Among the functionally important protein components of these microdomains are transmembrane adaptor proteins, containing in their intracellular domains tyrosine residues that can be phos
Trang 1LAT – an important raft-associated transmembrane
adaptor protein
Delivered on 6 July 2009 at the 34th FEBS Congress in Prague,
Czech Republic
Va´clav Horˇejsˇı´, Pavel Ota´hal and Toma´sˇ Brdicˇka
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 Horˇejsˇı´, Institute of Molecular Genetics,
AS CR, Vı´denˇska´ 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.
Trang 2Several 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 albet-ternative, as it produces much more stable and reproducible DRMs, presumably corresponding much
Trang 3better 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
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 Ca2+ 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
Trang 4phosphatase 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 immunophysio-logical 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.
Trang 5recruited 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 activaInhibi-tion 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
Trang 6of 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]
Trang 7Moreover, 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 tyroTyro-sines 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 FceRI activation, FceRI and LAT are present in distinct membrane
Trang 8domains 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 funcassocia-tional
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 maimma-ture 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 palmitoylaregula-tion 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 AV0Z50520514 awarded by the Academy of Sciences
of the Czech Republic, GACR (project MEM⁄
09⁄ E011) and by the Center of Molecular and Cellular Immunology (project 1M0506, Ministry of Education, Youth and Sports of the Czech Republic)
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