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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: [email protected]

The adapter molecule LATis a nucleating site for multiprotein signaling complexes that are vital for the function and differentiation of T cells. Extensive investigation of LATin 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 critical insights into mechanisms of signal transduction (Hunter 2009). Early studies inker for activation of T cells (LAT) was showed that a number of proteins became phos- Lcloned a little more than a decade ago. Since phorylated on tyrosine residues following TCR then, a multitude of studies have revealed that stimulation in Jurkat T-cell leukemia cells and LAT-based complexes catalyze critical TCR- in normal T cells (June et al. 1990). Many of mediated signaling reactions and enable activa- these proteins, such as ZAP-70, SLP-76, and tion of multiple downstream pathways that con- PLC-g1, have been shown to be critical elements trol almost all TCR-initiated cellular responses. for TCR signal transduction (Kane et al. 2000). This article spotlights diverse experimental sys- A protein with an apparent molecular weight tems in which LAT function has been studied. of 36 and 38 kDa was also prominently phos- Information gained from these studies has led phorylated on tyrosine in response to TCR to an integrated understanding of the cellular stimulation. Several preliminary studies showed function of LAT. that this protein, known then as pp36/38, was membrane-associated and capable of binding Cloning and Structural Features of LAT SH2 domains of Grb2, Grap, PLC-g1, and the The study of the tyrosine phosphorylation of p85 subunit of phosphatidylinositol 3-kinase proteins induced by immunoreceptor and (PI3K) (June et al. 1990; Gilliland et al. 1992; growth factor receptor stimulation has led to Buday et al. 1994; Sieh et al. 1994; Fukazawa

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et al. 1995a; Trub et al. 1997). Although pp36/ and are subject to posttranslational palmitoyla- 38 was first observed in 1990, it proved challeng- tion (Zhang et al. 1998b). Palmitoylation of LAT ing to isolate. It was not until 1998 that the is necessary for LAT function (Lin et al. 1999; Samelson laboratory cloned it by large-scale Zhang et al. 1999a), but the role of palmitoyla- membrane purification of activated Jurkat cells tion in specific localization of LAT within the (Zhang et al. 1998a). Shortly after, Weber et al. plasma membrane has been controversial and reported the cloning of the rat and human pro- is discussed below. More recently, studies have teins from thymocytes (Weber et al. 1998). The shown that LATis also subject to ubiquitylation Samelson lab named the protein product LAT, (Brignatz et al. 2005; Balagopalan et al. 2007), a for Linker for Activation of T cells based on sev- modification that might be involved in activa- eral of its characteristics. LAT is expressed in T tion-induced internalization of LAT complexes cells and in a limited number of other immune and regulation of LAT protein levels. Inspection cell types (mast cells, natural killer cells, mega- of LATamino acid sequence reveals two lysines karyocytes, platelets, and immature B cells) (K52 and K204 in human LAT), which might (Facchetti et al. 1999; Oya et al. 2003). Further- serve as potential sites for ubiquitylation. A more, as detailed below, LAT facilitates the re- schematic of LAT structural features is shown cruitment of many signaling proteins to the in Figure 1A. plasma membrane where it links receptors, tyrosine kinases and their substrates and other Palmitoylation and Membrane Localization effector molecules together, functioning as a of LAT critical activator of T cells. Sequencing of human LAT cDNA identified Palmitoylation is thought to enhance the asso- an open reading frame encoding a protein pre- ciation of transmembrane proteins, including dicted to contain 233 amino acids. The mouse LAT, with regions of membrane heterogeneity and rat homologs of LATencode 242 and 241 sometimes called lipid rafts (Brown 2006). In amino acid proteins, respectively, and have T cells, several signaling molecules in addition 65%–70% sequence identity with human LAT. to LAT, including the TCR, Lck, Vav, Grb2, The predicted molecular mass of LAT is PLC-g1, and Ras are associated with lipid rafts 25 kDa. However, LAT is strikingly acidic and (Brdicka et al. 1998; Montixi et al. 1998; Zhang its charge may account for slower migration et al. 1998b). Thus, rafts have been postulated on SDS-PAGE leading to its apparent molecular to function as important platforms to initiate weight of 36/38 kDa. Structurally, LAT is a signaling cascades (Brown and London 1998). type III transmembrane protein. It has a cyto- However, the physiological role of this mem- solic carboxyl terminus (like type I proteins), brane heterogeneity has been intensely debated but lacks a signal sequence (Brown 2006). LAT (Munro 2003; He and Marguet 2008; Kenwor- contains only a four-amino-acid extracellular thy 2008). Reflecting this debate, the impor- region, a single transmembrane spanning re- tance of raft localization for LAT function has gion and a long intracellular region with no been controversial. apparent intrinsic enzymatic activity or Early studies reported that a cysteine- protein–protein interaction domains. How- mutated form of LATwas not recruited to lipid ever, consistent with the strong tyrosine phos- rafts and could not reconstitute signaling in a phorylation of pp36/38 observed upon TCR LAT-deficient Jurkat cell line, leading to the stimulation, the intracellular domain of LAT conclusion that localization of LATto lipid rafts contains nine tyrosines conserved between was required for its function (Lin et al. 1999; humans, mice, and rats. Examination of LAT Zhang et al. 1999a). However, more recent stud- amino-acid sequence also revealed two con- ies indicated that palmitoylation of LAT was served cysteine residues (C26 and C29 in required for the protein to be transported human LAT), which are located adjacent to efficiently to the plasma membrane and that, the predicted transmembrane domain of LAT in the absence of palmitoylation, LAT was

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Signaling through LAT

A Ubiquitin Ubiquitin

O– O–

O OP–O OP–O O O O C26 Y36 K52 Y110 Y132 Y171 K204

N LAT C TM C29 Y45 Y64 Y127 T155 Y191 Y226 O O– O– O– O– O OP–O OP–O OP–O OP–O O O O O

B a TCR b

PIP PIP PIP g e e d 2 3 2 DAG DAG LCK RAS PI3K PKC SLP-76 PLC-g1 RASGRP Nck

z z WASp VAV Itk LAT InsP3 ZAP-70 GADS

GRB2 SOS1 Release of Ca++ NFAT Cytoskeletal ADAP reorganization translocation

MAPK Activate activation effectors Adhesion NF-kB & AP1

Figure 1. LATin TCR signal transduction. (A) Human LATis 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. Collectively, these data suggest that targeting 2000; Hundt et al. 2006; Tanimura et al. 2006; of LAT to the plasma membrane of the cell is Hundt et al. 2009). Furthermore, LAT fusion sufficient for its function, regardless of specific proteins targeted to nonraft domains recon- localization within the membrane. stituted LAT function in LAT-deficient Jurkat cells or LAT-deficient mice (Zhu et al. 2005; LAT is Central to T-Cell Signaling Hundt et al. 2009). These data raise the possi- bility that the signaling defects initially observed The essential role of LAT in T-cell signal trans- for a LAT palmitoylation mutant might result duction has been demonstrated in a variety of from defects in plasma membrane transport, experimental settings. Initial work showed that rather than displacement from lipid domains. Jurkat T-cell lines lacking expression of LAT

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were defective in several TCR-mediated signal- LAT,however CD148 has been implicated (Baker ing events including Ca2þ mobilization, Ras et al. 2001). activation, CD69 expression, Erk activation, and AP-1/NFAT-directed gene transcription (Finco PLC-g1 Binds Y132 of LAT and Mediates et al. 1998; Zhang et al. 1999a; Samelson 2002). Transcriptional Activation in T Cells Reintroduction of LATrescued all these defects indicating that LAT is indispensable for T-cell The phospholipase PLC-g1 is an important activation via the TCR. Soon after, targeted dis- mediator of TCR signal transduction. PLC-g1 ruption of the LAT gene in mice revealed a hydrolyzes phosphatidylinositol 4,5-bisphos- requirement for LAT in T-cell development. phate (PIP2) to produce inositol 1,4,5-trisphos- Animals that lack LATexhibited an early arrest phate (IP3) and diacylglycerol (DAG) (Rhee and of thymocyte development and no mature ab Bae 1997). DAG stimulates the Ras activator T cells were found in their peripheral lymphoid RasGrp (Ras guanyl-releasing protein) and the organs (Zhang et al. 1999b). Additional reports serine/threonine kinase PKC (protein kinase highlighted the adapter function of LAT by C). The production of IP3 by PLC-g1 promotes showing that multiple signaling proteins bind the release of stored intracellular calcium, caus- phosphorylated LATonce the TCR is engaged. ing an extracellular Ca2þ influx and a sustained These LAT binding proteins subsequently increase in intracellular Ca2þ concentrations attract multiple cytosolic protein partners and important for NFATactivation (Oh-hora 2009). further waves of tyrosine phosphorylation and PLC-g1 is a multidomain enzyme that protein interactions occur. Thus LAT functions includes a pleckstrin homology (PH) domain as a crucial nucleating site at the plasma mem- that can bind membrane phosphoinositides, brane for multiprotein signaling complexes. two phosphotyrosine-binding SH2 (src homol- ogy 2) domains and an SH3 (src homology 3) domain, which binds specific proline-rich LAT AS A NUCLEATION CENTER FOR T-CELL sequences. PLC-g1 binding to LATwas observed SIGNAL TRANSDUCTION even before LATwas cloned and was shown to be LAT functions as a classic adapter protein by dependent on the amino-terminal SH2 domain facilitating the formation of multiprotein of PLC-g1 (Gilliland et al. 1992; Weber et al. complexes (Fig. 1B). Numerous studies have 1992; Stoica et al. 1998; Irvin et al. 2000). Inter- investigated the composition of LAT-nucleated action with LAT was shown to be required for complexes. This section will describe the direct both PLC-g1 activation and localization near and indirect associations of enzymes, adapters its substrate PIP2 at the plasma membrane and effectors to LAT and provide insights into (Zhang et al. 1999a; Zhang et al. 2000; Lin and how these interactions lead to activation of spe- Weiss 2001). Subsequently, the amino-terminal cific intracellular pathways. SH2 domain of PLC-g1 was shown to bind phosphorylated LAT Y132 with high affinity (Paz et al. 2001; Houtman et al. 2004). Further- Phosphorylation of LAT more, a single Y132F mutation abolished the LAT is rapidly phosphorylated on tyrosine resi- association between LATand PLC-g1 in Jurkat dues upon TCR engagement. Though a full T cells (Zhang et al. 2000). However, further understanding of how the kinases phosphory- experimentation revealed that full PLC-g1 acti- late the individual tyrosines on LAT in vivo vation required additional binding interactions remains unresolved, overexpression and in vitro to LATand LAT-associated molecules. studies have implicated primarily ZAP-70, but Several studies showed that LAT Y171 and also Lck and Itk in LATphosphorylation (Zhang Y191 also contributed to PLC-g1 binding and et al. 1998a; Paz et al. 2001; Perez-Villar et al. that all four distal LAT tyrosines (132, 171, 2002; Jiang and Cheng 2007). Even less is known 191, and 226) were required for optimal PLC- about the phosphatases that dephosphorylate g1 phosphorylation and downstream calcium

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Signaling through LAT

influx (Zhang et al. 2000; Lin and Weiss 2001; 1998a). Studies of T cells from Grap-deficient Zhu et al. 2003). The adapter protein SLP-76 mice indicate that Grap may be a negative regu- (that binds to Gads, which binds to LAT Y171 lator of TCR signal transduction (Shen et al. and Y191) was also shown to be required for 2002). PLC-g1 activation (Yablonski et al. 1998). Brai- Sos1 (Son of Sevenless homolog) is a multi- man and colleagues demonstrated that multiple domain GEF that promotes activation of the domains of PLC-g1 were required for associa- small G protein Ras, which in turn activates tion with LAT and all three SH domains were the MAP kinase Erk (Quilliam 2007). Sos1 essential for enzyme activation. Furthermore, binds Grb2 constitutively (Wittekind et al. PLC-g1 activity depended on interactions with 1994; Houtman et al. 2006). Following TCR other LAT-associated proteins including SLP- ligation, Grb2 recruits Sos1 to the site of Ras 76, the guanine nucleotide exchange factor localization at the membrane. Early experi- Vav and the E3-ubiquitin ligase c-Cbl (Braiman ments suggested that a trimolecular complex et al. 2006). formed between Grb2, Sos1, and LAT following TCR engagement (Buday et al. 1994; Sieh et al. 1994). Consistent with this, Zhang and col- Grb2 Family Members Bind Y171, Y191, and leagues reported that Sos1 did not associate Y226 of Phosphorylated LAT and Recruit a with the LAT mutant Y171/191F, which also Number of Signaling Proteins to the LAT did not associatewith Grb2 (Zhang et al. 1998a). Complex Interestingly, the significance of a Grb2/Sos1 The Grb2 family members Grb2, Gads, and complex likely extends beyond the activation Grap can potentially bind phosphorylated LAT of Ras. Houtman and colleagues have described at the distal three tyrosines: Y171, Y191, and a mechanism whereby a 2:1 Grb2:Sos1 complex Y226 (Wange 2000). Grb2 (Growth-factor- can facilitate clustering of LAT-based signaling receptor-bound protein 2) is a ubiquitously ex- complexes (to be discussed in more detail later) pressed adapter protein that contains an SH2 (Houtman et al. 2006). domain flanked by amino- and carboxy-termi- The proto-oncogene c-Cbl encodes a 120 nal SH3 domains. The Grb2 adapter is involved kDa multidomain E3-ubiquitin ligase, which in Ras and MAP kinase pathway activation in is phosphorylated and associates with LAT fol- many cell surface receptor systems, primarily lowing TCR activation. c-Cbl also binds Grb2 by association with the guanine nucleotide ex- constitutively (Donovan et al. 1994; Fukazawa change factor (GEF) Sos1. Interestingly, Grb2 et al. 1995b). Grb2 seems to be required for association with LATrequires the SH2 domain the interaction between c-Cbl and LAT, because of Grb2 and pairs of LAT phosphotyrosines: c-Cbl does not associate with the LAT mutant Y171/Y191, Y171/Y226, or Y191/Y226 (Zhang Y171/191F, which fails to bind Grb2 (Zhang et al. 2000; Zhu et al. 2003). Gads (Grb2-related et al. 1998a). However, there is evidence that adapter protein downstream of Shc) contains a c-Cbl is also stabilized by interactions with structure similar to Grb2, but also has a unique other LAT-associated molecules, such as Nck glutamine- and proline-rich domain between (Rivero-Lezcano et al. 1994; Wunderlich et al. the SH2 domain and the carboxy-terminal 1999), the p85 subunit of PI3K (Fukazawa SH3 domain. In addition to binding LAT, et al. 1995b), and PLC-g1 (Rellahan et al. Gads binds SLP-76, which recruits a number 2003; Chiang et al. 2004; Braiman et al. 2006). of other signaling proteins including PLC-g1 By stabilizing the associations of molecules (see Fig. 1B) (Liu et al. 1999; Zhang et al. that contribute to signal activation, c-Cbl could 2000; Yablonskiet al. 2001). Grap (Grb2-related promote positive TCR-induced signals; how- adapter protein) is similar in structure to Grb2, ever, c-Cbl also appears to play a negative regu- but its role in T-cell signal transduction is not as latory role in signaling. well defined. It also associates with LATthrough In LAT-deficient Jurkat cells, c-Cbl is hyper- its SH2 domain (Trub et al. 1997; Zhang et al. phosphorylated upon TCR stimulation (Finco

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et al. 1998; Mingueneau et al. 2009) and phos- recruitment of proteins that contain PH do- phorylated c-Cbl has been implicated in nega- mains, which recognize PIP3 (Fruman et al. tive regulation of TCR signal transduction 1999). In T cells, PI3K regulates the recruitment (Boussiotis et al. 1997; Murphy et al. 1998). In of proteins that facilitate TCR signal trans- addition, consistent with its role as an E3-ubiq- duction such as PLC-g1, Sos1, RasGAP, Itk, uitin ligase in several signaling systems, c-Cbl Tec, and Vav. Early studies showed that PI3K has been implicated in ubiquitylation of the bound directly to LAT (Fukazawa et al. 1995a; TCRz and CD3d chains of the TCR as well as Lahesmaa et al. 1995; Paz et al. 2001); however the signaling proteins ZAP-70, LAT, PLC-g1, more recent studies showed binding of p85 to PI3K, Vav, and PKC-u in T cells (Weissman tyrosine-phosphorylated SLP-76 (Shim et al. 2001; Duan et al. 2004; Balagopalan et al. 2007). 2004). Inhibition of PI3K in T cells resulted in The effects of LATubiquitylation on controlling reduced TCR-induced calcium influx, Rac1 internalization and cellular LAT levels are dis- activation, and Erk1/2 phosphorylation. cussed in the imaging section later. However, PI3K inhibition did not reduce the phosphorylation of PH domain-containing proteins such as PLC-g1 and Vav (Cruz-Orcutt Molecules Recruited to LAT via SLP-76 and Houtman 2009). Therefore PI3K plays a Promote T-Cell Activation role in TCR activation, but its exact mechanism SLP-76 is an adapter protein containing three is still unclear. tyrosine motifs, a central proline-rich domain Other SLP-76 interactors have clearer roles and a carboxy-terminal SH2 domain (Koretzky on T-cell activation. Vav is a guanine nucleotide et al. 2006). The proline-rich domain of SLP-76 exchange factor that activates the Rho family binds SH3 domains of Grb2 and Gads; however G-proteins Rac1, RhoA, and Cdc42. A number SLP-76 binds Gads with a 40–50-fold stronger of studies have demonstrated the importance affinity than Grb2 (Berry et al. 2002; Harkiolaki of the association of the Vav SH2 domain et al. 2003). Gads recruits SLP-76 to LAT by way with SLP-76 phosphotyrosines (Koretzky et al. of the interaction of its SH2 domain with 2006). In addition to its catalytic role, Vav acts phospho-LAT (Liu et al. 1999). SLP-76 recruits as a scaffold to stabilize PLC-g1 and Itk interac- multiple effector molecules to the LATcomplex. tions with SLP-76. In Vav-deficient T cells, the The three phosphotyrosines of SLP-76 bind interaction between SLP-76 and PLC-g1was PI3K, Vav, Itk, and Nck. The central proline- substantially reduced and Itk was not phos- rich region binds to Gads with high affinity phorylated (Reynolds et al. 2002; Braiman and to PLC-g1 with low affinity (Yablonski et al. 2006). The Tecfamily kinase Itk phosphor- et al. 2001; Houtman et al. 2004). However, ylates PLC-g1 (Readinger et al. 2009) and LAT SLP-76 only associates weakly with LAT in the (Perez-Villar et al. 2002). Itk is recruited to the absence of the LAT residue Y132, suggesting LAT complex by binding of its SH2 domain to that SLP-76 binding to LAT is stabilized by the tyrosine phosphorylated SLP-76 (Bunnell presence of PLC-g1 (Zhang et al. 2000). The et al. 2000). Itk plays a major role in T-cell acti- carboxy-terminal SH2 domain of SLP-76 inter- vation by virtue of its phosphorylation of acts with the proteins HPK1, ADAP, and Shb. PLC-g1, which is required for the activation of These interactions result in positive and nega- PLC-g1. tive effects on TCR signaling. Interactions that Vav and the adapters Nck and WASp coop- lead to negative effects on TCR signaling will erate to effect cytoskeletal changes in activated T be discussed in the following section. cells. Nck binds phosphorylated SLP-76 (Wun- PI3K is comprised of a 110 kDa catalytic derlich et al. 1999) and WASp(Wiskott-Aldrich subunit and an 85 kDa regulatory subunit (p85) syndrome protein) (Rohatgi et al. 2001; Barda- and catalyzes the phosphorylation of PIP2 to Saad et al. 2005), bringing WASp in proximity generate PIP3 (Fruman and Bismuth 2009). to Vav. Vav recruits and activates Cdc42, which The accumulation of PIP3 facilitates membrane is necessary for WASp activation. Activated

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WASp interacts with the Arp2/3 complex to adapter SLAP (Src-like adapter protein) (Myers initiate actin polymerization and cytoskeletal et al. 2006), the tyrosine phosphatase SHP-1 changes (Zeng et al. 2003). (Lorenz 2009) and 4.1R (Diakowski et al. 2006). Two other adapter molecules that bind SLP- 4.1R binds LAT directly and is thought to pre- 76 and mediate positive signal transduction in T vent LAT phosphorylation by ZAP-70 (Kang cells are ADAP and Shb. Phosphorylated ADAP et al. 2009). binds the SH2 domain of SLP-76 (da Silva et al. 1997; Musci et al. 1997). This interaction is cru- cial for adhesion and integrin function in T cells COOPERATIVITY AMONG LAT-ASSOCIATED PROTEINS STABILIZES (Baker et al. 2009; Horn et al. 2009; Wang et al. SIGNALING COMPLEXES 2009). Tyrosine phosphorylated Shb also binds the SH2 domain of SLP-76 and promotes LAT, It is clear that LAT plays a central role in T-cell SLP-76, Vav, and PLC-g1 phosphorylation activation downstream of the TCR by directly (Lindholm et al. 1999; Lindholm et al. 2002). or indirectly recruiting kinases, effectors, and HPK1 is another molecule that binds the SH2 adapters to facilitate the coordination of highly domain of SLP-76 but mediates negative signal regulated signal transduction pathways. Bio- transduction. physical approaches have begun to reveal im- portant insights into some of the molecular mechanisms that govern the stabilization of LAT Interactions also Inhibit TCR Signal LAT-associated complexes. These studies indi- Transduction cate that numerous weak interactions coopera- Several molecules that bind to the LAT complex tively coordinate the formation of spatially can inhibit TCR signal transduction. HPK1 (he- and temporally specific signaling complexes. matopoietic progenitor kinase 1) is a MAP4 kinase (Hu et al. 1996; Kiefer et al. 1996) that Qualitative Results Indicate Cooperativity down-regulates activation of the MAP kinase Among LAT-Associated Proteins Erk2 and the transcription factor AP-1 in a kinase-dependent manner (Liou et al. 2000). Cooperativity is an altered affinity between pro- HPK-1 has a complex relationship with SLP- teins because of interaction with other mole- 76. It can phosphorylate SLP-76; however, this cules and/or posttranslational modifications. ultimately leads to decreased SLP-76 and PLC- A number of results suggest that LATcomplexes g1 phosphorylation and IL-2 production (Di are stabilized by cooperative interactions. SH2 Bartolo et al. 2007; Shui et al. 2007). domain-containing proteins including PLC- SHIP-1 is a lipid phosphatase that medi- g1, Grb2, and Gads bind directly to LAT phos- ates Fc receptor-dependent negative signaling photyrosine motifs with a preference for (Daeron and Lesourne 2006). In T cells, SHIP- PLC-g1 at pY132, Gads at pY191 and pY171, 1 also acts as an adapter to recruit Dok-1 and and Grb2 at pY171, pY191, and pY226 (Samel- Dok-2 to LAT complexes, which results in atte- son 2002). Nevertheless, mutations of LAT nuated TCR signaling. SHIP-1-associated Dok- Gads/Grb2-binding residues (Y171, Y191, and 1 and Dok-2 negatively regulate ZAP-70 activity Y226) reduced PLC-g1 binding (Zhang et al. (Dong et al. 2006; Yasudaet al. 2007) and recruit 2000; Hartgroves et al. 2003). At a minimum, RasGAP,which opposes Ras-mediated signaling Y132 and Y191 were required for an association (Tamir et al. 2000; Ott et al. 2002). The adapter between LAT and PLC-g1, indicating that protein Gab2 also has several potential mecha- PLC-g1 association with LATwas stabilized by nisms for inhibiting signaling. It can bind Gads binding to LAT(Zhu et al. 2003). Likewise, SHP-2 (Yamasaki et al. 2001) and it may com- there was evidence that PLC-g1 stabilized the pete with SLP-76 for binding to Grb2 and binding of Grb2 to LAT. LAT Y132F mutation Gads (Yamasaki et al. 2003). Other molecules resulted in substantial losses of both Grb2 that may mediate inhibitory signaling are the and PLC-g1 binding (Hartgroves et al. 2003).

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Experiments with LAT-nucleated signaling com- that the interaction required structural changes plexes on liposomes by Sangani and colleagues within SLP-76. They showed that the high affin- also indicated cooperativity. In these experi- ity interaction between Gads and SLP-76 sta- ments Grb2 alone did not stably bind lipo- bilized the PLC-g1-binding conformation of some-associated LAT; however, after incubation SLP-76. Therefore, positive cooperativity medi- with Jurkat lysate, Grb2 associated along with ated by the Gads/SLP-76 pair allowed PLC-g1 Gads, PLC-g1, SLP-76, Itk, and Sos1 (Sangani to bind SLP-76 (Houtman et al. 2004). Interest- et al. 2009). Interestingly, other studies showed ingly, LAT residue Y191 was phosphorylated that the LATphosphorylation sites were required substantially earlier than Y132 in T cells. within a single molecule and could not be added Therefore, it is possible that the Gads/SLP-76 in trans among multiple LAT proteins (Lin and complex associates with LAT before PLC-g1. Weiss 2001). This suggests that signaling com- Moreover, SLP-76 is among the earliest proteins plexes are stabilized around individual LAT to be phosphorylated (Houtman et al. 2005). molecules. Phosphorylated SLP-76 binds Itk, which has been shown to phosphorylate the PLC-g1 resi- due Y783 (Bunnell et al. 2000; Serrano et al. LAT Binding Preferences are not Solely 2005; Beach et al. 2007). Y783 of PLC-g1is Driven by Affinities phosphorylated before LAT Y132, suggesting Binding studies were performed to better that PLC-g1 associates with the Gads/SLP-76/ understand the basis for the specificity of the Itk complex before binding to pY132 (Hout- interactions of PLC-g1, Grb2, and Gads with man et al. 2005). It was suggested that these individual LAT motifs. Interestingly, Grb2 and cooperative binding events preceding PLC-g1 Gads did not have substantially different affin- binding to LAT could allow for tight control ities for the pY171, pY191, or pY226 motifs, of PLC-g1 activity. results that did not explain the preference of Gads for pY171 and pY191. However, the pro- Cooperative Interactions Promote LAT teins had a 50–100-fold weaker affinity for Oligomerization pY132, which explained the lack of binding to that site. The PLC-g1 SH2 domain bound the Because LAT can potentially bind two or more pY132 motif with stronger affinity than the Grb2 molecules and because the Grb2 SH3 pY171, pY191, or pY226 motif; however, it ligands Sos1 and c-Cbl also contain multiple was thought that the affinity difference alone Grb2 binding sites, Houtman and colleagues would not account for its selectivity (Houtman investigated whether Grb2 complexes could et al. 2004). Overall, these studies indicated that link multiple LAT molecules into higher-order the binding affinities alone were not sufficient structures (Fig. 2). Biophysical studies were car- to drive the apparent specificity of LAT motifs ried out to investigate the binding, composition for PLC-g1 and Gads. and stiochiometry of complexes formed from In addition to affinity, other factors likely mixtures of LAT with Grb2 and Sos1 or c-Cbl contribute to the specificity of PLC-g1 for the (Houtman et al. 2006; Houtman et al. 2007). LAT residue Y132. For example, the PLC-g1 The studies provided direct evidence that LAT PH domain would position PLC-g1 near the was able to bind multiple Grb2 molecules and membrane, likely favoring interactions with that the proline-rich regions of Sos1 and c-Cbl pY132, as it is the most membrane proximal each bound two Grb2 molecules. Interestingly, of the four motifs (Houtman et al. 2004). How- Grb2 bound the two regions of Sos1 with either ever, SLP-76 may also contribute to the stabili- significantly different affinities or a twofold zation of PLC-g1 binding to LAT. Binding negative cooperativity in binding to the second studies had revealed a weak interaction be- site, whereby binding of the first Grb2 weakened tween the SLP-76 proline-rich region and PLC- the affinity of Sos1 for a second Grb2 molecule. g1; however, Houtman and colleagues showed These results indicated that the 2:1 complex was

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Signaling through LAT

PIP3 PIP3

SLP-76 Nck PLC-g1 Nck

SLP-76 VAV PLC-g1 VAV

Itk Itk LAT LAT LAT

GADS GRB2 GADS c-Cbl SOS1 ADAP GRB2 GRB2 GRB2 ADAP

Figure 2. LAT complexes. Multiple LATmolecules are oligomerized by complexes of 2:1 Grb2:Sos1 or Grb2:Cbl in vitro and in cells. The clustering of LATcomplexes 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 a high degree of organization of signaling mole- was further demonstrated that Grb2 is in cules into macromolecular complexes visualized 1000-fold excess of Sos1 in T cells, suggesting as “microclusters” using imaging techniques as that the 2:1 complex of Grb2 and Sos1, which described later. forms in vitro could also be physiologically rel- evant in T cells (Houtman et al. 2006; Houtman IMAGING OF LAT SIGNALING COMPLEXES et al. 2007). Next, it was shown that Grb2/Sos AND CLUSTERS complexes could oligomerize LAT into large molecular complexes. Detailed studies of the In the past decade, advanced microscopic ap- energetics contributing to the complex forma- proaches have provided striking images of the tion showed that intermediate complexes, early events in T-cell activation. This section such as a 2:1 complex of Grb2:LAT,were slightly will review the contribution of imaging studies destabilized, consistent with negative coopera- in developing a clearer understanding of the ini- tivity in their formation. In contrast, positive tiation, composition, dynamics, and spatio- cooperative interactions resulted in a large temporal regulation of LAT-nucleated signaling stabilized molecular complex of LAT, Grb2, complexes. and Sos1. It was suggested that the presence of both negative and positive cooperativity in the LAT Microclusters: Sites of Nucleation of formation of these complexes promoted the Signaling Complexes assembly of physiologically relevant complexes, while suppressing intermediates (Houtman A little over a decade ago the immunological et al. 2007). The relevance of Grb2-mediated synapse (IS) was identified as a specialized junc- LAT clustering was verified in vivo and was tion between T cells and antigen presenting shown to have functional consequences on sig- surfaces, which is characterized by a central naling and T-cell activation (Houtman et al. cluster enriched for TCR-CD3 (cSMAC) and 2006). an integrin-rich peripheral cluster (pSMAC) The biophysical analyses of LAT and LAT- (Monks et al. 1998). Subsequent studies identi- associated proteins have revealed a role for in- fied the distal SMAC (dSMAC), an outer region termolecular cooperativity in stabilizing LAT- that contains large molecules such as CD43 and nucleated complexes. Cooperativity allows for CD45 (Delon et al. 2001; Freiberg et al. 2002).

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Real time visualization of the early events of interface and were transported and consoli- T-cell activation has given greater temporal and dated at the center of the contact to form a spatial accuracy. Studies in which images were cSMAC (Varma et al. 2006). Microclusters collected at the onset of T-cell activation showed play a predominant role in the generation of that within the first 30 s of contact of a T cell sustained signals. Current models also indicate with a stimulatory surface, small TCR-rich that cluster centralization plays an important structures form at the periphery of the T-cell role in signal termination and that the cSMAC contact zone (Grakoui et al. 1999; Krummel is a site both for TCR down-regulation yet also et al. 2000). These discrete structures enriched for amplification of weak signals (Lee et al. for TCR have subsequently been called “micro- 2003; Mossman et al. 2005; Varma et al. 2006; clusters.” Balagopalan et al. 2007; Cemerski et al. 2008; The first high resolution images of LAT Nguyen et al. 2008). A recent study using a pho- microclusters came from a method in which T toactivatable agonist in a lipid bilayer system has cells were dropped onto stimulatory coverslips provided even greater precision in the temporal coated with CD3 antibody and visualized using resolution of these events and demonstrated confocal microscopy (Bunnell et al. 2002). that adapters are recruited to microclusters Using this technique, LAT colocalized with within four seconds (Huse et al. 2007). TCR-rich microclusters within seconds of acti- Fine spatial imaging studies indicated that vation. In addition to LAT, a large number of signaling protein clusters may actually represent components of the TCR signaling pathway discrete interdigitating domains (Douglass including protein tyrosine kinases ZAP-70 and and Vale 2005; Nguyen et al. 2008). These struc- Lck, adapters SLP-76, Grb2, Gads, Nck and tures resembled the distinct protein and lipid WASp, and enzymes including PLC-g1, Vav, domains observed by transmission electron c-Cbl, and Sos1 were rapidly recruited to micro- microscopy (TEM) (Wilson et al. 2001; Lilleme- clusters. Larger glycoproteins such as CD43 ier et al. 2006). TEM studies in mast cells and T and the protein tyrosine phosphatase CD45 cells have shown formation of “primary signal- were excluded from these structures (Bunnell ing domains” that include the receptor, and et al. 2002; Barda-Saad et al. 2005; Braiman “secondary signaling domains” that include et al. 2006; Houtman et al. 2006). These rapidly LAT and PLC-g1, but not the receptor. Appa- assembled clusters of signaling complexes rently, these domains, submicron in size, pre- were the predominant sites of TCR-induced exist before receptor activation. Upon receptor tyrosine phosphorylation and their appearance activation, these domains were shown to con- was coincident with cytosolic Ca2þ elevations, catenate to form larger patches that are likely establishing TCR microclusters as sites of signal equivalent to the microclusters observed by initiation (Krummel et al. 2000; Bunnell et al. light microscopy (Wilson et al. 2001; Lillemeier 2002; Seminario and Bunnell 2008). Thus et al. 2006; Lillemeier et al. 2009). microclusters are the smallest structures visible Because the resolution limit of the micro- by confocal microscopy capable of driving scope (ffi200 nm) is much larger than the size effective TCR signaling and are made up of sig- of individual proteins or protein complexes naling complexes that contain adapters and (on the order of a few nanometers), colocaliza- effectors required for T-cell activation (Fig. 3). tion observed by fluorescence microscopy does Subsequent studies using peptide-MHC- not prove that proteins interact. Instead, Forster containing lipid bilayers and TIRF microscopy resonance energy transfer (FRET) can be verified and extended these results (Campi employed to study colocalization on the scale et al. 2005; Yokosuka et al. 2005; Varma et al. of a few nanometers and therefore provides 2006; Yokosuka et al. 2008; Kaizuka et al. information about molecular interactions 2009). Under these experimental conditions, (Gascoigne et al. 2009). Using this technique, microclusters were continuously generated FRET was detected between PLC-g1 and either at the periphery of the T-cell peptide-MHC LAT, c-Cbl, Vav or SLP-76, as well as SLP-76

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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 immobility and rapid diffusion (Douglass and interactions of these proteins within microclus- Vale 2005). Transient immobilization of LAT ters (Barda-Saad et al. 2005; Braiman et al. correlated strongly with encounter with clus- 2006).Incomparison,measurable butlowFRET ters. These data indicate that LATmolecules dif- was observed between LATand either SLP-76 or fuse between clusters with occasional trapping Nck, consistent with previous data showing that within clusters. these molecules interact indirectly (Barda-Saad et al. 2005). Mechanisms of Assembly of LAT Clusters Another microscopic technique, fluores- and LAT-Nucleated Complexes cence recovery after photobleaching (FRAP), has been used to evaluate LAT dynamics at the Data from various experimental systems have population level. This method allows evalua- shown that microclusters play a crucial role in tion of mobility and population-level diffusion TCR-mediated signaling pathways because dynamics of molecules (Tanimura et al. 2003a). inhibition of cluster formation resulted in Studies on LAT dynamics at the membrane re- reduced levels of TCR signaling (Singer et al. vealed the fast exchange of molecules between 2004; Bunnell et al. 2006; Houtman et al. LAT clusters or patches localized at sites of 2006). The importance of clustering may be to stimulation (Tanimura et al. 2003b; Douglass concentrate activators and exclude inhibitors and Vale 2005). Single-molecule imaging stud- creating microdomains that shift the equili- ies have revealed that LATmolecules at the plas- brium to favor downstream signaling in T ma membrane display abrupt changes between cells. However, the mechanisms that govern the

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assembly of these structures have remained elu- raft association of LAT through mutations did sive. Because LATis both a lipid raft resident and not alter its diffusion behavior (Douglass and an adapter with multiple tyrosines that serve as Vale 2005). docking sites for signaling proteins, the contri- The earlier-described results are most con- bution of both lipid rafts as well as protein scaf- sistent with LAT clusters being formed and folds in LAT clustering has been investigated. maintained by a network of protein–protein Early studies indicated that lipid raft pat- interactions. The combination of biochemical ches form at the sites of TCR activation and and biophysical studies has shown that cooper- copatched with proteins essential for T-cell acti- ative and multivalent interactions drive the vation (Harder and Simons 1999; Janes et al. assembly of productive signaling complexes 1999). Hence it was proposed that lipid rafts and imaging approaches have supported this provide a platform for assembly of signaling scenario. Houtman et al. used confocal micro- domains during T-cell activation. However, scopy to verify the physiological relevance of data from multiple studies indicated that mem- Grb2-Sos1-mediated oligomerization of LAT, brane recruitment, not raft localization was observed by biophysical techniques (Houtman required for LAT function (Zhu et al. 2005; et al. 2006). A LAT mutant unable to bind Hundt et al. 2009). Furthermore, Harder and Grb2 did not localize to signaling clusters. In Kuhn showed that LAT, but not other raft-asso- addition, expression of a Sos1 fragment unable ciated molecules such as Lck and GM1, was to oligomerize LAT inhibited LAT clustering selectively enriched within plasma membrane and downstream signaling. Thus, multivalent fragmentscontainingactivatedTCR,callinginto interactions between these three molecules ap- question the view that upon TCR activation coa- peared to promote the assembly of multiprotein lescenceofraft-associatedmembraneproteinsin complexes important for TCR activation. In the vicinity of activated TCR leads to T-cell trig- another study using confocal approaches, Bun- gering(HarderandKuhn2000).Imagingstudies nell and colleagues demonstrated that coopera- also showed that LAT did not colocalize with a tive interactions between LAT, Gads and SLP-76 general raft marker GFP-GPI (Bunnell et al. stabilized SLP-76 microclusters (Bunnell et al. 2002). Together, these studies indicate that lipid 2006). Another report demonstrated that al- rafts are not a primary factor in the assembly of though the SH3 and C-terminal SH2 domains LAT-containing signaling complexes. of PLC-g1 do not bind directly to LAT,they par- Instead, several studies have indicated that ticipate in the stabilization of PLC-g1-LATasso- phosphotyrosine-dependent protein interac- ciation via other proteins in the LAT-nucleated tions play the predominant role in organization signaling complex (Braiman et al. 2006). of TCR-dependent signaling complexes. First, Finally, several studies have argued that LATwas not recruited to TCR-enriched immu- actin is required for segregating molecules on noisolates in cells treated with the potent Src the T-cell surface (Wulfing and Davis 1998; kinase inhibitor PP2 (Harder and Kuhn 2000). Delon et al. 2001). F actin was present at micro- Second, imaging approaches using planar sub- clusters and the contact sites of T cells and stim- strates demonstrated that LAT variants with ulatory surfaces at early time points (Bunnell mutated tyrosines in the cytoplasmic tail were et al. 2001; Barda-Saad et al. 2005). TEM studies not recruited to signaling microclusters of Wilson et al. in mast cells also support the (Douglass and Vale 2005; Bunnell et al. 2006; interaction of the actin cytoskeleton with re- Houtman et al. 2006). Finally, single particle ceptor clusters (Wilson et al. 2001). Further- tracking techniques as well as FRAP methods more, inhibitor studies showed that actin is that measured diffusion rates of LAT revealed essential for the formation of microclusters that LAT cytoplasmic tyrosines are required (Campi et al. 2005; Douglass and Vale 2005; for confinement of LATwithin transient subdo- Varma et al. 2006; Nguyen et al. 2008). An intact mains (Tanimura et al. 2003b; Douglass and cytoskeleton is also required for dynamic trans- Vale 2005). In contrast, interfering with lipid location of clusters once they are formed and it

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Signaling through LAT

has been proposed that retrograde actin flow intimately involved in the sorting of LAT and drives centripetal cluster movement (Barr et al. SLP-76 into mobile endocytic structures. 2006; Varma et al. 2006; Kaizuka et al. 2007; In addition to containing tyrosines, the LAT Nguyen et al. 2008; Ilani et al. 2009). In this cytoplasmic domain contains two lysines that regard, microclusters must be connected to actin could serve as sites of ubiquitylation. Indeed filaments via linker proteins, and LAT-mediated LAT ubiquitylation has been observed both in recruitment of actin-regulating adapters Nck Jurkat T cells and in COS-7 cells (Brignatz and WASpto these structures may play a central et al. 2005; Balagopalan et al. 2007). Consistent role in this process (Barda-Saad et al. 2005). with c-Cbl being an E3 ligase for LATubiquity- lation, expression of c-Cbl caused a modest in- crease in LAT ubiquitylation. In contrast, the Dynamics of LAT Clusters, LAT Internalization RING finger mutant caused a significant de- and LAT Ubiquitylation crease in ubiquitylated LATspecies. These data The dynamic nature and changing composition are consistent with a model in which c-Cbl- of TCR microclusters after they are generated mediated ubiquitylation is required for rapid has been extensively demonstrated. Across all internalization of LAT-nucleated signaling clus- modeling systems, following initial recruitment ters. Given the essential scaffolding role of the in close proximity of the TCR, components of adapter protein LAT in T cell activation, the the complexes rapidly exit microclusters with regulated internalization of activated LAT sig- distinct dynamics (Bunnell et al. 2002; Barda- naling complexes may be one efficient strategy Saad et al. 2005; Braiman et al. 2006). The by which to control the duration and localiza- adapters LAT and SLP-76 departed the initial tion of signaling from microclusters and, thus, complexes in what appeared to be small vesicu- regulate the kinetics, intensity, and specificity lar intermediates and dissipated soon thereafter of T-cell signaling. For a detailed discussion (Bunnell et al. 2002). Recent studies have shown on endocytic regulation of T-cell microclusters, that microclusters containing LAT and SLP-76 see Balagopalan et al. 2009. undergo endocytosis upon TCR stimulation (Barr et al. 2006; Balagopalan et al. 2007). ANIMAL STUDIES REVEAL THE IN VIVO SLP-76 is endocytosed in a lipid-raft dependent FUNCTIONS OF LAT pathway that requires the association of the endocytic machinery with ubiquitylated pro- Although T-cell lines have been enormously teins. LAT endocytosis is a complex process useful for discerning the role of LAT in T-cell and internalized LAT is found in several intra- function within single T cells, animal model sys- cellular compartments (Bonello et al. 2004; tems provide the opportunity to study the func- Brignatz et al. 2005; Balagopalan et al. 2007). tion of LATduring T-cell development as well as Insights into the molecular mechanisms that its function in T cells in the context of the entire regulate endocytosis of microcluster-associated immune system. proteins came from studies in which internal- ization of the microclusters was inhibited. First, Studies of LAT Null Mice expression of versions of c-Cbl that are defective in the RING domain that mediates ubiquitin Studies of LAT null mice show the crucial ligase activity severely inhibited LAT and SLP- importance of LATin pre-TCR and TCR signal- 76 movement and endocytosis. Furthermore, ing. ab T-cell development proceeds through the ubiquitin-interacting motif (UIM) of eps15, ordered stages that can be characterized by which is known to block clathrin-independent expression of the cell surface markers CD4 internalization of the EGFR (Sigismund et al. and CD8: DN (double negative for CD4 and 2005), severely inhibited the internalization CD8)!DP (double positive for CD4 and of SLP-76 clusters (Barr et al. 2006). Thus, CD8)!SP (single positive for either CD4 or the E3 ligase c-Cbl and ubiquitin appear to be CD8). The DN stage can further be divided

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into four stages based on the expression of the knockout bone marrow cells were infected with cell surface markers CD44 and CD25: DN1 retroviruses containing mutant LAT C26/29A. (CD44þCD252)!DN2 (CD44þCD25þ)! The infected bone marrow cells were trans- DN3 (CD442CD25þ)!DN4 (CD442 CD252). ferred to irradiated wild type B6 mice and Signaling through the immature, nonclonotypic the mutant LAT was unable to mediate T-cell pre-TCR drives development from the DN3 to development (Hundt et al. 2009). To assess the DN4 stage. Mature TCR signaling drives whether raft localization (in addition to plasma T-cell development from the DP to SP stage. membrane localization) was necessary for the During the DP!SP transition, thymocytes are in vivo function of LAT, several approaches tested based on their TCR specificities and are have been taken using LATfusion proteins. LAT- selected for survival (by positive selection, based LAX and Src-LAT fusion proteins localized to on MHC specificity and nonself reactivity) or the plasma membrane but not to rafts. Both of death (by negative selection, based on potential these fusion proteins mediated T-cell develop- self-reactivity) (Sommers et al. 2004). ment in LAT-deficient environments (Zhu In LAT knockout mice, the endogenous et al. 2005; Hundt et al. 2009). Therefore C26 wild-type LAT gene was replaced with a mutant and C29 of LATwere necessary for plasma mem- form of the gene that could not be expressed brane localization and for the in vivo function by homologous recombination (Zhang et al. of LAT, but raft localization was not necessary 1999b). LAT knockout mice showed a complete for T-cell development. block in ab T-cell development at the imma- In vitro experiments have shown the impor- ture, DN3 stage indicating an essential role for tance of the distal four phosphotyrosines for the LAT in pre-TCR signaling. No peripheral ab T function of LAT. A “knock-in” LAT mutant in cells were observed. To investigate the role of which the endogenous wild type LAT gene was LAT in mature TCR signaling, Shen et al. used replaced with a mutant form of LAT containing a conditional knockout approach (Shen et al. Y!F substitutions at the four distal tyrosines 2009). Their approach used Cre-Lox technology revealed that those four distal tyrosines were (Wang 2009), whereby conditional expression required for T-cell development (Sommers of the Cre recombinase allowed deletion of a et al. 2001). Knock-in mutants of Y136! F LoxP-flanked sequence that resulted in T cell- (corresponding to human Y132! F) showed specific disruption of expression of LAT at the an incomplete block in early ab T cell develop- DP stage. The authors observed a severe block ment and a fatal lymphoproliferative disease in the DP!SP stage of thymocyte development involving marked lymphadenopathy, spleno- indicating a strong role for LAT in TCR-medi- megaly, and multiorgan lymphocyte infiltration ated thymocyte selection. However, some CD4 (Fig. 4) (Aguado et al. 2002; Sommers et al. and CD8 SP T cells developed in these mice and 2002). A knock-in LATmutant containing Y!F it was unclear whether these T cells developed mutations at Y175, Y195, and Y235, (corre- because of residual LAT in some developing sponding to human Y171, Y191, and Y226) DPs or because LAT is not completely essential also developed lymphoproliferative disease for the DP!SP transition (Shen et al. 2009). (Nunez-Cruz et al. 2003). Although the lym- phoproliferative phenotypes of these two mouse models were similar, the intrathymic T-cell In Vivo Functions of LAT Phosphotyrosines development differed between these two mod- and Cysteines els. In LAT Y136F knock-in mice, some ab T Previous in vitro studies with C26/29A LAT cells developed and expanded, whereas in LAT mutants had shown that the mutant LATmole- Y175/195/235F knock-in mice, cells of the gd cules could not be tyrosine phosphorylated, T cells lineage developed and expanded. did not localize to plasma membrane rafts In addition to the developmental block in and could not mediate TCR signaling (Zhang LAT Y136F mice, thymocyte selection was pro- et al. 1998b). In the animal experiments, LAT foundly affected (Sommers et al. 2005) and Treg

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Signaling through LAT

BC

A 24232221201918

LAT-KI WT

D E

Figure 4. Lymphoproliferative disease in LATY136F 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 other events downstream of PLC-g1 (Sommers (Koonpaew et al. 2006; Wang et al. 2008). et al. 2002). Reports of Erk1/2 activation in Abnormal cytokine production was observed LAT Y136F T cells have been variable (Sommers in these mice, especially elevated levels of IL-4. et al. 2002; Mingueneau et al. 2009; Miyaji et al. In fact the mice showed many aspects of Th2 2009), most likely because of different methods lymphoproliferative disease including IgG1 of in vitro activation and age of the animals and IgE hypergammaglobulinemia, elevated from which the T cells were derived. However, a serum IL-4, eosinophilia, and lymphocyte pro- report by Miyaji et al. showed that Erk activation liferation in lungs (Aguado et al. 2002; Som- played an important role in lymphoproliferative mers et al. 2002; Genton et al. 2006; Miyaji disease in LAT Y136F mice (Miyaji et al. 2009). et al. 2009). The contributing factors leading to Precisely how other aspects of altered signaling development of lymphoproliferative disease are (especially altered calcium signaling) lead to still under investigation, but some are known. hyperproliferation, decreased cell death and Altered or crippled signaling leads to a block alteredcytokineproduction inLATY136FTcells in T-cell development and a lymphopenic envi- is a subject for future investigations. ronment. Altered signaling in the T cells de- veloping in this lymphopenic environment LAT-Independent TCR Signaling results in T-cell hyperproliferation, defective activation-induced cell death and altered cyto- A recent report by Mingueneau et al. highlights kine production (IL-4,IL-2). Lack of de- a mouse model in which LAT-independent T velopment and function of Treg cells may be cell signaling was demonstrated (Mingueneau secondary to decreased IL-2 production. Al- et al. 2009). In these experiments, CD4þ T cells tered signaling in LAT Y136F T cells consists that had developed in a LAT-sufficient envi- of decreased TCR-induced calcium flux and ronment were rendered LAT-deficient following

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expression of Cre recombinase, which was ac- REFERENCES complished by retroviral infection. After ad- Aguado E, Richelme S, Nunez-Cruz S, Miazek A, Mura AM, ditional culturing with IL-2, the cells were Richelme M, Guo XJ, Sainty D, He HT, Malissen B, et al. stimulated through the TCR and various re- 2002. Induction of T helper type 2 immunity by a point sponses were assessed or the T cells were mutation in the LATadaptor. Science 296: 2036–2040. adoptively transferred to T cell-deficient hosts. Baker RG, Hsu CJ, Lee D, Jordan MS, Maltzman JS, Hammer DA, Baumgart T, Koretzky GA. 2009. The Although phosphorylation of several key signal- adapter protein SLP-76 mediates "outside-in" integrin ing molecules was not observed in the LAT-defi- signaling and function in T cells. Mol Cell Biol 29: cient mature T cells (including PLC-g1 and Erk 5578–5589. Baker JE, Majeti R, Tangye SG, Weiss A. 2001. Protein tyro- 1/2), phosphorylation of some signaling mole- sine phosphatase CD148-mediated inhibition of T-cell cules was observed (e.g., SLP-76 and Akt). receptor signal transduction is associated with reduced TCR-induced calcium flux and interferon-g LAT and phospholipase Cg1 phosphorylation. Mol Cell production were also profoundly diminished Biol 21: 2393–2403. Balagopalan L, Barr VA, Samelson LE. 2009. Endocytic in these cells. However, these experiments lend events in TCR signaling: Focus on adapters in microclus- credence to the hypothesis that although LAT ters. Immunol Rev 232: 84–98. is critical for TCR signaling, some TCR- Balagopalan L, Barr VA,Sommers CL, Barda-Saad M, Goyal induced, LAT-independent signaling can occur A, Isakowitz MS, Samelson LE. 2007. c-Cbl-mediated regulation of LAT-nucleated signaling complexes. Mol in mature T cells. Furthermore, LAT-deficient Cell Biol 27: 8622–8636. mature T cells that had developed in a LAT- Barda-Saad M, Braiman A, Titerence R, Bunnell SC, Barr sufficient environment could initiate lympho- VA,Samelson LE. 2005. Dynamic molecular interactions linking the T cell antigen receptor to the actin cytoskele- proliferative disease when transferred into ton. Nat Immunol 6: 80–89. T-cell-deficient (CD31 knockout) hosts. This Barr VA, Balagopalan L, Barda-Saad M, Polishchuk R, raises the fascinating possibility that TCR- Boukari H, Bunnell SC, Bernot KM, Toda Y, Nossal R, induced, LAT-independent signaling mecha- Samelson LE. 2006. T-cell antigen receptor-induced sig- naling complexes: Internalization via a cholesterol- nisms (in a lymphopenic context) can lead to dependent endocytic pathway. Traffic 7: 1143–1162. lymphoproliferative disease. It will be interest- Beach D, Gonen R, Bogin Y, Reischl IG, Yablonski D. 2007. ing to compare the LAT-independent signaling Dual role of SLP-76 in mediating T cell receptor-induced mechanisms from this situation with LAT-inde- activation of phospholipase C-g1. J Biol Chem 282: 2937–2946. pendent signaling described in T-cell clones Berry DM, Nash P, Liu SK, Pawson T, McGlade CJ. 2002. A (Chau and Madrenas 1999) and in Jurkat T cells high-affinity Arg-X-X-Lys SH3 binding motif confers (Ku et al. 2001; Shan et al. 2001). specificity for the interaction between Gads and SLP-76 in T cell signaling. Curr Biol 12: 1336–1341. Bonello G, Blanchard N, Montoya MC, Aguado E, Langlet CONCLUDING REMARKS C, He HT, Nunez-Cruz S, Malissen M, Sanchez-Madrid F, Olive D, et al. 2004. Dynamic recruitment of the adap- As described in this review, identification of the tor protein LAT: LATexists in two distinct intracellular pools and controls its own recruitment. J Cell Sci 117: critical adapter molecule LATover a decade ago 1009–1016. has led to a multitude of studies. Our under- Boussiotis VA, Freeman GJ, Berezovskaya A, Barber DL, standing of TCR-mediated signaling and T cell Nadler LM. 1997. Maintenance of human T cell anergy: biology in general has been thereby enriched. Blocking of IL-2 gene transcription by activated Rap1. Science 278: 124–128. We expect that continued application of state- Braiman A, Barda-Saad M, Sommers CL, Samelson LE. of-the-art approaches will lead to further in- 2006. Recruitment and activation of PLCg1 in T cells: sight into how LAT serves as the focal point of A new insight into old domains. EMBO J 25: 774–784. TCR-mediated activation. Brdicka T,Cerny J, Horejsi V.1998. T cell receptor signalling results in rapid tyrosine phosphorylation of the linker protein LAT present in detergent-resistant membrane microdomains. Biochem Biophys Res Commun 248: ACKNOWLEDGMENTS 356–360. The authors would like to thank Robert Brignatz C, Restouin A, Bonello G, Olive D, Collette Y.2005. Evidences for ubiquitination and intracellular trafficking Kortum, Valarie Barr and Ronald Wange for of LAT, the linker of activated T cells. Biochim Biophys helpful suggestions. Acta 1746: 108–115.

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Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a005512 21 Downloaded from http://cshperspectives.cshlp.org/ on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press

The LAT Story: A Tale of Cooperativity, Coordination, and Choreography

Lakshmi Balagopalan, Nathan P. Coussens, Eilon Sherman, Lawrence E. Samelson and Connie L. Sommers

Cold Spring Harb Perspect Biol published online July 7, 2010

Subject Collection Immunoreceptor Signaling

The Coordination of T-cell Function by Perspectives for Computer Modeling in the Study Serine/Threonine Kinases of T Cell Activation David Finlay and Doreen Cantrell Jesse Coward, Ronald N. Germain and Grégoire Altan-Bonnet ITAM-mediated Signaling by the T-Cell Antigen Structural Biology of the T-cell Receptor: Insights Receptor into Receptor Assembly, Ligand Recognition, and Paul E. Love and Sandra M. Hayes Initiation of Signaling Kai W. Wucherpfennig, Etienne Gagnon, Melissa J. Call, et al. Coordination of Receptor Signaling in Multiple Src-family and Syk Kinases in Activating and Hematopoietic Cell Lineages by the Adaptor Inhibitory Pathways in Innate Immune Cells: Protein SLP-76 Signaling Cross Talk Martha S. Jordan and Gary A. Koretzky Clifford A. Lowell The Cytoskeleton Coordinates the Early Events of The LAT Story: A Tale of Cooperativity, B-cell Activation Coordination, and Choreography Naomi E. Harwood and Facundo D. Batista Lakshmi Balagopalan, Nathan P. Coussens, Eilon Sherman, et al. An Enigmatic Tail of CD28 Signaling Antigen Receptor Signaling to NF-κB via Jonathan S. Boomer and Jonathan M. Green CARMA1, BCL10, and MALT1 Margot Thome, Jean Enno Charton, Christiane Pelzer, et al. Mediation of T-Cell Activation by Actin It's All About Change: The Antigen-driven Meshworks Initiation of B-Cell Receptor Signaling Peter Beemiller and Matthew F. Krummel Wanli Liu, Hae Won Sohn, Pavel Tolar, et al.

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Copyright © 2010 Cold Spring Harbor Laboratory Press; all rights reserved Downloaded from http://cshperspectives.cshlp.org/ on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press

T-Cell Signaling Regulated by the Tec Family ZAP-70: An Essential Kinase in T-cell Signaling Kinase, Itk Haopeng Wang, Theresa A. Kadlecek, Byron B. Amy H. Andreotti, Pamela L. Schwartzberg, Raji E. Au-Yeung, et al. Joseph, et al. Lipid Signaling in T-Cell Development and Understanding the Structure and Function of the Function Immunological Synapse Yina H. Huang and Karsten Sauer Michael L. Dustin, Arup K. Chakraborty and Andrey S. Shaw

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Copyright © 2010 Cold Spring Harbor Laboratory Press; all rights reserved