The LAT Story: A Tale of Cooperativity,
Coordination, and Choreography
Lakshmi Balagopalan, Nathan P. Coussens, Eilon Sherman, Lawrence E. Samelson,
and Connie L. Sommers
LCMB/CCR/NCI/NIH, Bethesda, Maryland 20892-4256
Correspondence: samelson@helix.nih.gov
The adapter molecule LAT is a nucleating site for multiprotein signaling complexes that are
vital for the function and differentiation of T cells. Extensive investigation of LAT in multiple
experimental systems has led to an integrated understanding of the formation, composition,
regulation, dynamic movement, and function of LAT-nucleated signaling complexes. This
review discusses interactions of signaling molecules that bind directly or indirectly to LAT
and the role of cooperativity in stabilizing LAT-nucleated signaling complexes. In addition,
it focuses on how imaging studies visualize signaling assemblies as signaling clusters and
demonstrate their dynamic nature and cellular fate. Finally, this review explores the function
of LAT based on the interpretation of mouse models using various LAT mutants.
INTRODUCTION
inker for activation of T cells (LAT) was
cloned a little more than a decade ago. Since
then, a multitude of studies have revealed that
LAT-based complexes catalyze critical TCRmediated signaling reactions and enable activation of multiple downstream pathways that control almost all TCR-initiated cellular responses.
This article spotlights diverse experimental systems in which LAT function has been studied.
Information gained from these studies has led
to an integrated understanding of the cellular
function of LAT.
L
Cloning and Structural Features of LAT
The study of the tyrosine phosphorylation of
proteins induced by immunoreceptor and
growth factor receptor stimulation has led to
critical insights into mechanisms of signal
transduction (Hunter 2009). Early studies
showed that a number of proteins became phosphorylated on tyrosine residues following TCR
stimulation in Jurkat T-cell leukemia cells and
in normal T cells (June et al. 1990). Many of
these proteins, such as ZAP-70, SLP-76, and
PLC-g1, have been shown to be critical elements
for TCR signal transduction (Kane et al. 2000).
A protein with an apparent molecular weight
of 36 and 38 kDa was also prominently phosphorylated on tyrosine in response to TCR
stimulation. Several preliminary studies showed
that this protein, known then as pp36/38, was
membrane-associated and capable of binding
SH2 domains of Grb2, Grap, PLC-g1, and the
p85 subunit of phosphatidylinositol 3-kinase
(PI3K) (June et al. 1990; Gilliland et al. 1992;
Buday et al. 1994; Sieh et al. 1994; Fukazawa
Editors: Lawrence E. Samelson and Andrey Shaw
Additional Perspectives on Immunoreceptor Signaling available at www.cshperspectives.org
Copyright # 2010 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a005512
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1
L. Balagopalan et al.
et al. 1995a; Trub et al. 1997). Although pp36/
38 was first observed in 1990, it proved challenging to isolate. It was not until 1998 that the
Samelson laboratory cloned it by large-scale
membrane purification of activated Jurkat cells
(Zhang et al. 1998a). Shortly after, Weber et al.
reported the cloning of the rat and human proteins from thymocytes (Weber et al. 1998). The
Samelson lab named the protein product LAT,
for Linker for Activation of T cells based on several of its characteristics. LAT is expressed in T
cells and in a limited number of other immune
cell types (mast cells, natural killer cells, megakaryocytes, platelets, and immature B cells)
(Facchetti et al. 1999; Oya et al. 2003). Furthermore, as detailed below, LAT facilitates the recruitment of many signaling proteins to the
plasma membrane where it links receptors,
tyrosine kinases and their substrates and other
effector molecules together, functioning as a
critical activator of T cells.
Sequencing of human LAT cDNA identified
an open reading frame encoding a protein predicted to contain 233 amino acids. The mouse
and rat homologs of LAT encode 242 and 241
amino acid proteins, respectively, and have
65% – 70% sequence identity with human LAT.
The predicted molecular mass of LAT is
25 kDa. However, LAT is strikingly acidic and
its charge may account for slower migration
on SDS-PAGE leading to its apparent molecular
weight of 36/38 kDa. Structurally, LAT is a
type III transmembrane protein. It has a cytosolic carboxyl terminus (like type I proteins),
but lacks a signal sequence (Brown 2006). LAT
contains only a four-amino-acid extracellular
region, a single transmembrane spanning region and a long intracellular region with no
apparent intrinsic enzymatic activity or
protein – protein interaction domains. However, consistent with the strong tyrosine phosphorylation of pp36/38 observed upon TCR
stimulation, the intracellular domain of LAT
contains nine tyrosines conserved between
humans, mice, and rats. Examination of LAT
amino-acid sequence also revealed two conserved cysteine residues (C26 and C29 in
human LAT), which are located adjacent to
the predicted transmembrane domain of LAT
2
and are subject to posttranslational palmitoylation (Zhang et al. 1998b). Palmitoylation of LAT
is necessary for LAT function (Lin et al. 1999;
Zhang et al. 1999a), but the role of palmitoylation in specific localization of LAT within the
plasma membrane has been controversial and
is discussed below. More recently, studies have
shown that LAT is also subject to ubiquitylation
(Brignatz et al. 2005; Balagopalan et al. 2007), a
modification that might be involved in activation-induced internalization of LAT complexes
and regulation of LAT protein levels. Inspection
of LAT amino acid sequence reveals two lysines
(K52 and K204 in human LAT), which might
serve as potential sites for ubiquitylation. A
schematic of LAT structural features is shown
in Figure 1A.
Palmitoylation and Membrane Localization
of LAT
Palmitoylation is thought to enhance the association of transmembrane proteins, including
LAT, with regions of membrane heterogeneity
sometimes called lipid rafts (Brown 2006). In
T cells, several signaling molecules in addition
to LAT, including the TCR, Lck, Vav, Grb2,
PLC-g1, and Ras are associated with lipid rafts
(Brdicka et al. 1998; Montixi et al. 1998; Zhang
et al. 1998b). Thus, rafts have been postulated
to function as important platforms to initiate
signaling cascades (Brown and London 1998).
However, the physiological role of this membrane heterogeneity has been intensely debated
(Munro 2003; He and Marguet 2008; Kenworthy 2008). Reflecting this debate, the importance of raft localization for LAT function has
been controversial.
Early studies reported that a cysteinemutated form of LAT was not recruited to lipid
rafts and could not reconstitute signaling in a
LAT-deficient Jurkat cell line, leading to the
conclusion that localization of LAT to lipid rafts
was required for its function (Lin et al. 1999;
Zhang et al. 1999a). However, more recent studies indicated that palmitoylation of LAT was
required for the protein to be transported
efficiently to the plasma membrane and that,
in the absence of palmitoylation, LAT was
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Signaling through LAT
Ubiquitin
A
Ubiquitin
O–
O
O–
–O P O
–O P O
O
O
Y132
Y171
O
C26
Y36
K52
Y110
C
LAT
N
TM
C29
Y45
O
Y64
Y127
T155
O–
O
B
K204
Y191
O–
Y226
O–
O–
–O P O
–O P O
–O P O
–O P O
O
O
O
O
a TCR b
PI3K
Itk
ZAP-70
RASGRP
PKC
InsP3
GADS
GRB2
Cytoskeletal
reorganization
DAG
1
C-g
76
VAV
PL
WASp
P-
z
SL
Nck
z
DAG
RAS
SOS1
LCK
PIP2
PIP3
PIP2
e d
LAT
g e
ADAP
MAPK
activation
Adhesion
Release of
Ca++
NFAT
translocation
Activate
effectors
NF-kB & AP1
Figure 1. LAT in TCR signal transduction. (A) Human LAT is a 233 amino-acid type III transmembrane protein
with four extracellular amino acids, a single transmembrane region, and a cytosolic region that undergoes
multiple posttranslational modifications. Modifications include palmitoylation at cysteine residues C26 and
C29 and possible ubiquitylation at lysine residues K52 and K204 (Ubiquitin [Protein Data Bank ID 1UBQ]
is represented as a ribbon diagram generated with the program PyMOL). Nine tyrosine residues are
conserved between human and mouse LAT sequences, five of which have been shown to undergo
phosphorylation (Y127, Y132, Y171, Y191, and Y226). Threonine 155 is also phosphorylated by Erk. (B)
Ligation of the TCR induces tyrosine phosphorylation of numerous adapter and effector proteins leading to
the activation of multiple signaling pathways important for gene transcription, cytoskeletal reorganization,
and cell adhesion. LAT is central to this process by nucleating multiprotein signaling complexes that are
important for enzyme activation and signal propagation.
susceptible to degradation (Gringhuis et al.
2000; Hundt et al. 2006; Tanimura et al. 2006;
Hundt et al. 2009). Furthermore, LAT fusion
proteins targeted to nonraft domains reconstituted LAT function in LAT-deficient Jurkat
cells or LAT-deficient mice (Zhu et al. 2005;
Hundt et al. 2009). These data raise the possibility that the signaling defects initially observed
for a LAT palmitoylation mutant might result
from defects in plasma membrane transport,
rather than displacement from lipid domains.
Collectively, these data suggest that targeting
of LAT to the plasma membrane of the cell is
sufficient for its function, regardless of specific
localization within the membrane.
LAT is Central to T-Cell Signaling
The essential role of LAT in T-cell signal transduction has been demonstrated in a variety of
experimental settings. Initial work showed that
Jurkat T-cell lines lacking expression of LAT
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3
L. Balagopalan et al.
were defective in several TCR-mediated signaling events including Ca2þ mobilization, Ras
activation, CD69 expression, Erk activation, and
AP-1/NFAT-directed gene transcription (Finco
et al. 1998; Zhang et al. 1999a; Samelson 2002).
Reintroduction of LAT rescued all these defects
indicating that LAT is indispensable for T-cell
activation via the TCR. Soon after, targeted disruption of the LAT gene in mice revealed a
requirement for LAT in T-cell development.
Animals that lack LAT exhibited an early arrest
of thymocyte development and no mature ab
T cells were found in their peripheral lymphoid
organs (Zhang et al. 1999b). Additional reports
highlighted the adapter function of LAT by
showing that multiple signaling proteins bind
phosphorylated LAT once the TCR is engaged.
These LAT binding proteins subsequently
attract multiple cytosolic protein partners and
further waves of tyrosine phosphorylation and
protein interactions occur. Thus LAT functions
as a crucial nucleating site at the plasma membrane for multiprotein signaling complexes.
LAT AS A NUCLEATION CENTER FOR T-CELL
SIGNAL TRANSDUCTION
LAT functions as a classic adapter protein by
facilitating the formation of multiprotein
complexes (Fig. 1B). Numerous studies have
investigated the composition of LAT-nucleated
complexes. This section will describe the direct
and indirect associations of enzymes, adapters
and effectors to LAT and provide insights into
how these interactions lead to activation of specific intracellular pathways.
Phosphorylation of LAT
LAT is rapidly phosphorylated on tyrosine residues upon TCR engagement. Though a full
understanding of how the kinases phosphorylate the individual tyrosines on LAT in vivo
remains unresolved, overexpression and in vitro
studies have implicated primarily ZAP-70, but
also Lck and Itk in LAT phosphorylation (Zhang
et al. 1998a; Paz et al. 2001; Perez-Villar et al.
2002; Jiang and Cheng 2007). Even less is known
about the phosphatases that dephosphorylate
4
LAT, however CD148 has been implicated (Baker
et al. 2001).
PLC-g1 Binds Y132 of LAT and Mediates
Transcriptional Activation in T Cells
The phospholipase PLC-g1 is an important
mediator of TCR signal transduction. PLC-g1
hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (Rhee and
Bae 1997). DAG stimulates the Ras activator
RasGrp (Ras guanyl-releasing protein) and the
serine/threonine kinase PKC ( protein kinase
C). The production of IP3 by PLC-g1 promotes
the release of stored intracellular calcium, causing an extracellular Ca2þ influx and a sustained
increase in intracellular Ca2þ concentrations
important for NFATactivation (Oh-hora 2009).
PLC-g1 is a multidomain enzyme that
includes a pleckstrin homology (PH) domain
that can bind membrane phosphoinositides,
two phosphotyrosine-binding SH2 (src homology 2) domains and an SH3 (src homology 3)
domain, which binds specific proline-rich
sequences. PLC-g1 binding to LATwas observed
even before LATwas cloned and was shown to be
dependent on the amino-terminal SH2 domain
of PLC-g1 (Gilliland et al. 1992; Weber et al.
1992; Stoica et al. 1998; Irvin et al. 2000). Interaction with LAT was shown to be required for
both PLC-g1 activation and localization near
its substrate PIP2 at the plasma membrane
(Zhang et al. 1999a; Zhang et al. 2000; Lin and
Weiss 2001). Subsequently, the amino-terminal
SH2 domain of PLC-g1 was shown to bind
phosphorylated LAT Y132 with high affinity
(Paz et al. 2001; Houtman et al. 2004). Furthermore, a single Y132F mutation abolished the
association between LAT and PLC-g1 in Jurkat
T cells (Zhang et al. 2000). However, further
experimentation revealed that full PLC-g1 activation required additional binding interactions
to LAT and LAT-associated molecules.
Several studies showed that LAT Y171 and
Y191 also contributed to PLC-g1 binding and
that all four distal LAT tyrosines (132, 171,
191, and 226) were required for optimal PLCg1 phosphorylation and downstream calcium
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Signaling through LAT
influx (Zhang et al. 2000; Lin and Weiss 2001;
Zhu et al. 2003). The adapter protein SLP-76
(that binds to Gads, which binds to LAT Y171
and Y191) was also shown to be required for
PLC-g1 activation (Yablonski et al. 1998). Braiman and colleagues demonstrated that multiple
domains of PLC-g1 were required for association with LAT and all three SH domains were
essential for enzyme activation. Furthermore,
PLC-g1 activity depended on interactions with
other LAT-associated proteins including SLP76, the guanine nucleotide exchange factor
Vav and the E3-ubiquitin ligase c-Cbl (Braiman
et al. 2006).
Grb2 Family Members Bind Y171, Y191, and
Y226 of Phosphorylated LAT and Recruit a
Number of Signaling Proteins to the LAT
Complex
The Grb2 family members Grb2, Gads, and
Grap can potentially bind phosphorylated LAT
at the distal three tyrosines: Y171, Y191, and
Y226 (Wange 2000). Grb2 (Growth-factorreceptor-bound protein 2) is a ubiquitously expressed adapter protein that contains an SH2
domain flanked by amino- and carboxy-terminal SH3 domains. The Grb2 adapter is involved
in Ras and MAP kinase pathway activation in
many cell surface receptor systems, primarily
by association with the guanine nucleotide exchange factor (GEF) Sos1. Interestingly, Grb2
association with LAT requires the SH2 domain
of Grb2 and pairs of LAT phosphotyrosines:
Y171/Y191, Y171/Y226, or Y191/Y226 (Zhang
et al. 2000; Zhu et al. 2003). Gads (Grb2-related
adapter protein downstream of Shc) contains a
structure similar to Grb2, but also has a unique
glutamine- and proline-rich domain between
the SH2 domain and the carboxy-terminal
SH3 domain. In addition to binding LAT,
Gads binds SLP-76, which recruits a number
of other signaling proteins including PLC-g1
(see Fig. 1B) (Liu et al. 1999; Zhang et al.
2000; Yablonski et al. 2001). Grap (Grb2-related
adapter protein) is similar in structure to Grb2,
but its role in T-cell signal transduction is not as
well defined. It also associates with LAT through
its SH2 domain (Trub et al. 1997; Zhang et al.
1998a). Studies of T cells from Grap-deficient
mice indicate that Grap may be a negative regulator of TCR signal transduction (Shen et al.
2002).
Sos1 (Son of Sevenless homolog) is a multidomain GEF that promotes activation of the
small G protein Ras, which in turn activates
the MAP kinase Erk (Quilliam 2007). Sos1
binds Grb2 constitutively (Wittekind et al.
1994; Houtman et al. 2006). Following TCR
ligation, Grb2 recruits Sos1 to the site of Ras
localization at the membrane. Early experiments suggested that a trimolecular complex
formed between Grb2, Sos1, and LAT following
TCR engagement (Buday et al. 1994; Sieh et al.
1994). Consistent with this, Zhang and colleagues reported that Sos1 did not associate
with the LAT mutant Y171/191F, which also
did not associate with Grb2 (Zhang et al. 1998a).
Interestingly, the significance of a Grb2/Sos1
complex likely extends beyond the activation
of Ras. Houtman and colleagues have described
a mechanism whereby a 2:1 Grb2:Sos1 complex
can facilitate clustering of LAT-based signaling
complexes (to be discussed in more detail later)
(Houtman et al. 2006).
The proto-oncogene c-Cbl encodes a 120
kDa multidomain E3-ubiquitin ligase, which
is phosphorylated and associates with LAT following TCR activation. c-Cbl also binds Grb2
constitutively (Donovan et al. 1994; Fukazawa
et al. 1995b). Grb2 seems to be required for
the interaction between c-Cbl and LAT, because
c-Cbl does not associate with the LAT mutant
Y171/191F, which fails to bind Grb2 (Zhang
et al. 1998a). However, there is evidence that
c-Cbl is also stabilized by interactions with
other LAT-associated molecules, such as Nck
(Rivero-Lezcano et al. 1994; Wunderlich et al.
1999), the p85 subunit of PI3K (Fukazawa
et al. 1995b), and PLC-g1 (Rellahan et al.
2003; Chiang et al. 2004; Braiman et al. 2006).
By stabilizing the associations of molecules
that contribute to signal activation, c-Cbl could
promote positive TCR-induced signals; however, c-Cbl also appears to play a negative regulatory role in signaling.
In LAT-deficient Jurkat cells, c-Cbl is hyperphosphorylated upon TCR stimulation (Finco
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5
L. Balagopalan et al.
et al. 1998; Mingueneau et al. 2009) and phosphorylated c-Cbl has been implicated in negative regulation of TCR signal transduction
(Boussiotis et al. 1997; Murphy et al. 1998). In
addition, consistent with its role as an E3-ubiquitin ligase in several signaling systems, c-Cbl
has been implicated in ubiquitylation of the
TCRz and CD3d chains of the TCR as well as
the signaling proteins ZAP-70, LAT, PLC-g1,
PI3K, Vav, and PKC-u in T cells (Weissman
2001; Duan et al. 2004; Balagopalan et al. 2007).
The effects of LATubiquitylation on controlling
internalization and cellular LAT levels are discussed in the imaging section later.
Molecules Recruited to LAT via SLP-76
Promote T-Cell Activation
SLP-76 is an adapter protein containing three
tyrosine motifs, a central proline-rich domain
and a carboxy-terminal SH2 domain (Koretzky
et al. 2006). The proline-rich domain of SLP-76
binds SH3 domains of Grb2 and Gads; however
SLP-76 binds Gads with a 40 – 50-fold stronger
affinity than Grb2 (Berry et al. 2002; Harkiolaki
et al. 2003). Gads recruits SLP-76 to LAT by way
of the interaction of its SH2 domain with
phospho-LAT (Liu et al. 1999). SLP-76 recruits
multiple effector molecules to the LAT complex.
The three phosphotyrosines of SLP-76 bind
PI3K, Vav, Itk, and Nck. The central prolinerich region binds to Gads with high affinity
and to PLC-g1 with low affinity (Yablonski
et al. 2001; Houtman et al. 2004). However,
SLP-76 only associates weakly with LAT in the
absence of the LAT residue Y132, suggesting
that SLP-76 binding to LAT is stabilized by the
presence of PLC-g1 (Zhang et al. 2000). The
carboxy-terminal SH2 domain of SLP-76 interacts with the proteins HPK1, ADAP, and Shb.
These interactions result in positive and negative effects on TCR signaling. Interactions that
lead to negative effects on TCR signaling will
be discussed in the following section.
PI3K is comprised of a 110 kDa catalytic
subunit and an 85 kDa regulatory subunit ( p85)
and catalyzes the phosphorylation of PIP2 to
generate PIP3 (Fruman and Bismuth 2009).
The accumulation of PIP3 facilitates membrane
6
recruitment of proteins that contain PH domains, which recognize PIP3 (Fruman et al.
1999). In T cells, PI3K regulates the recruitment
of proteins that facilitate TCR signal transduction such as PLC-g1, Sos1, RasGAP, Itk,
Tec, and Vav. Early studies showed that PI3K
bound directly to LAT (Fukazawa et al. 1995a;
Lahesmaa et al. 1995; Paz et al. 2001); however
more recent studies showed binding of p85 to
tyrosine-phosphorylated SLP-76 (Shim et al.
2004). Inhibition of PI3K in T cells resulted in
reduced TCR-induced calcium influx, Rac1
activation, and Erk1/2 phosphorylation.
However, PI3K inhibition did not reduce the
phosphorylation of PH domain-containing
proteins such as PLC-g1 and Vav (Cruz-Orcutt
and Houtman 2009). Therefore PI3K plays a
role in TCR activation, but its exact mechanism
is still unclear.
Other SLP-76 interactors have clearer roles
on T-cell activation. Vav is a guanine nucleotide
exchange factor that activates the Rho family
G-proteins Rac1, RhoA, and Cdc42. A number
of studies have demonstrated the importance
of the association of the Vav SH2 domain
with SLP-76 phosphotyrosines (Koretzky et al.
2006). In addition to its catalytic role, Vav acts
as a scaffold to stabilize PLC-g1 and Itk interactions with SLP-76. In Vav-deficient T cells, the
interaction between SLP-76 and PLC-g1 was
substantially reduced and Itk was not phosphorylated (Reynolds et al. 2002; Braiman
et al. 2006). The Tec family kinase Itk phosphorylates PLC-g1 (Readinger et al. 2009) and LAT
(Perez-Villar et al. 2002). Itk is recruited to the
LAT complex by binding of its SH2 domain to
tyrosine phosphorylated SLP-76 (Bunnell
et al. 2000). Itk plays a major role in T-cell activation by virtue of its phosphorylation of
PLC-g1, which is required for the activation of
PLC-g1.
Vav and the adapters Nck and WASp cooperate to effect cytoskeletal changes in activated T
cells. Nck binds phosphorylated SLP-76 (Wunderlich et al. 1999) and WASp (Wiskott-Aldrich
syndrome protein) (Rohatgi et al. 2001; BardaSaad et al. 2005), bringing WASp in proximity
to Vav. Vav recruits and activates Cdc42, which
is necessary for WASp activation. Activated
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Signaling through LAT
WASp interacts with the Arp2/3 complex to
initiate actin polymerization and cytoskeletal
changes (Zeng et al. 2003).
Two other adapter molecules that bind SLP76 and mediate positive signal transduction in T
cells are ADAP and Shb. Phosphorylated ADAP
binds the SH2 domain of SLP-76 (da Silva et al.
1997; Musci et al. 1997). This interaction is crucial for adhesion and integrin function in T cells
(Baker et al. 2009; Horn et al. 2009; Wang et al.
2009). Tyrosine phosphorylated Shb also binds
the SH2 domain of SLP-76 and promotes LAT,
SLP-76, Vav, and PLC-g1 phosphorylation
(Lindholm et al. 1999; Lindholm et al. 2002).
HPK1 is another molecule that binds the SH2
domain of SLP-76 but mediates negative signal
transduction.
LAT Interactions also Inhibit TCR Signal
Transduction
Several molecules that bind to the LAT complex
can inhibit TCR signal transduction. HPK1 (hematopoietic progenitor kinase 1) is a MAP4
kinase (Hu et al. 1996; Kiefer et al. 1996) that
down-regulates activation of the MAP kinase
Erk2 and the transcription factor AP-1 in a
kinase-dependent manner (Liou et al. 2000).
HPK-1 has a complex relationship with SLP76. It can phosphorylate SLP-76; however, this
ultimately leads to decreased SLP-76 and PLCg1 phosphorylation and IL-2 production (Di
Bartolo et al. 2007; Shui et al. 2007).
SHIP-1 is a lipid phosphatase that mediates Fc receptor-dependent negative signaling
(Daeron and Lesourne 2006). In T cells, SHIP1 also acts as an adapter to recruit Dok-1 and
Dok-2 to LAT complexes, which results in attenuated TCR signaling. SHIP-1-associated Dok1 and Dok-2 negatively regulate ZAP-70 activity
(Dong et al. 2006; Yasuda et al. 2007) and recruit
RasGAP, which opposes Ras-mediated signaling
(Tamir et al. 2000; Ott et al. 2002). The adapter
protein Gab2 also has several potential mechanisms for inhibiting signaling. It can bind
SHP-2 (Yamasaki et al. 2001) and it may compete with SLP-76 for binding to Grb2 and
Gads (Yamasaki et al. 2003). Other molecules
that may mediate inhibitory signaling are the
adapter SLAP (Src-like adapter protein) (Myers
et al. 2006), the tyrosine phosphatase SHP-1
(Lorenz 2009) and 4.1R (Diakowski et al. 2006).
4.1R binds LAT directly and is thought to prevent LAT phosphorylation by ZAP-70 (Kang
et al. 2009).
COOPERATIVITY AMONG
LAT-ASSOCIATED PROTEINS STABILIZES
SIGNALING COMPLEXES
It is clear that LAT plays a central role in T-cell
activation downstream of the TCR by directly
or indirectly recruiting kinases, effectors, and
adapters to facilitate the coordination of highly
regulated signal transduction pathways. Biophysical approaches have begun to reveal important insights into some of the molecular
mechanisms that govern the stabilization of
LAT-associated complexes. These studies indicate that numerous weak interactions cooperatively coordinate the formation of spatially
and temporally specific signaling complexes.
Qualitative Results Indicate Cooperativity
Among LAT-Associated Proteins
Cooperativity is an altered affinity between proteins because of interaction with other molecules and/or posttranslational modifications.
A number of results suggest that LAT complexes
are stabilized by cooperative interactions. SH2
domain-containing proteins including PLCg1, Grb2, and Gads bind directly to LAT phosphotyrosine motifs with a preference for
PLC-g1 at pY132, Gads at pY191 and pY171,
and Grb2 at pY171, pY191, and pY226 (Samelson 2002). Nevertheless, mutations of LAT
Gads/Grb2-binding residues (Y171, Y191, and
Y226) reduced PLC-g1 binding (Zhang et al.
2000; Hartgroves et al. 2003). At a minimum,
Y132 and Y191 were required for an association
between LAT and PLC-g1, indicating that
PLC-g1 association with LAT was stabilized by
Gads binding to LAT (Zhu et al. 2003). Likewise,
there was evidence that PLC-g1 stabilized the
binding of Grb2 to LAT. LAT Y132F mutation
resulted in substantial losses of both Grb2
and PLC-g1 binding (Hartgroves et al. 2003).
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7
L. Balagopalan et al.
Experiments with LAT-nucleated signaling complexes on liposomes by Sangani and colleagues
also indicated cooperativity. In these experiments Grb2 alone did not stably bind liposome-associated LAT; however, after incubation
with Jurkat lysate, Grb2 associated along with
Gads, PLC-g1, SLP-76, Itk, and Sos1 (Sangani
et al. 2009). Interestingly, other studies showed
that the LAT phosphorylation sites were required
within a single molecule and could not be added
in trans among multiple LAT proteins (Lin and
Weiss 2001). This suggests that signaling complexes are stabilized around individual LAT
molecules.
LAT Binding Preferences are not Solely
Driven by Affinities
Binding studies were performed to better
understand the basis for the specificity of the
interactions of PLC-g1, Grb2, and Gads with
individual LAT motifs. Interestingly, Grb2 and
Gads did not have substantially different affinities for the pY171, pY191, or pY226 motifs,
results that did not explain the preference of
Gads for pY171 and pY191. However, the proteins had a 50 – 100-fold weaker affinity for
pY132, which explained the lack of binding to
that site. The PLC-g1 SH2 domain bound the
pY132 motif with stronger affinity than the
pY171, pY191, or pY226 motif; however, it
was thought that the affinity difference alone
would not account for its selectivity (Houtman
et al. 2004). Overall, these studies indicated that
the binding affinities alone were not sufficient
to drive the apparent specificity of LAT motifs
for PLC-g1 and Gads.
In addition to affinity, other factors likely
contribute to the specificity of PLC-g1 for the
LAT residue Y132. For example, the PLC-g1
PH domain would position PLC-g1 near the
membrane, likely favoring interactions with
pY132, as it is the most membrane proximal
of the four motifs (Houtman et al. 2004). However, SLP-76 may also contribute to the stabilization of PLC-g1 binding to LAT. Binding
studies had revealed a weak interaction between the SLP-76 proline-rich region and PLCg1; however, Houtman and colleagues showed
8
that the interaction required structural changes
within SLP-76. They showed that the high affinity interaction between Gads and SLP-76 stabilized the PLC-g1-binding conformation of
SLP-76. Therefore, positive cooperativity mediated by the Gads/SLP-76 pair allowed PLC-g1
to bind SLP-76 (Houtman et al. 2004). Interestingly, LAT residue Y191 was phosphorylated
substantially earlier than Y132 in T cells.
Therefore, it is possible that the Gads/SLP-76
complex associates with LAT before PLC-g1.
Moreover, SLP-76 is among the earliest proteins
to be phosphorylated (Houtman et al. 2005).
Phosphorylated SLP-76 binds Itk, which has
been shown to phosphorylate the PLC-g1 residue Y783 (Bunnell et al. 2000; Serrano et al.
2005; Beach et al. 2007). Y783 of PLC-g1 is
phosphorylated before LAT Y132, suggesting
that PLC-g1 associates with the Gads/SLP-76/
Itk complex before binding to pY132 (Houtman et al. 2005). It was suggested that these
cooperative binding events preceding PLC-g1
binding to LAT could allow for tight control
of PLC-g1 activity.
Cooperative Interactions Promote LAT
Oligomerization
Because LAT can potentially bind two or more
Grb2 molecules and because the Grb2 SH3
ligands Sos1 and c-Cbl also contain multiple
Grb2 binding sites, Houtman and colleagues
investigated whether Grb2 complexes could
link multiple LAT molecules into higher-order
structures (Fig. 2). Biophysical studies were carried out to investigate the binding, composition
and stiochiometry of complexes formed from
mixtures of LAT with Grb2 and Sos1 or c-Cbl
(Houtman et al. 2006; Houtman et al. 2007).
The studies provided direct evidence that LAT
was able to bind multiple Grb2 molecules and
that the proline-rich regions of Sos1 and c-Cbl
each bound two Grb2 molecules. Interestingly,
Grb2 bound the two regions of Sos1 with either
significantly different affinities or a twofold
negative cooperativity in binding to the second
site, whereby binding of the first Grb2 weakened
the affinity of Sos1 for a second Grb2 molecule.
These results indicated that the 2:1 complex was
Cite this article as Cold Spring Harb Perspect Biol 2010;2:a005512
Signaling through LAT
ADAP
GRB2
GRB2
GRB2
76
-g1
LAT
PLC
c-Cbl
GADS
SOS1
Itk
LAT
LAT
VAV
-g1
PLC
76
PSL
Nck
PIP3
SL
P-
PIP3
Nck
VAV
Itk
GADS
GRB2
ADAP
Figure 2. LAT complexes. Multiple LAT molecules are oligomerized by complexes of 2:1 Grb2:Sos1 or Grb2:Cbl
in vitro and in cells. The clustering of LAT complexes is thought to be crucial for signal transduction and possibly
for the control of TCR sensitivity to strong or weak stimuli.
only formed when Grb2 was in excess of Sos1. It
was further demonstrated that Grb2 is in
≏1000-fold excess of Sos1 in T cells, suggesting
that the 2:1 complex of Grb2 and Sos1, which
forms in vitro could also be physiologically relevant in T cells (Houtman et al. 2006; Houtman
et al. 2007). Next, it was shown that Grb2/Sos
complexes could oligomerize LAT into large
molecular complexes. Detailed studies of the
energetics contributing to the complex formation showed that intermediate complexes,
such as a 2:1 complex of Grb2:LAT, were slightly
destabilized, consistent with negative cooperativity in their formation. In contrast, positive
cooperative interactions resulted in a large
stabilized molecular complex of LAT, Grb2,
and Sos1. It was suggested that the presence of
both negative and positive cooperativity in the
formation of these complexes promoted the
assembly of physiologically relevant complexes,
while suppressing intermediates (Houtman
et al. 2007). The relevance of Grb2-mediated
LAT clustering was verified in vivo and was
shown to have functional consequences on signaling and T-cell activation (Houtman et al.
2006).
The biophysical analyses of LAT and LATassociated proteins have revealed a role for intermolecular cooperativity in stabilizing LATnucleated complexes. Cooperativity allows for
a high degree of organization of signaling molecules into macromolecular complexes visualized
as “microclusters” using imaging techniques as
described later.
IMAGING OF LAT SIGNALING COMPLEXES
AND CLUSTERS
In the past decade, advanced microscopic approaches have provided striking images of the
early events in T-cell activation. This section
will review the contribution of imaging studies
in developing a clearer understanding of the initiation, composition, dynamics, and spatiotemporal regulation of LAT-nucleated signaling
complexes.
LAT Microclusters: Sites of Nucleation of
Signaling Complexes
A little over a decade ago the immunological
synapse (IS) was identified as a specialized junction between T cells and antigen presenting
surfaces, which is characterized by a central
cluster enriched for TCR-CD3 (cSMAC) and
an integrin-rich peripheral cluster ( pSMAC)
(Monks et al. 1998). Subsequent studies identified the distal SMAC (dSMAC), an outer region
that contains large molecules such as CD43 and
CD45 (Delon et al. 2001; Freiberg et al. 2002).
Cite this article as Cold Spring Harb Perspect Biol 2010;2:a005512
9
L. Balagopalan et al.
Real time visualization of the early events of
T-cell activation has given greater temporal and
spatial accuracy. Studies in which images were
collected at the onset of T-cell activation showed
that within the first 30 s of contact of a T cell
with a stimulatory surface, small TCR-rich
structures form at the periphery of the T-cell
contact zone (Grakoui et al. 1999; Krummel
et al. 2000). These discrete structures enriched
for TCR have subsequently been called “microclusters.”
The first high resolution images of LAT
microclusters came from a method in which T
cells were dropped onto stimulatory coverslips
coated with CD3 antibody and visualized using
confocal microscopy (Bunnell et al. 2002).
Using this technique, LAT colocalized with
TCR-rich microclusters within seconds of activation. In addition to LAT, a large number of
components of the TCR signaling pathway
including protein tyrosine kinases ZAP-70 and
Lck, adapters SLP-76, Grb2, Gads, Nck and
WASp, and enzymes including PLC-g1, Vav,
c-Cbl, and Sos1 were rapidly recruited to microclusters. Larger glycoproteins such as CD43
and the protein tyrosine phosphatase CD45
were excluded from these structures (Bunnell
et al. 2002; Barda-Saad et al. 2005; Braiman
et al. 2006; Houtman et al. 2006). These rapidly
assembled clusters of signaling complexes
were the predominant sites of TCR-induced
tyrosine phosphorylation and their appearance
was coincident with cytosolic Ca2þ elevations,
establishing TCR microclusters as sites of signal
initiation (Krummel et al. 2000; Bunnell et al.
2002; Seminario and Bunnell 2008). Thus
microclusters are the smallest structures visible
by confocal microscopy capable of driving
effective TCR signaling and are made up of signaling complexes that contain adapters and
effectors required for T-cell activation (Fig. 3).
Subsequent studies using peptide-MHCcontaining lipid bilayers and TIRF microscopy
verified and extended these results (Campi
et al. 2005; Yokosuka et al. 2005; Varma et al.
2006; Yokosuka et al. 2008; Kaizuka et al.
2009). Under these experimental conditions,
microclusters were continuously generated
at the periphery of the T-cell peptide-MHC
10
interface and were transported and consolidated at the center of the contact to form a
cSMAC (Varma et al. 2006). Microclusters
play a predominant role in the generation of
sustained signals. Current models also indicate
that cluster centralization plays an important
role in signal termination and that the cSMAC
is a site both for TCR down-regulation yet also
for amplification of weak signals (Lee et al.
2003; Mossman et al. 2005; Varma et al. 2006;
Balagopalan et al. 2007; Cemerski et al. 2008;
Nguyen et al. 2008). A recent study using a photoactivatable agonist in a lipid bilayer system has
provided even greater precision in the temporal
resolution of these events and demonstrated
that adapters are recruited to microclusters
within four seconds (Huse et al. 2007).
Fine spatial imaging studies indicated that
signaling protein clusters may actually represent
discrete interdigitating domains (Douglass
and Vale 2005; Nguyen et al. 2008). These structures resembled the distinct protein and lipid
domains observed by transmission electron
microscopy (TEM) (Wilson et al. 2001; Lillemeier et al. 2006). TEM studies in mast cells and T
cells have shown formation of “primary signaling domains” that include the receptor, and
“secondary signaling domains” that include
LAT and PLC-g1, but not the receptor. Apparently, these domains, submicron in size, preexist before receptor activation. Upon receptor
activation, these domains were shown to concatenate to form larger patches that are likely
equivalent to the microclusters observed by
light microscopy (Wilson et al. 2001; Lillemeier
et al. 2006; Lillemeier et al. 2009).
Because the resolution limit of the microscope (ffi200 nm) is much larger than the size
of individual proteins or protein complexes
(on the order of a few nanometers), colocalization observed by fluorescence microscopy does
not prove that proteins interact. Instead, Forster
resonance energy transfer (FRET) can be
employed to study colocalization on the scale
of a few nanometers and therefore provides
information about molecular interactions
(Gascoigne et al. 2009). Using this technique,
FRET was detected between PLC-g1 and either
LAT, c-Cbl, Vav or SLP-76, as well as SLP-76
Cite this article as Cold Spring Harb Perspect Biol 2010;2:a005512
Signaling through LAT
GRB2
LAT
GADS
MERGE
5 mm
Figure 3. LAT signaling complexes and microclusters. A confocal image of the surface of a Jurkat E6.1 T cell
dropped onto a TCR stimulatory coverslip shows LAT clusters, which colocalize with the LAT-binding
proteins Grb2 and Gads. Grb2 (upper left) is in blue, LAT (upper middle) is in green and Gads (upper right)
is in red. LAT signaling complexes (represented in the cartoon) form a pronounced network of
heterogeneous and dynamic microclusters on the plasma membrane (see merge).
and Nck within microclusters, suggesting direct
interactions of these proteins within microclusters (Barda-Saad et al. 2005; Braiman et al.
2006). In comparison, measurable but low FRET
was observed between LATand either SLP-76 or
Nck, consistent with previous data showing that
these molecules interact indirectly (Barda-Saad
et al. 2005).
Another microscopic technique, fluorescence recovery after photobleaching (FRAP),
has been used to evaluate LAT dynamics at the
population level. This method allows evaluation of mobility and population-level diffusion
dynamics of molecules (Tanimura et al. 2003a).
Studies on LAT dynamics at the membrane revealed the fast exchange of molecules between
LAT clusters or patches localized at sites of
stimulation (Tanimura et al. 2003b; Douglass
and Vale 2005). Single-molecule imaging studies have revealed that LAT molecules at the plasma membrane display abrupt changes between
immobility and rapid diffusion (Douglass and
Vale 2005). Transient immobilization of LAT
correlated strongly with encounter with clusters. These data indicate that LATmolecules diffuse between clusters with occasional trapping
within clusters.
Mechanisms of Assembly of LAT Clusters
and LAT-Nucleated Complexes
Data from various experimental systems have
shown that microclusters play a crucial role in
TCR-mediated signaling pathways because
inhibition of cluster formation resulted in
reduced levels of TCR signaling (Singer et al.
2004; Bunnell et al. 2006; Houtman et al.
2006). The importance of clustering may be to
concentrate activators and exclude inhibitors
creating microdomains that shift the equilibrium to favor downstream signaling in T
cells. However, the mechanisms that govern the
Cite this article as Cold Spring Harb Perspect Biol 2010;2:a005512
11
L. Balagopalan et al.
assembly of these structures have remained elusive. Because LAT is both a lipid raft resident and
an adapter with multiple tyrosines that serve as
docking sites for signaling proteins, the contribution of both lipid rafts as well as protein scaffolds in LAT clustering has been investigated.
Early studies indicated that lipid raft patches form at the sites of TCR activation and
copatched with proteins essential for T-cell activation (Harder and Simons 1999; Janes et al.
1999). Hence it was proposed that lipid rafts
provide a platform for assembly of signaling
domains during T-cell activation. However,
data from multiple studies indicated that membrane recruitment, not raft localization was
required for LAT function (Zhu et al. 2005;
Hundt et al. 2009). Furthermore, Harder and
Kuhn showed that LAT, but not other raft-associated molecules such as Lck and GM1, was
selectively enriched within plasma membrane
fragments containing activated TCR, calling into
question the view that upon TCR activation coalescence of raft-associated membrane proteins in
the vicinity of activated TCR leads to T-cell triggering (Harderand Kuhn 2000). Imaging studies
also showed that LAT did not colocalize with a
general raft marker GFP-GPI (Bunnell et al.
2002). Together, these studies indicate that lipid
rafts are not a primary factor in the assembly of
LAT-containing signaling complexes.
Instead, several studies have indicated that
phosphotyrosine-dependent protein interactions play the predominant role in organization
of TCR-dependent signaling complexes. First,
LAT was not recruited to TCR-enriched immunoisolates in cells treated with the potent Src
kinase inhibitor PP2 (Harder and Kuhn 2000).
Second, imaging approaches using planar substrates demonstrated that LAT variants with
mutated tyrosines in the cytoplasmic tail were
not recruited to signaling microclusters
(Douglass and Vale 2005; Bunnell et al. 2006;
Houtman et al. 2006). Finally, single particle
tracking techniques as well as FRAP methods
that measured diffusion rates of LAT revealed
that LAT cytoplasmic tyrosines are required
for confinement of LATwithin transient subdomains (Tanimura et al. 2003b; Douglass and
Vale 2005). In contrast, interfering with lipid
12
raft association of LAT through mutations did
not alter its diffusion behavior (Douglass and
Vale 2005).
The earlier-described results are most consistent with LAT clusters being formed and
maintained by a network of protein – protein
interactions. The combination of biochemical
and biophysical studies has shown that cooperative and multivalent interactions drive the
assembly of productive signaling complexes
and imaging approaches have supported this
scenario. Houtman et al. used confocal microscopy to verify the physiological relevance of
Grb2-Sos1-mediated oligomerization of LAT,
observed by biophysical techniques (Houtman
et al. 2006). A LAT mutant unable to bind
Grb2 did not localize to signaling clusters. In
addition, expression of a Sos1 fragment unable
to oligomerize LAT inhibited LAT clustering
and downstream signaling. Thus, multivalent
interactions between these three molecules appeared to promote the assembly of multiprotein
complexes important for TCR activation. In
another study using confocal approaches, Bunnell and colleagues demonstrated that cooperative interactions between LAT, Gads and SLP-76
stabilized SLP-76 microclusters (Bunnell et al.
2006). Another report demonstrated that although the SH3 and C-terminal SH2 domains
of PLC-g1 do not bind directly to LAT, they participate in the stabilization of PLC-g1-LATassociation via other proteins in the LAT-nucleated
signaling complex (Braiman et al. 2006).
Finally, several studies have argued that
actin is required for segregating molecules on
the T-cell surface (Wulfing and Davis 1998;
Delon et al. 2001). F actin was present at microclusters and the contact sites of T cells and stimulatory surfaces at early time points (Bunnell
et al. 2001; Barda-Saad et al. 2005). TEM studies
of Wilson et al. in mast cells also support the
interaction of the actin cytoskeleton with receptor clusters (Wilson et al. 2001). Furthermore, inhibitor studies showed that actin is
essential for the formation of microclusters
(Campi et al. 2005; Douglass and Vale 2005;
Varma et al. 2006; Nguyen et al. 2008). An intact
cytoskeleton is also required for dynamic translocation of clusters once they are formed and it
Cite this article as Cold Spring Harb Perspect Biol 2010;2:a005512
Signaling through LAT
has been proposed that retrograde actin flow
drives centripetal cluster movement (Barr et al.
2006; Varma et al. 2006; Kaizuka et al. 2007;
Nguyen et al. 2008; Ilani et al. 2009). In this
regard, microclusters must be connected to actin
filaments via linker proteins, and LAT-mediated
recruitment of actin-regulating adapters Nck
and WASp to these structures may play a central
role in this process (Barda-Saad et al. 2005).
Dynamics of LAT Clusters, LAT Internalization
and LAT Ubiquitylation
The dynamic nature and changing composition
of TCR microclusters after they are generated
has been extensively demonstrated. Across all
modeling systems, following initial recruitment
in close proximity of the TCR, components of
the complexes rapidly exit microclusters with
distinct dynamics (Bunnell et al. 2002; BardaSaad et al. 2005; Braiman et al. 2006). The
adapters LAT and SLP-76 departed the initial
complexes in what appeared to be small vesicular intermediates and dissipated soon thereafter
(Bunnell et al. 2002). Recent studies have shown
that microclusters containing LAT and SLP-76
undergo endocytosis upon TCR stimulation
(Barr et al. 2006; Balagopalan et al. 2007).
SLP-76 is endocytosed in a lipid-raft dependent
pathway that requires the association of the
endocytic machinery with ubiquitylated proteins. LAT endocytosis is a complex process
and internalized LAT is found in several intracellular compartments (Bonello et al. 2004;
Brignatz et al. 2005; Balagopalan et al. 2007).
Insights into the molecular mechanisms that
regulate endocytosis of microcluster-associated
proteins came from studies in which internalization of the microclusters was inhibited. First,
expression of versions of c-Cbl that are defective
in the RING domain that mediates ubiquitin
ligase activity severely inhibited LAT and SLP76 movement and endocytosis. Furthermore,
the ubiquitin-interacting motif (UIM) of eps15,
which is known to block clathrin-independent
internalization of the EGFR (Sigismund et al.
2005), severely inhibited the internalization
of SLP-76 clusters (Barr et al. 2006). Thus,
the E3 ligase c-Cbl and ubiquitin appear to be
intimately involved in the sorting of LAT and
SLP-76 into mobile endocytic structures.
In addition to containing tyrosines, the LAT
cytoplasmic domain contains two lysines that
could serve as sites of ubiquitylation. Indeed
LAT ubiquitylation has been observed both in
Jurkat T cells and in COS-7 cells (Brignatz
et al. 2005; Balagopalan et al. 2007). Consistent
with c-Cbl being an E3 ligase for LAT ubiquitylation, expression of c-Cbl caused a modest increase in LAT ubiquitylation. In contrast, the
RING finger mutant caused a significant decrease in ubiquitylated LAT species. These data
are consistent with a model in which c-Cblmediated ubiquitylation is required for rapid
internalization of LAT-nucleated signaling clusters. Given the essential scaffolding role of the
adapter protein LAT in T cell activation, the
regulated internalization of activated LAT signaling complexes may be one efficient strategy
by which to control the duration and localization of signaling from microclusters and, thus,
regulate the kinetics, intensity, and specificity
of T-cell signaling. For a detailed discussion
on endocytic regulation of T-cell microclusters,
see Balagopalan et al. 2009.
ANIMAL STUDIES REVEAL THE IN VIVO
FUNCTIONS OF LAT
Although T-cell lines have been enormously
useful for discerning the role of LAT in T-cell
function within single T cells, animal model systems provide the opportunity to study the function of LAT during T-cell development as well as
its function in T cells in the context of the entire
immune system.
Studies of LAT Null Mice
Studies of LAT null mice show the crucial
importance of LAT in pre-TCR and TCR signaling. ab T-cell development proceeds through
ordered stages that can be characterized by
expression of the cell surface markers CD4
and CD8: DN (double negative for CD4 and
CD8)!DP (double positive for CD4 and
CD8)!SP (single positive for either CD4 or
CD8). The DN stage can further be divided
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13
L. Balagopalan et al.
into four stages based on the expression of the
cell surface markers CD44 and CD25: DN1
(CD44þCD25þ)!
(CD44þCD252)!DN2
2
þ
DN3 (CD44 CD25 )!DN4 (CD442 CD252).
Signaling through the immature, nonclonotypic
pre-TCR drives development from the DN3 to
the DN4 stage. Mature TCR signaling drives
T-cell development from the DP to SP stage.
During the DP!SP transition, thymocytes are
tested based on their TCR specificities and are
selected for survival (by positive selection, based
on MHC specificity and nonself reactivity) or
death (by negative selection, based on potential
self-reactivity) (Sommers et al. 2004).
In LAT knockout mice, the endogenous
wild-type LAT gene was replaced with a mutant
form of the gene that could not be expressed
by homologous recombination (Zhang et al.
1999b). LAT knockout mice showed a complete
block in ab T-cell development at the immature, DN3 stage indicating an essential role for
LAT in pre-TCR signaling. No peripheral ab T
cells were observed. To investigate the role of
LAT in mature TCR signaling, Shen et al. used
a conditional knockout approach (Shen et al.
2009). Their approach used Cre-Lox technology
(Wang 2009), whereby conditional expression
of the Cre recombinase allowed deletion of a
LoxP-flanked sequence that resulted in T cellspecific disruption of expression of LAT at the
DP stage. The authors observed a severe block
in the DP!SP stage of thymocyte development
indicating a strong role for LAT in TCR-mediated thymocyte selection. However, some CD4
and CD8 SP T cells developed in these mice and
it was unclear whether these T cells developed
because of residual LAT in some developing
DPs or because LAT is not completely essential
for the DP!SP transition (Shen et al. 2009).
In Vivo Functions of LAT Phosphotyrosines
and Cysteines
Previous in vitro studies with C26/29A LAT
mutants had shown that the mutant LAT molecules could not be tyrosine phosphorylated,
did not localize to plasma membrane rafts
and could not mediate TCR signaling (Zhang
et al. 1998b). In the animal experiments, LAT
14
knockout bone marrow cells were infected with
retroviruses containing mutant LAT C26/29A.
The infected bone marrow cells were transferred to irradiated wild type B6 mice and
the mutant LAT was unable to mediate T-cell
development (Hundt et al. 2009). To assess
whether raft localization (in addition to plasma
membrane localization) was necessary for the
in vivo function of LAT, several approaches
have been taken using LAT fusion proteins. LATLAX and Src-LAT fusion proteins localized to
the plasma membrane but not to rafts. Both of
these fusion proteins mediated T-cell development in LAT-deficient environments (Zhu
et al. 2005; Hundt et al. 2009). Therefore C26
and C29 of LATwere necessary for plasma membrane localization and for the in vivo function
of LAT, but raft localization was not necessary
for T-cell development.
In vitro experiments have shown the importance of the distal four phosphotyrosines for the
function of LAT. A “knock-in” LAT mutant in
which the endogenous wild type LAT gene was
replaced with a mutant form of LAT containing
Y!F substitutions at the four distal tyrosines
revealed that those four distal tyrosines were
required for T-cell development (Sommers
et al. 2001). Knock-in mutants of Y136! F
(corresponding to human Y132! F) showed
an incomplete block in early ab T cell development and a fatal lymphoproliferative disease
involving marked lymphadenopathy, splenomegaly, and multiorgan lymphocyte infiltration
(Fig. 4) (Aguado et al. 2002; Sommers et al.
2002). A knock-in LATmutant containing Y!F
mutations at Y175, Y195, and Y235, (corresponding to human Y171, Y191, and Y226)
also developed lymphoproliferative disease
(Nunez-Cruz et al. 2003). Although the lymphoproliferative phenotypes of these two mouse
models were similar, the intrathymic T-cell
development differed between these two models. In LAT Y136F knock-in mice, some ab T
cells developed and expanded, whereas in LAT
Y175/195/235F knock-in mice, cells of the gd
T cells lineage developed and expanded.
In addition to the developmental block in
LAT Y136F mice, thymocyte selection was profoundly affected (Sommers et al. 2005) and Treg
Cite this article as Cold Spring Harb Perspect Biol 2010;2:a005512
Signaling through LAT
C
D
E
18
19
20
21
22
LAT-KI
WT
23
24
A
B
Figure 4. Lymphoproliferative disease in LAT Y136F KI mice. (A) Spleens from 10-week-old wild type C57BL/6
and LAT Y136F KI mice are shown. The ruler depicts centimeters. (B–E) Lymphocyte infiltration into lung.
H&E-stained sections of lung from wild type C57BL/6 (B,C ) and LAT Y136F KI mice (D,E) are shown.
Images were photographed using a 2X (B,D) or 10X (C,E) objective.
cells (Lu and Rudensky 2009) did not develop
(Koonpaew et al. 2006; Wang et al. 2008).
Abnormal cytokine production was observed
in these mice, especially elevated levels of IL-4.
In fact the mice showed many aspects of Th2
lymphoproliferative disease including IgG1
and IgE hypergammaglobulinemia, elevated
serum IL-4, eosinophilia, and lymphocyte proliferation in lungs (Aguado et al. 2002; Sommers et al. 2002; Genton et al. 2006; Miyaji
et al. 2009). The contributing factors leading to
development of lymphoproliferative disease are
still under investigation, but some are known.
Altered or crippled signaling leads to a block
in T-cell development and a lymphopenic environment. Altered signaling in the T cells developing in this lymphopenic environment
results in T-cell hyperproliferation, defective
activation-induced cell death and altered cytokine production (IL-4,IL-2). Lack of development and function of Treg cells may be
secondary to decreased IL-2 production. Altered signaling in LAT Y136F T cells consists
of decreased TCR-induced calcium flux and
other events downstream of PLC-g1 (Sommers
et al. 2002). Reports of Erk1/2 activation in
LAT Y136F T cells have been variable (Sommers
et al. 2002; Mingueneau et al. 2009; Miyaji et al.
2009), most likely because of different methods
of in vitro activation and age of the animals
from which the T cells were derived. However, a
report by Miyaji et al. showed that Erk activation
played an important role in lymphoproliferative
disease in LAT Y136F mice (Miyaji et al. 2009).
Precisely how other aspects of altered signaling
(especially altered calcium signaling) lead to
hyperproliferation, decreased cell death and
altered cytokine production in LAT Y136F T cells
is a subject for future investigations.
LAT-Independent TCR Signaling
A recent report by Mingueneau et al. highlights
a mouse model in which LAT-independent T
cell signaling was demonstrated (Mingueneau
et al. 2009). In these experiments, CD4þ T cells
that had developed in a LAT-sufficient environment were rendered LAT-deficient following
Cite this article as Cold Spring Harb Perspect Biol 2010;2:a005512
15
L. Balagopalan et al.
expression of Cre recombinase, which was accomplished by retroviral infection. After additional culturing with IL-2, the cells were
stimulated through the TCR and various responses were assessed or the T cells were
adoptively transferred to T cell-deficient hosts.
Although phosphorylation of several key signaling molecules was not observed in the LAT-deficient mature T cells (including PLC-g1 and Erk
1/2), phosphorylation of some signaling molecules was observed (e.g., SLP-76 and Akt).
TCR-induced calcium flux and interferon-g
production were also profoundly diminished
in these cells. However, these experiments lend
credence to the hypothesis that although LAT
is critical for TCR signaling, some TCRinduced, LAT-independent signaling can occur
in mature T cells. Furthermore, LAT-deficient
mature T cells that had developed in a LATsufficient environment could initiate lymphoproliferative disease when transferred into
T-cell-deficient (CD31 knockout) hosts. This
raises the fascinating possibility that TCRinduced, LAT-independent signaling mechanisms (in a lymphopenic context) can lead to
lymphoproliferative disease. It will be interesting to compare the LAT-independent signaling
mechanisms from this situation with LAT-independent signaling described in T-cell clones
(Chau and Madrenas 1999) and in Jurkat T cells
(Ku et al. 2001; Shan et al. 2001).
CONCLUDING REMARKS
As described in this review, identification of the
critical adapter molecule LATover a decade ago
has led to a multitude of studies. Our understanding of TCR-mediated signaling and T cell
biology in general has been thereby enriched.
We expect that continued application of stateof-the-art approaches will lead to further insight into how LAT serves as the focal point of
TCR-mediated activation.
ACKNOWLEDGMENTS
The authors would like to thank Robert
Kortum, Valarie Barr and Ronald Wange for
helpful suggestions.
16
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The LAT Story: A Tale of Cooperativity, Coordination, and Choreography
Cold Spring Harbor Perspectives in Biology, 2010
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