Tài liệu Báo cáo khoa học: LAT – an important raft-associated transmembrane adaptor protein pptx

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:
(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
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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
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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.
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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
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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
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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
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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.
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).
LAT, a key membrane raft-associated protein V. Hor
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