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 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 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. Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a005512 1 Downloaded from http://cshperspectives.cshlp.org/ on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press L. Balagopalan et al. 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 2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a005512 Downloaded from http://cshperspectives.cshlp.org/ on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press 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.