI of mast cells, it becomes tyrosine phosphorylated by Syk kinase and associates with several cytoplasmic signalling proteins [103,136–138]; the lipid environment of membrane rafts appears to be important in these processes [139]. The extent of LAT involvement in FceRI signalling seems to be linked to the strength of the stimulus [140]. Tyro- sine 136 and the three distal tyrosines differentially contribute to exocytosis and the secretion of cytokines; in addition to the positive signalling roles they are also apparently involved in complex negative regulations, which is probably based on the assembly of signalling complexes composed of a set of intracellular molecules with antagonistic properties [137]. Electron micro- scopic studies found that following Fc eRI activation, FceRI and LAT are present in distinct membrane V. Hor ˇ ejs ˇ ı ´ et al. LAT, a key membrane raft-associated protein FEBS Journal 277 (2010) 4383–4397 ª 2010 The Authors Journal compilation ª 2010 FEBS 4389 domains containing associated cytoplasmic signalling molecules [141]. In addition to LAT, mast cells also express a similar adaptor, NTAL (also called LAB or LAT2). LAT and NTAL are phosphorylated after ligation of FceRI and co-operate positively and negatively in regulation of the response [10,12,142,143]. In the absence of LAT, NTAL can partially take over the positive signalling role of LAT, whereas if both adaptors are present, NTAL rather serves as a negative regulator of the activation process. Participation of LAT in signalling by NK cell receptors CD2 cross-linking in NK cells induced tyrosine phos- phorylation of LAT, resulting in SH2-based associa- tion with PI3K and PLC-c1 [144,145]. This functional relationship was dependent on intact membrane rafts, as cholesterol depletion inhibited LAT tyrosine phos- phorylation and NK cell cytotoxicity and degranula- tion [144]. LAT is tyrosine phosphorylated upon stimulation of NK cells through FccRIII receptors and following direct contact with NK-sensitive target cells. This NK stimulation induces the association of LAT with sev- eral phosphotyrosine-containing proteins, including PLCc. Over-expression of LAT in NK cells enhances their cytotoxic responses [146]. 2B4 (CD244), a receptor belonging to the Ig super- family expressed on NK cells and a subset of T cells, was reported to be constitutively associated with LAT in membrane rafts. 2B4-mediated cytotoxicity is defec- tive in the absence of LAT, indicating that LAT is an important component in the 2B4 signal transduction pathway. Engagement of 2B4 results in tyrosine phos- phorylation of both 2B4 and the associated LAT, recruitment of PLCc and Grb2 [147–149]. The 2B4- LAT association was independent of the cytoplasmic tail of 2B4, but required a CxC cysteine motif (pre- sumably palmitoylated) found in the transmembrane region [148]. Therefore, the association is probably indirect, based on the association of both molecules with membrane rafts. Similar to myeloid cells, in addition to LAT, NK cells also express the abovementioned similar adaptor, NTAL (also called LAB or LAT2) [12]. LAT and NTAL are phosphorylated after ligation of an NK cell, activating receptors CD16 (FccRIIIa, associated with the FcRc signalling chain containing ITAM motifs) and NK1.1 (associated with the DAP12 signal- ling chain containing ITAM motifs). Mice lacking either LAT or NTAL have abnormalities in the reper- toire of the inhibitory receptors of the Ly49 family and respond poorly to stimulation through NK1.1. The absence of both LAT and NTAL markedly reduces NK1.1 signalling in both resting and activated NK cells [150]. LAT in B cell lineage In contrast to T cells, BCR does not seem to use a LAT-like molecule as a critical component of its signalling machinery; LAT is actually absent in imma- ture and mature B cells. A cytoplasmic adapter SLP- 65 (BLNK) of B cells, functionally analogous to SLP-76 in T cells, appears to associate directly with the activated BCR complex [151]. However, LAT is expressed in mouse pro-B and pre-B cells; in pre-B cells it becomes tyrosine phosphorylated upon cross- linking of the pre-BCR. LAT may thus play a role in the regulation of early phases of B cell development at the transition from pre-B to immature B cell stage [42,152]. LAT and SLP-76 are recruited to the pre- BCR after its experimental cross-linking; LAT is spontaneously associated with SLP-76 in untreated pre-B cells [43]. Four distal tyrosines (Y136, Y175, Y195, Y235) are required for LAT activity in murine and human pre-B cells [153]. LAT is also found in B-ALL cells, probably reflecting their developmental origin [154]. Concluding remarks Although LAT is one of the most thoroughly studied leukocyte signalling molecules, some of its aspects remain poorly understood. As discussed above, its possible role in negative TCR regulatory pathways remains to be clarified, as well as the details of the importance of its association with raft-like microdo- mains. Another exciting field of research is the regula- tion of LAT palmitoylation and its role in membrane microdomain distribution and the outcome of TCR signalling. Furthermore, details of mutual functional interactions between LAT and the structurally related NTAL (LAB) in myeloid cells remain to be eluci- dated. Acknowledgements This work was supported in part by project no. 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THE SIR HANS KREBS LECTURE LAT – an important raft-associated transmembrane adaptor protein Delivered on 6 July 2009 at the. that can be phosphorylated and bind other cytoplasmic signalling proteins. The most important leukocyte transmembrane adaptor protein is LAT (linker for activation