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(Journal of Leukocyte Biology. 2001;69:317-330.)
© 2001 by Society for Leukocyte Biology

Molecular motors involved in T cell receptor clusterings

Connie Krawczyk and Josef M. Penninger

Amgen Institute/Ontario Cancer Institute, Departments of Medical Biophysics and Immunology, University of Toronto, Ontario, Canada

Correspondence: Josef Penninger, Amgen Institute/Ontario Cancer Institute, Departments of Medical Biophysics and Immunology, University of Toronto, 620 Toronto Ave., Ontario, Canada M5G 2C1. E-mail: jpenning{at}amgen.com


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ABSTRACT
 
Engagement of antigen receptors on T and B cells triggers reorganization of the cytoskeleton and ordered clustering of cell surface receptors. These receptor clusters constitute spatially organized signaling machines and form the immune synapse with antigen-presenting cells. Formation of supramolecular activation clusters appear to be essential to induce functional lymphocyte responses and have been implicated as molecular mechanisms of costimulation. The Vav1-Rho-GTPase-WASP pathway constitutes a molecular motor that relays antigen receptor stimulation to changes in the cytoskeleton and receptor clustering.

Key Words: lymphocytes • actin • Vav • cytoskeleton • T-cell activation • immune synapse


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INTRODUCTION
 
"In one of his gloomier moments Pascal said that man’s unhappiness stemmed from a single cause, his inability to remain quietly in a room. ‘Notre nature,’ he wrote, ‘est dans le mouvement. ... La seule chose qui nous console de nos misères est le divertissement.’ Diversion. Distraction. Fantasy. Change in fashion, food, love, and landscape. We need them as the air we breathe. Without change our brains rot." (Bruce Chatwin, Anatomy of Restlessness). Without change, our lymphocytes rot. All stages in lymphocyte life and activation are associated with profound changes in cell morphology that depend on a functional cytoskeleton. This review examines the role of the actin cytoskeleton as an integral component of signal transduction and cell fate in lymphocytes, and discusses the molecular mechanisms that link antigen receptor engagement to the actomyosin cytoskeleton and the formation of the supramolecular immune synapse.

Lymphocyte development, activation, and function are reliant on a dynamic cytoskeleton. During lymphocyte maturation, developing thymocytes and B-cell precursors are in continuous contact with stromal cells of the microenvironment, and these physical contacts are crucial prerequisites for lymphocyte maturation and selection [1 ]. Mature lymphocytes migrate through blood and lymph vessels, attach to endothelial cells, transmigrate through blood vessel walls, and extracellular matrix spaces, home into secondary lymphoid organs, interact with antigen-presenting cells (APCs) and stromal cells, and adhere to target cells. These extracellular interactions relay intracellular signals, which influence lymphocyte homeostasis, inducing proliferation, activation, survival, apoptosis, and/or unresponsiveness. Precise lymphocyte responses rely on effective signal transduction cascades, which are influenced by cytoskeletal remodeling. Many signaling molecules are associated with cytoskeletal scaffolds, and the cytoskeletal structure and scaffold geometries can directly regulate molecular dynamics of signaling and biochemical responses [2 ]. Similarly, cytoskeletal reorganizations and subcellular distribution of signaling molecules and scaffolded signaling modules help to fine-tune cellular responses. Moreover, it has been shown that under certain experimental conditions cell shape can regulate cell fate decisions for survival or apoptosis [3 , 4 ]. Compartmentalization of signaling cascades through protein-protein or protein-lipid interactions is a fundamental mechanism for the initiation, fine-tuning, and propagation of effective signal transduction and cellular responses [5 , 6 ]. In lymphocytes, a highly organized supramolecular activation complex (SMAC) has been described that forms at the site of contact between an APC and a T cell. Based on the structural similarity to synapses in the nervous system, SMACs have also been termed the "immune synapse." SMAC formation is dependent on the actin cytoskeleton and can be induced by costimulatory signals [7 ].


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CYTOSKELETAL HARD-WIRING AND METAMORPHOSIS
 
Actin microfilaments, myosin intermediate filaments, and microtubulin bundles are the organizers of the cytoskeleton. Actin is expressed in all eukaryotic cells, whereas myosin expression is restricted to multicellular organisms. Recent experimental findings suggest that cells use tensional integrity architecture for their organization, similar to geometric principles in geodesic Buckminster domes or the carbon 30 structure [8 ]. Tensegrity implies that cells are hard-wired and can rapidly respond to mechanical changes. It has been shown that this hard-wired cytoskeleton can mediate immediate mechanochemical responses from cell surface receptors to the nucleus [4 , 9 ]. Mechanical tension within the cytoskeleton appears to regulate biological functions, including vertebrate brain morphogenesis, chromosome movement, and gene transcription [10 ]. Moreover, modulation of cell shape using micrometer-scale islands of extracellular matrix proteins, different-sized microbeads, or different densities of extracellular matrix, has demonstrated that cell shape and cell geometry play important roles in determining apoptosis as well as growth [11 ]. Cell spreading can define apoptosis or growth, independent of the type of matrix proteins/integrin receptors engaged, suggesting that local geometric control of cell growth and viability may represent a fundamental mechanism for developmental regulation within defined organ microenvironments. Remarkably, integrin binding and mechanical tension can induce movement of mRNA and ribosomes to the focal adhesion complex, indicating that protein translation can occur near the site of signal reception and that gene expression can be locally controlled by targeting mRNAs to specialized cytoskeletal domains [12 ].

Although hard-wired actin cables and tension-regulated cell shape changes can contribute to biochemical responses, an extraordinary feature of the cytoskeleton is that it is also highly adaptable and changes rapidly in a dynamic fashion. After biochemical or mechanical stimulation, various reversible and rapid changes occur in the actin cytoskeleton: monomeric, globular G-actin polymerizes into filamentous F-actin (microfilaments) constructed of identical strings of actin monomers. Strings of F-actin assemble into F-actin bundles and form higher-order structures such as stress fibers, which allow cultured cells to attach to surfaces. Actin filaments also associate with the plasma membrane and determine cell shape, motility, or formation of filopodias and lamellipodias. Similarly, intermediate myosin filaments together with F-actin form highly organized structures such as sarcomeres in muscles. Moreover, polymeric microtubules are structures that are in a dynamic balance with monomeric subunits. It is important to note that cytoskeletal organization can be different in distinct compartments of the same cells [13 , 14 ]. Because many cytoplasmic molecules, including signaling molecules, are associated with the cytoskeleton, and the cytoskeleton provides a scaffold on which organelles and proteins bind, subcellular differences in the actin cytoskeleton have profound functional consequences for a cell.


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LYMPHOCYTE FUNCTION AND ACTIVATION: IMPLICATIONS OF THE CYTOSKELETON
 
Whereas most dynamic cytoskeletal changes have been deciphered in adherent cells, experiments on the role, structure, and inducibility of the cytoskeleton in T and B lymphocytes are much more complex due to their cell shape and large nuclei. Engagement of antigen receptors in T cells and B cells triggers of an initial wave of signaling events including tyrosine phosphorylation, phospholipase C{gamma} (PLC{gamma}) activation and Ca2+ flux, activation of protein kinase C (PKC) isoforms, MAPK, and SAPK/JNK signaling pathways, and translocation of transcription factors into the nucleus [15 16 17 ] (Fig. 1 ). Activation of antigen receptors in lymphocytes also leads to changes in the actin cytoskeleton defined by actin polymerization, dynamic reorganization of the actin matrixes, membrane ruffling, or formation of filopodias and uropods [18 , 19 ]. In addition, antigen-receptor engagement leads to reorientation of the microtubule organizing centers (MTOCs) and membrane-associated cytoskeleton in order to accomplish unidirectional killing of target cells by cytotoxic T cells and natural killer (NK) cells or during physical interactions between T helper cells and B cells to regulate polar secretion of cytokines [20 ]. Moreover, in T cells the Golgi apparatus is reoriented toward antigen-presenting cells. Butanedione monoxime can prevent CD43-induced polarization and homotypic cell aggregation indicating the involvement of the myosin cytoskeleton in these phenomena [21 ]. Engagement of antigen receptors on T cells also leads to the recruitment of signaling molecules and TCR/CD3 complexes into so-called membrane rafts [22 23 24 25 26 ]. Rafts are defined anatomical microdomains in the cell membrane that are enriched in glycosphingolipids and cholesterol [22 , 26 ].



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  		Figure 1. Schematic of defined pathways involved in T-cell activation. Activation of T cells requires both a TCR-mediated signal and a costimulatory signal. TCR ligation induces the activation of src-family kinases such as Lck and Fyn. These kinases phosphorylate ITAM motifs on CD3{zeta}, which subsequently recruit and activate Zap70, a syk family kinase, via its SH2 domain. ZAP70 phosphorylates many downstream targets, including Vav1, SLP-76, and LAT. Many adaptor proteins influence and are required for the activation of downstream signaling cascades that lead to cell cycle progression and cytokine production. The Grb2 protein activates the exchange factor Sos, which activates Ras and thus the MAPK cascade. Grb2 also plays a role in the activation of phosholipase C gamma (PLC{gamma}1), which hydrolyzes PIP2 to produce diacylglycerol (DAG) and inositide triphosphate (IP3). DAG and IP3 induce protein kinase C (PKC) activation and calcium release, respectively. PKC can also activate the MAPK pathway. In addition, PKC activation, in particular PKC{theta}, contributes to the activation of NF-{kappa}B, which targets survival and inflammatory genes. Calcium mobilization activates a phosphatase, calcineurin, which dephosphorylates the cytoplasmic nuclear factor of activated T cells (NF-ATc). NF-ATc, when dephosphorylated, translocates to the nucleus and activates several genes, including the IL-2 gene. SAPK/JNK activation is induced by both signals from CD28 as well as Rho family GTPases. SAPK/JNK phosphorylates c-Jun, a component of the transcriptionally active AP1 complex. Another component of the AP1 complex is Fos, which is activated by the MAPK pathway. Signals from both the TCR and CD28 result in the Vav1 phosphorylation. Active Vav1 induced Rho-family GTPase-mediated cytoskeletal remodeling via the effector WASP. The cytoskeletal remodeling induces the formation of TCR caps and SMACs. Formation of the SMAC is essential for sustained tyrosine phosphorylation and signaling, and thus T-cell activation. Other, as yet undefined, signaling pathways must exist.

Activation of the T cell antigen receptor causes a rapid increase in filamentous actin and activation-dependent association of filamentous actin with the TCR{zeta} chain [27 ]. However, approximately 30–40% of total TCR{zeta} molecules on the surface of resting T cells are already associated with the actin cytoskeleton [28 ]. The cytoskeletal-linked TCR{zeta} chains display distinct patterns of phosphorylation after TCR activation and could play a negative role in signal transduction [29 ]. In addition, various cytoskeleton-organizing molecules such as ezrin, talin, vinculin, paxillin, fimbrin, the focal adhesion kinase (FAK), its homologue Pyk2 (CAKß or RAFTK), and the myosin light chain have been shown to be phosphorylated and activated after antigen receptor activation [30 ]. These studies suggest that TCR-mediated signal transduction cascades lead to cytoskeletal remodeling, which is essential for TCR signal propagation and T-cell activation. It should be noted that experiments performed on the Jurkat T cell line using pharmacological inhibitors revealed contradictory evidence that a dynamic actin and microtubule cytoskeleton were indeed necessary for proximal T cell signaling events. TCR-induced tyrosine phosphorylation in Jurkat T cells after a 5-min incubation with anti-CD3 antibody was not affected by cytochalasin D and colchicine, which disrupt actin polymerization and microtubule polymerization, respectively [31 ]. Although this study suggests that immediate TCR-induced tyrosine phosphorylation events may not require actin and tubulin polymerization, it does not indicate that the signaling events induced by the TCR in the presence of cytoskeletal-disrupting pharmacological agents are sufficient to induce T-cell proliferation, activation, and cytokine production. Recent studies have demonstrated a correlation between induction of cytoskeletal remodeling and sustenance of protein tyrosine phosphorylation, both events crucial for full T-cell activation [7 , 32 ]. In naive T cells, these events require costimulatory signals and are evident 5–15 min after receptor engagement. It is therefore plausible that, after TCR stimulation, cytoskeletal reorganization inhibitors would abrogate sustained tyrosine phosphorylation rather than immediate tyrosine phosphorylation events and result in a failure to induce sufficient signal transduction events resulting in full T-cell activation. Actin cytoskeletal reorganization has been shown to be necessary for T-cell proliferation and cytokine production, however, precise contribution of the microtubule cytoskeleton to propagation of TCR signaling events is not certain. Although microtubule blocking agent colchicine does not appear to have an effect on immediate proximal TCR signals, its relevance in sustained TCR signaling is unknown [31 ]. However, tubulin has been shown to interact with and be a substrate of ZAP70, a tyrosine kinase important in TCR signaling [33 ]. In addition, tubulin was detected in co-immunoprecipitates with Vav1, a crucial mediator of cytostructure remodeling events after TCR signaling [34 ]. Finally, as mentioned, reorientation of the MTOC is an early activation event after signals that induce full T-cell activation. Collectively, both tubulin and actin polymerization appear important to cellular reorganization after TCR signaling to induce, sustain, and propagate effective signal transduction cascades sufficient for T-cell activation.

More recently, the necessity of a dynamic cytoskeleton for lymphocyte activation and function was solidified through the use of genetically targeted mice. Mutations that interfere with antigen receptor-mediated cytoskeletal reorganization, including mutations in the guanosine nucleotide exchange factor Vav1 [35 , 36 ] Rho-family proteins [19 ], or the Wiscott Aldrich syndrome protein (WASP) [37 ] severely impair lymphocyte activation and lymphocyte development. The studies on genetically targeted mice have not only identified important molecular links between signaling cascades and induction of cytoskeletal remodeling, but have allowed the dissection of cytoskeletal-dependent and -independent signaling cascades downstream of the TCR.

Elegant studies have revealed an essential role of actin remodeling on T-cell activation. Most cell surface receptors that regulate cell growth or death form supramolecular activation clusters. In lymphocytes, antigen receptor stimulation leads to the formation of antigen receptor Caps, which are asymmetrical aggregations of antigen receptors at the site of receptor engagement [38 ]. Engagement of the TCR in the presence of costimulation induces the formation of the immunological synapse, which is an organized assembly of antigen receptor clusters, adhesion molecules, co-receptors, membrane rafts, and intracellular signaling molecules located at the site of receptor engagement [39 ]. The immune synapse is further organized into structurally discrete SMACs [40 ]. The process of receptor and lipid raft recruitment and SMAC formation is dependent on dynamic reorganization of the actin and myosin cytoskeleton [39 ]. For a more complete and detailed review of the reorganization events observed during the formation of the immune synapse and the contribution of lipid rafts to TCR signaling, please see previously published reviews [40 41 42 ].


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THE IMMUNE SYNAPSE
 
SMACs and caps/patches have been recognized for a long time [43 44 45 ] and specify highly organized structures generated by the recruitment and exclusion of antigen receptors, coreceptors, integrin receptors, signaling molecules, and cytoskeletal components such as the actin anchors Vinculin and Talin. The notion that the actin cytoskeleton might be involved in intracellular signaling was first raised in the 1970s in studies which showed that treatment of cells with cytochalasin, a fungal metabolite that inhibits actin reorganization, could block the formation of the Cap structure [45 ]. Because the Cap includes many molecules that are involved in lymphocyte activation (including the antigen receptor), it was thought that the Cap might be required for conveying signals to the cellular interior. In the following years, conflicting data accumulated regarding the role of this actin-dependent collection of signaling molecules. The significance of the Cap was called into question when several groups realized that Cap formation required 3–5 min, whereas the tyrosine phosphorylation characteristic of early activation events occurred within 10 s of antigen receptor engagement [38 ]. If one believed that tyrosine phosphorylation was all there was to signal transduction, the question seemed settled: the slow-forming, actin-dependent Cap had no role in signaling during lymphocyte activation. However, in the last few years, very elegant genetic, functional, and morphological studies have shown that formation of the antigen receptor cluster appears to be crucial to initiate physiological responses in lymphocytes [35 , 36 ]. These physiological responses include gene transcription, cytokine production, cell cycle progression and proliferation, lymphocyte selection, or antigen-receptor-triggered apoptosis.

In resting T and B cells, antigen receptors appear to be evenly distributed throughout the cell surface. Activation of antigen receptors on T lymphocytes leads to the organization of SMACs at the interfaces of physical contact between T cells and antigen-presenting cells [46 ]. SMACs constitute highly organized structures that assemble antigen receptors, adhesion and co-receptors, and signaling molecules within a focal interaction site (Fig. 2 ). Whereas by definition SMACs form at sites of antigen recognition, antibody cross linking of antigen receptors can also trigger the formation of asymmetric and polar receptor patches and caps. Antibody-induced patches and caps might be similar in their functional relevance to SMACs, although important structural differences must exist due to the nature of these interactions. Formation of SMACs and Caps are relatively late events in TCR activation and depend on a functional actomyosin cytoskeleton. The reorganized cytoskeleton probably pulls the antigen receptors in the lipid membranes to form Caps and SMACs and provides a scaffold for the focal assembly for structural and signaling molecules. Antigen receptor activation also leads to modulation of integrin affinity, a phenomenon known as inside-out signaling [47 ]. Induction of high-affinity integrin binding enhances the overall avidity of interactions between lymphocyte and antigen-presenting cells and further links cell surface receptors to the cytoskeleton. Actin-cytoskeleton-dependent clustering of antigen receptor together with other membrane and cytosolic proteins is critical for sustained TCR signaling [48 , 49 ]. Full T-cell activation requires not only a TCR-mediated signal but also a costimulatory signal. It is intriguing that costimulatory signals have been shown to be required for both sustained TCR-induced protein tyrosine phosphorylation and SMAC formation [7 ]. Thus, if costimulation is essential for SMAC formation, SMAC formation may coordinate the molecular mechanisms that regulate immunological tolerance.



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  		Figure 2. The organization of the SMAC facilitates the interaction between relatively small membrane proteins, including CD3, TCR, CD28, CD2, and CD4 and their ligands on the APC. Adhesive forces by the small CD2/CD48 and the larger LFA-1/ICAM-1 interaction provide the initial stability of the the T cell:APC conjugate. After TCR ligation with MHC-peptide complex, integrins are induced into a high-affinity conformation, which further stabilizes the conjugate. Actin remodeling reorganizes the membrane proteins and intracellular signaling molecules such that the TCR stimulus can be sustained for the duration for required for T-cell activation. Specific organization of the intracellular signaling molecules is thought to be facilitated by lipid raft microdomains. Distinct assemblies of signaling complexes associate within the lipid rafts, which accumulate at the site of TCR engagement (represented by the green line). The SMAC organization is thought to exclude large glycoproteins such as CD43 and CD45, which may obstruct the association of the TCR with MHC/peptide because of their size. Structural exclusion of a protein tyrosine phosphatase, such as CD45, from tyrosine kinases should also allow sustained signaling.

Recent experiments using three-dimensional immunofluorescence to visualize the contact areas between T cells and peptide-pulsed antigen-presenting cells indicate that SMACs are not uniform but are highly segregated and organized into distinct spatial domains [46 ]. The central areas of these clusters contain TCR, CD3, p56lck, p59fyn, and PKC{theta}. The peripheral SMAC regions are enriched in LFA-1 and Talin. Segregation of MHC class II molecules and the LFA-1 ligand ICAM1 in the contact zone of the antigen-presenting cell mirrors SMAC segregation on T cells. Based on physical dimensions and molecular interactions, it has been proposed that CD4, CD8, CD28, and CD2 molecules cosegregate with the central SMAC areas, whereas CD45 and CD43 might also be excluded from the central contact zone [49 ]. Spatial segregation of activated receptors and signal transducing molecules might provide a unique environment for signal transduction. When T cells were activated with altered peptides that do not trigger cytokine production or T cell proliferation, CD3 and talin formed clusters but failed to segregate. Thus, it appears that segregation of receptors and signaling molecules within SMACs is critical for the initiation of physiological responses.

The critical question remains how and why SMACs segregate into two structurally distinct regions. One hint came from the cloning of a novel SH3-domain-containing adaptor molecule called CD2-associated protein (CD2AP). Engagement of the adhesion molecule CD2 initiates a process of protein segregation, receptor clustering, and polarization of the T cell cytoskeleton [50 ]. Similarly, CD2AP can mediate receptor clustering and cytoskeletal polarization. Besides three SH3 domains, CD2AP also contains proline-rich stretches and a sequence that has homology to the monomeric actin-binding protein thymosine-ß4. Because association between CD2 and CD2AP are dependent on T-cell activation, it has been suggested that CD2AP links TCR activation to the adhesion molecule CD2 and subsequent receptor patterning and T-cell polarization. Similarly, CD2AP might scaffold T-cell polarization and receptor clustering. However, CD2 gene-deficient mice have no apparent defects in T-cell activation and lymphocyte development, suggesting that surface receptors other than CD2 can mediate receptor patterning and/or CD2AP can associate with different molecules. The CD2AP knockout mice are reported to develop a severe polycystic kidney disease, however, the effect of a loss of function mutation of CD2AP on formation of the immune synapse has not been reported [51 ].


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THE MOLECULAR MOTOR OF ANTIGEN RECEPTOR CLUSTERING
 
The connection between signaling and Cap formation has been revealed in recent studies of genetically engineered mouse mutants. The assembly of the Cap is defective in T and B cells deficient for the guanine nucleotide exchange factor Vav1 [35 , 36 ]. Vav1 links TCR stimulation to the activation of the Rho-family kinases Rac1, CDC42, and RhoA [52 , 53 ]. Vav1-deficient T cells and thymocytes exhibit defects in antigen receptor-induced actin polymerization and the recruitment of actin to the TCR{zeta} chain [36 , 54 ]. Cap formation is also impaired in the CDC42-associated WASP-deficient mice [37 ]. In addition to defects in antigen receptor capping, human Wiscott Aldrich syndrome is characterized by immunodeficiency, thrombocytopenia, and lymphoid malignancies. The transfection of the gene for a dominant-negative Rac1 mutation also results in a failure of Cap assembly in lymphocytes [35 ]. These experiments indicate that Vav1, Rac1, and WASP constitute at least part of a signaling pathway that links antigen receptor engagement to cytoskeletal reorganization, receptor clustering, and Cap formation.


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RHO-FAMILY GTPASES AND GUANINE NUCLEOTIDE EXCHANGE FACTORS
 
The Rho-like GTPases CDC42, Rac, and Rho orchestrate distinct cytoskeletal changes in response to receptor stimulation and regulate the actin cytoskeleton, which controls morphology, adhesion, and motility of cells [55 ]. For example, in fibroblasts, Rho regulates stress fiber formation in response to LPA or bombesin; Rac mediates formation of lamellipodias and filamentous actin in response to insulin, activated-Ras, or platelet-derived growth factor (PDGF); and CDC42 activity is required for the formation of filopodias in response to bradykinin [56 , 57 ]. Besides organization of the actin cytoskeleton, extensive studies have uncovered roles of Rho-family GTPases in the activation of stress-activated protein kinases (SAPKs or c-Jun amino-terminal kinases JNK), p21-activated kinases (PAK) signaling, cell cycle progression, and transcription of SRF genes and cyclin D [58 59 60 61 ]. It appears that GTPases function as molecular switches in cells that coordinate cellular responses as diverse as cytoskeletal changes and DNA synthesis.

Multiple Rac1, CDC42, and/or RhoA target molecules have been described that differ in their functions and cellular distribution [62 ]. For example, Rho can associate with and/or potentially activate PIP5 kinase, which regulates PIP2 production, protein kinase N (PKN), p164 Rho-associated kinase (Rho-K), p160 Rho-associated coiled-coil-containing protein serine/threonine kinase (ROCK), PLD1, or the myosin light chain (MLC) phosphatase. Rac associates with the p21-activated kinase PAK, PLD1, POSH, POR1, MLK2, MLK3, p60 S6 kinase, p67phox, IQGAP1/2, and possibly PIP5 kinase. Interplay between Rac1 and Rac2 and PKB/Akt has been reported. Rac2-deficient mast cells have a defect in PKB/Akt activation, whereas Rac1 has been implicated as a regulator of PKB/Akt activation [63 64 65 ]. CDC42 interacts with WASP, p21-activated kinase PAK, MLK2, MLK3, IQGAP1/2, or p60 S6 kinase. This list of Rho, Rac, and CDC42 binding partners and molecular targets is expanding [66 ].

Although many of the described Rho, CDC42, and Rac targets might be involved in the regulation of the cytoskeleton, production of PIP2 via PIP5 kinase activation might be a crucial link between surface receptor engagement, Rho-GTPases, and reorganization of the cytoskeleton. PIP2 can bind to the actin capping molecules gelsolin and profilin. This association releases the capping molecules from actin and thus promotes polymerization [67 , 68 ]. PIP2 can also associate with and unfold vinculin and regulate vinculin/talin/actin interactions in focal sites of adhesion [69 , 70 ]. PIP2 has also been implicated in the translocation and association of the chromatin remodeling complex SWI/SNF (or BAF complex) to the chromatin in T cells [71 ]. In addition to PIP2, it has been proposed that the Rho-regulated MLC phosphatase regulates actin bundling and stress fiber formation and links Rho-activation to the myosin-actin cytoskeleton [72 ]. Moreover, PAK1, which is expressed in lymphocytes, has been shown to regulate actin polymerization in mammalian cells [73 ].

The Rho family of GTPases has been most characterized as crucial regulators of cytoskeletal remodeling. However, the complex regulation of cytoskeletal architecture is not under exclusive control by the Rho family GTPases. ARF6, a member of the ARF family of Ras-related GTPases, redistributes to the cortical cytoskeleton [74 ] and can induce cortical actin polymerization possibly by regulating Rac1 distribution [75 ]. ARF1 potentiates Rho-induced assembly of actin stress fibers and formation of paxillin-rich focal adhesions [76 ]. As well, overexpression of the ARF-activating protein, ASAP, in cell lines alters the cellular morphology and influences cytoskeletal remodeling in response to PDGF stimulation [77 ]. Gcs1p, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, binds actin filaments and stimulates actin polymerization in vitro [78 ].

Lymphocytes express many small G proteins including p21Ras, Rap1, and the Rho-family GTPases RhoA, Rac1, Rac2, and CDC42 [19 ]. All of these molecules have a crucial role in the development and function of lymphocytes. For example, a functional Ras signaling pathway is required in B cell development before formation of the pre-B cell receptor and Ras signaling is important for TCR-mediated positive, but not negative selection [79 ]. Rap1 has been implicated in the induction of clonal anergy and regulation of interleukin-2 (IL-2) production in Jurkat cells [80 ]. Early thymocyte differentiation requires Rho for the survival of pro-T cells and for the cell cycle progression of late pre-T cells [81 , 82 ]. Constitutive activation of Rac2, a hematopoietic-specific small GTPase, or Cdc42 in thymocytes or T cell lines can induce apoptosis [83 ]. Cdc42 controls T cell polarity toward antigen-presenting cells and is required for IL-2 production [84 , 85 ]. Rac1 also has a role in antigen-receptor and IL-2 receptor-mediated membrane ruffling [86 ] and, importantly, TCR-mediated cap formation [87 ]. Moreover, Rac1 is important in NK cells to reorient cytotoxic granules toward their target cells and abrogation of Rac1 activity in NK cells activity impairs cytotoxic activity [88 ]. In mast cells, Rac and CDC42 activity has also been implicated in Fc{varepsilon}RI-mediated phagocytosis and kit-mediated proliferation. CDC42 is also a crucial regulator of dendritic cell (DC) endocytosis. Immature DCs actively internalize antigen and express active CDC42, whereas active CDC42 is undetectable in poorly endocytic mature DCs [89 ]. In short, Rho-like GTPases play a crucial role in orchestrating physiological responses in hematopoietic cells and T and B lymphocytes in response to receptor engagement. The challenge for the future of lymphocyte activation will be to dissect expression and complex formation of different Rho-GTPases with different downstream targets.

Similar to Ras, Rho-like GTPases are being activated when bound GDP is exchanged for GTP, a process catalyzed by guanine nucleotide exchange factors (GEF; or GDP-dissociation stimulator GDS proteins). GEFs are a growing family of cellular second messengers characterized by the canonical coupling of a Dbl-homology (DH) domain, which confers guanine-nucleotide exchange activity, to an adjacent pleckstrin-homology (PH) domain. The DH domain of the proto-oncogene Dbl confers the capacity for guanine nucleotide exchange for the Rho family of small GTPases [59 , 62 , 73 , 90 91 92 ]. Besides prototypic Dbl-homology family GEFs, an additional family of GEFs, the PIX (PAK-interacting exchange protein) family, which exhibit catalytic activity toward Rac1 have been isolated by virtue of binding to the PAK kinase family of Rac and Cdc42 effectors [93 ]. In yeast, binding of the GEF Cdc24 to the Cdc42 effector Ste20, which is a PAK homologue, regulates the growth of filamentous actin. In mammalians, GEFs exhibit a multitude of different functions. For example, the GEF Tiam1 and Tiam1-regulated Rac1 signaling regulate membrane ruffling and invasiveness of T cell lymphomas [94 ]. On the other hand Tiam1/Rac1 signaling can inhibit invasion of epithelial cells and can restore E-cadherin-mediated adhesion in Ras-transformed MDCK cells [95 ], suggesting that Tiam1-Rac1 can promote or suppress invasion of tumor cells depending on the cellular signals and cell types.


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VAV1: SIGNALING AND THE ACTIN CYTOSKELETON
 
Vav stands for the sixth letter of the Hebrew alphabet and was cloned as the sixth oncogene through gene transfer assays designed to isolate novel human transforming genes [96 , 97 ]. Vav1 is expressed in hematopoietic cells and is rapidly phosphorylated after activation of T cells and B cells by various growth factors or by cross-linking of antigen receptors [98 99 100 ]. Among all GEFs described so far, Vav1 contains a unique combination of protein-protein interaction modules in addition to its GEF domain (DH domain), which include an SH2 domain flanked by two SH3 domains which reflects the role of Vav1 in signaling pathways downstream of protein tyrosine kinases. Moreover, Vav1 contains a pleckstrin homology (PH) domain, known to facilitate membrane localization and a calponin homology (CH) domain [97 , 98 ]. CH domains mediate protein binding to filamentous actin (F-actin) and are found in several cytoskeletal and signaling proteins including the spectrin and filamin family of cytoskeletal proteins, and the Cdc42 target protein IQGAP. Binding of Vav1 to filamentous actin has not been shown yet; however, this domain of Vav1 must be an important regulatory region because deletion of this region (amino-terminal deletion of 67 amino acids), which probably destroys the actin-binding capability of the calponin-homology domain, results in the oncogenic activation of Vav1. If this is the case, the oncogenic transversion of Vav1 may result from inappropriate cellular localization.

Recently two structural Vav1 homologues have been identified, Vav2 and Vav3 [101 , 102 ]. Both Vav2 and Vav3 are expressed in T cells and could potentially function in a similar signaling pathway as Vav1. However, in in vitro experimental systems, it appears that at least Vav3 has different substrate specificity for Rho-family GTPases as compared to Vav1 [102 ]. Moreover, it is possible that different receptors and/or upstream signaling pathways differentially activate Vav1, Vav2, or Vav3.

Vav1 was originally implicated in Ras and MAPK signaling [98 , 103 , 104 ]. However, recent biochemical in vitro experiments and genetic studies have established that Vav1 functions as a GDP/GTP-exchange factor (GEF) for the Rho-like small GTPases RhoA, Rac1, and CDC42 [53 , 60 , 62 , 92 , 105 ]. Moreover, Vav1 expression can complement the temperature-sensitive Cdc24ts yeast strain, but Vav1 does not complement the Cdc25ts strain, which is the yeast exchange factor for Ras. In intact cells, it is, however, not known whether Vav1 activates all Rho-family GTPases or exhibits substrate specificity toward a selective member of the Rho-family GTPases [53 ]. Because RhoA, Rac, and CDC42 can interact and regulate distinct downstream signaling pathways, elucidation of the exact in vivo GTPase target of Vav1 is of crucial importance to identify specific effectors of Vav1-regulated signaling pathways.

For Vav1 to act as a GEF for Rac, RhoA, or CDC42, PI3’ kinase activity and the binding of PI3’K-generated phospholipid products to the PH domain of Vav1 are required [106 ]. PH domains mediate interactions with specific charged headgroups of phosphoinositides and regulate subcellular redistribution of signal molecules to regions of localized signaling at the plasma membrane as well as the activity of the target proteins. For instance, the exchange activity of Vav1 toward Rac1 is enhanced in the presence of the PI3’K products phosphatidylinositol-3-4-phosphate [PtdIns(3,4)P] or phosphatidylinositol-3-4-5-phosphate [PtdIns(3,4,5)P] but inhibited in the presence of the PI3’K substrate PI(4,5)P2 [106 ]. Thus it appears that PI3’ kinases may strongly stimulate Vav1 activity by converting inhibitory regulators of Vav1 to positive activators. In addition to the regulation by phospholipids, full activation of Vav1 is dependent on TCR-mediated tyrosine phosphorylation, which is mediated via p56lck and/or ZAP70 kinases [53 , 107 , 108 ]. These experiments suggest that the catalytic activity of Vav1 is regulated by a combination of enzymatic and positional signals and Vav1 catalytic activity requires two input signals originating from activated Lck/ZAP70 and membrane phosphoinositides generated by activated PI3’K. The differences in the levels of the two signals may contribute to the fine tuning of cellular responses to antigen-receptor signals via Vav1 activity. In addition, GTPases activated by Vav1 may also be located in or recruited to the microenvironment of the activated receptor. This prediction is in line with findings that RhoA, Rac1, and CDC42 colocalize with the TCR/CD3 complex to receptor patches and caps [36 ]. It is interesting that Vav1 may initiate a negative feedback loop via activation of Rac and Rho, both of which have been shown to bind to and activate phosphoinositide-4-phosphate 5 kinase, which catalyzes production of the Vav1 inhibitor PI(4,5)P2 (PIP2) [109 ]. PIP2 in cell membranes is highly rate limiting, and catalysis of PIP2 into IP3 and diacylglycerol by PLC{gamma} is a crucial prerequisite for the opening of intracellular Ca2+ channels and activation of various PKC isoforms. Moreover, PIP2 can promote actin polymerization and stabilizes focal adhesion complexes.

In addition to the GEF activity, Vav1 appears to function as a molecular scaffold with adaptor molecules such as Slp-76 or Nck, which participate in the assembly of multimolecular signaling complexes downstream of activated receptors [110 ]. Accordingly, it has been reported that Vav1 associates with a large variety of molecules via its SH2 and two SH3 domains. Whereas SH3 domains can be also found in two other mammalian GEFs, Dbs, and PIX, each of which contain one SH3 domain, Vav1 was the first known GTPase activator that contains an SH2 domain. The Vav1 SH2 domain binds Shp-1, Syk, Zap70, Slp-76, and c-Cbl [108 , 111 112 113 114 ]. Vav1 itself becomes rapidly phosphorylated on tyrosine residues upon activation by a variety of cell-surface receptors, and becomes a target of SH2-containing proteins, which include Syk, Fyn, PLC{gamma}, p120 RasGAP, Shc, and the p85 subunit of PI3’K. In Jurkat T cells, Vav1 and the Vav1-associated phosphoprotein Slp-76 synergize with Calcineurin to induce NF-AT transactivation and IL-2 production [112 , 114 , 115 ]. Slp-76 expression is fairly restricted to T cells and gene ablation of Slp-76 leads to a complete block in early thymocyte development [116 ].

The Vav1-SH3 domain interacts with a variety of quite unrelated proteins such as the heterogenous ribonucleoprotein K (hnRNPK), which has been proposed as a docking platform for the assembly of signaling modules, Ku-70, which forms part of the DNA-binding subunit of the nuclear DNA-dependent subunit of protein kinase DNA-PK, and Cbl-b, a member of the Sli/c-Cbl protein family implicated in negative regulation of signal transduction [117 ]. Moreover, the molecular adapter Grb2 associates Vav1 via the Grb2 SH3 domain. In B cells, it has been shown that Vav1 associates with Btk via the Btk-SH3 domain [118 ]. Intriguingly, hnRNPK, which associates with the Vav1-SH3 domain and WASP, also binds to the SH3 domain of Btk [119 ]. WASP is a cytoskeletal protein and effector of CDC42 required for IL-2 production and proliferation in T cells and TCR capping [37 ]. Moreover, the Vav1 carboxy-terminal SH3 domain has been shown to associate with the cytoskeletal protein zyxin.

To analyze the role of Vav1 on in vivo T cell development and activation, we and others have targeted the vav1 gene by homologous recombination [35 , 36 ]. vav1-/- mice are viable and healthy. However, in the absence of Vav1, early thymocyte development at the pre-TCR stage, positive and negative thymocyte selection, peptide/MHC- and antigen receptor-mediated thymocyte apoptosis, antigen receptor-induced cell cycle progression, calcium mobilization, and the activation of immune response genes such as the IL-2 gene are all impaired [35 , 36 , 54 , 120 ]. Although the TCRs do not cluster in response to antigenic stimulation, principal signaling pathways such as overall tyrosine phosphorylation, MAPK and SAPK activation can still operate. However, the level of signal transduction achieved is clearly not sufficient to produce a complete T cell response. Vav1 was also found to bind constitutively to the cytoskeletal membrane anchors talin and vinculin and in the absence of Vav1, phosphorylation Slp-76 and vinculin, Slp-76/talin interactions, and recruitment of the actin cytoskeleton to the CD3{zeta} chain were impaired. Consistent with a role for Vav1 in transducing TCR signals to the actin cytoskeleton, vav1-/- mice T cells displayed impaired actin polymerization in response to antigen receptor activation and exhibit defective clustering (patching and capping) of the TCR. Functionally, loss of Vav1 expression results in impaired, albeit not abolished, in vivo B and T cell immunity in response to multiple virus infections and challenges with protein antigens [121 ]. vav-/- B cells develop normally and can be activated by repetitive, polyvalent T cell-independent viral antigens that effectively cross link B cell receptors, but not by non-repetitive hapten antigens or anti-IgM antibody treatment [122 ]. Thus, it appears that, similar to in vitro T-cell activation, Vav1 is required for efficient antigen receptor clustering in vivo and viral antigens that can provide strong antigen receptor cross linking can overcome the functional defect.

The data from vav1-/- T cells demonstrates that the activation of non-clustered T cell receptors can still induce activation of principal signaling pathways, but are not sufficient to confer the full T cell response invoked by multimerized T cell receptors. Thus, we proposed that antigen receptor activation may lead to the organization of focal actin-scaffolded signaling highways [123 ]. These results may offer a biochemical rationale for receptor reorganization and clustering, i.e, actin-dependent receptor clustering results in signal transduction at regionally organized focal points as a prerequisite for the induction of physiological responses. These genetic data provided the first genetic evidence for a role for Vav1 in cytoskeletal reorganization in vivo and showed that Vav1 is a crucial and specific regulator of TCR-mediated cytoskeletal reorganization and receptor clustering and calcium flux required for T cell maturation, IL-2 production, and cell cycle progression.

Similarly to Vav1 mutant mice, deletion of the WASP gene severely impairs T cell proliferation and cytokine production [37 , 124 ]. At the molecular level, wasp-/- T cells display defective antigen receptor clustering after antigen receptor stimulation. Antigen receptor- and CD28-mediated activation of ERK1/ERK2, SAPKs/JNKs, as well as Ca2+-mobilization is normal in wasp-/- T cells. Moreover, WASP associates with activated CDC42 and WASP-regulated cytoskeletal changes require binding of WASP to the active form of CDC42 [125 ]. Thus, in T cells, the Rho-family GDP/GTP exchange factor Vav1, the Rho-family GTPases Rac1, CDC42, and RhoA, and the CDC42-associated WASP constitute a signaling pathway that links antigen receptor engagement to receptor clustering. Genetically Vav1, Rac1, and WASP regulated cytoskeletal reorganization and receptor clustering are essential prerequisites for lymphocyte maturation, cell cycle progression, IL-2 production, and the induction of physiological T cell responses.

Additional clues can be found in studies of thymocytes and mature peripheral T cells treated with cytochalasin D (CytD) and latrunculin, two inhibitors of actin polymerization. These drugs inhibit IL-2 production, proliferation, TCR capping, and peptide/MHC-mediated thymocyte apoptosis but do not impair known signaling pathways with the exception of Ca2+ mobilization [35 , 54 ]. Thus, the effects of actin polymerization blockers on T cell signaling and T cell functions mimic an absence of Vav1 or WASP or the overexpression of dominant-negative Rac1. Moreover, it has been reported that Nck, Slp-76, SLAP/Fyb contribute to the scaffolding and formation of the molecular motor complex that ultimately links to Arp2/3, Ena/VASP, and actin polymerization [126 ] (Fig. 3 ).



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  		Figure 3. Schematic of the multimolecular complex that induces actin polymerization induced by TCR stimulation. Upon TCR stimulation, SLP-76 is tyrosine phosphorylated and forms a complex with the Rho-family GTPase exchange factor Vav1, the adaptor proteins Nck and Fyb/SLAP. Vav1 catalyzes the exchange of GDP for GTP, thus activating CDC42. The CDC42 effector WASP directly binds to Nck, therefore enabling WASP to be in proximity to and activated by CDC42. Fyb/SLAP links Ena/VASP proteins to profilin and ultimately to the remodeling of the actin cytoskeleton. Binding of WASP to the Arp2/3 complex stimulates actin nucleation. The Arp2/3 complex contains five polypeptides in addition to Arp2 and Arp3. This multimolecular complex is detected at sites of actin polymerization after T-cell activation and is thought to coordinate the spatial and temporal organization of the cytoskeleton necessary for T-cell activation [126 ].


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MEMBRANE RAFTS
 
The plasma membrane of mammalian cells contains anatomically distinct membrane microdomains called rafts that are enriched in glycosphingolipids and cholesterol and remain intact in the presence of detergents [127 128 129 130 ]. These sphingolipid-cholesterol rafts are also known as glycolipid-enriched membrane domains (GEMs), detergent-insoluble glycolipid-enriched domains (DIGs), detergent-resistant membranes (DRMs), or low-density detergent-insoluble membranes (DIMs). Caveolae, small flask-shaped invaginations of the plasma membrane that are thought to participate in the internalization and distribution of phospholipid-anchored receptors, co-localize to raft microdomains [131 132 133 ]. Sphingolipids and cholesterol-enriched membrane raft have also been also demonstrated in lymphocytes [25 ]. Biophysically, rafts represent liquid ordered phases with a reduced membrane fluidity [134 , 135 ].

Functionally, it has been suggested that rafts might play a role in morphogenesis, membrane trafficking, and/or signal transduction [41 , 42 ]. For example, rafts are enriched for a variety of important signaling proteins, including glycosylphosphatidylinositol (GPI)-linked surface molecules, src-family and receptor tyrosine kinases, heterotrimeric and Ras-like G proteins, molecules involved in Ca2+ mobilization, including an IP3-responsive Ca2+ channel and a Ca2+ ATPase [25 ]. Moreover, high concentrations of phosphatidylinositol bisphosphate (PIP2) can be found in rafts, suggesting that rafts play a role in phospholipid signaling [136 ]. Recent experiments in mouse and human T cells have demonstrated that several molecules localize to the detergent-insoluble fraction of T cells including the CD4 co-receptor, GPI-linked molecules Thy-1 and ThB, the src-family kinases p56Lck and p59fyn, the phospholipid PIP2, the molecular adapter c-Cbl, the Syk tyrosine kinase, or the small G protein Ras and Rho-family proteins [128 , 130 , 137 ]. By contrast, it appears that the protein tyrosine phosphatase CD45 [137 ], paxillin, Grb2, Raf1, Rab5, {alpha}-tubulin, and the transferrin receptor CD71 are excluded from the detergent-insoluble fraction [138 ].

Recent experiments in human Jurkat T cell lines and mouse thymocytes have shown that T cell raft integrity is required for effective T cell receptor signal transduction [22 ]. It is intriguing to note that antigen receptor engagement triggers the dynamic recruitment of TCR/CD3 complexes to the rafts. Moreover, Vav1, the high (p85) and low (p55) PI3’K regulatory subunits, PLC{gamma}1, p36LAT, the TCR{zeta} chain, or SHC redistribute to rafts after TCR/CD3 activation. Redistribution of the TCR/CD3 complex and signaling molecules to the rafts is accompanied by the accumulation of a series of tyrosine-phosphorylated substrates, in particular hyperphosphorylated ZAP70, PLC{gamma}1, LAT, and p23 TCR{zeta} chains, and increased p56lck kinase activity and these molecules appear to form higher-order supramolecular structures. Recruitment of TCR/CD3 molecules to the rafts depends on the activity of Src family kinases [139 ]. It is important to note that recruitment of TCR{zeta} into rafts could also be observed in peptide-MHC complex-stimulated thymocytes from female transgenic mice that expressed a transgenic TCR recognizing the male H-Y antigen presented by MHC class I molecule H-2Db [139 ]. Conditions that reversibly disrupt raft structure either by dispersing their contents or by forcing their internalization reversibly disrupt the earliest steps of T-cell activation, including decreased phosphorylation of TCR{zeta} and PLC{gamma}1 and decreased Ca2+ flux after anti-CD3 stimulation [140 ].

The question remains whether these rafts and localization of signal transduction intermediates to these anatomically distinct microdomains are regulated by TCR-triggered reorganization of the cytoskeleton [140 ]. For example, 30–40% of total TCR{zeta} protein localizes to the insoluble fraction in resting T cells and around 90% of these TCR{zeta} chains are associated with insoluble fractions, rafts, and/or polymerized actin filaments after antigen receptor activation [28 , 141 ]. Moreover, TCR-engagement triggers association of phosphorylated TCR{zeta} chains with actin microfilaments, and inhibition of this association abrogates TCR/CD3-mediated cytokine production [27 , 28 ]. The formation and stabilization of supramolecular activation clusters at contact zones between T and B cells depends on a functional actin cytoskeleton [87 ]. Actin cytoskeleton-dependent recruitment of TCR/CD3 complexes to membrane rafts and receptor clustering are strategies utilized by T cells to ensure organization of higher-order molecular clusters of signaling proteins that concentrate second messenger molecules and adaptor proteins and exclude negative regulators such as the CD45, leading to effective TCR signaling and lymphocyte activation [49 , 137 , 140 , 142 ]. It is conceivable that anatomically and biochemically different raft structures exist that mediate distinct functions. The question also arises whether recruitment of antigen receptors and signaling molecules into rafts and organization of actin-scaffolded supramolecular activation complexes are regulated via the same signaling molecules such as Vav1 and Rho-family GTPases?


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FUNCTIONAL IMPLICATIONS
 
Full activation of T cells requires two distinct signals; signal one is conveyed through the TCR when it recognizes antigen in the context of MHC molecules and signal two, a costimulatory signal, is essential for naive T-cell activation [143 ]. Cooperative signals from the TCR and the costimulatory molecule CD28 are essential for T-cell activation. When a naive T cell receives signal one in the absence of signal two, induction of anergy or apoptosis may occur [144 ]. Regulation of the CD28 costimulatory signal is crucial for T-cell activation, tumor immunosurveillance, maintaining immunological tolerance, and avoiding autoimmunity. At the molecular level, CD28 costimulation triggers signaling for two key cellular events: SAPK/JNK and p38 kinase activation [145 ] and asymmetrical receptor clustering and raft aggregations at the site of antigen contact [7 , 146 ]. Thus, Cap and SMAC formation and cluster-dependent signaling might be an important molecular mechanism that regulates immunological tolerance and lymphocyte unresponsiveness.

The studies reviewed above provide compelling evidence that clustering of signaling molecules, possibly in specific architectures, is an essential step in lymphocyte activation. Antigen receptor activation along with costimulation may lead to the organization of focal actin-scaffolded signaling highways whose function is to sustain TCR signaling and coordinate downstream signaling events such that complete activation is achieved and late events such as proliferation and cytokine secretion can occur. Clustering of receptors could favor sustained signaling in three ways: (1) by increasing the likelihood of contacts between the TCR and the MHC-bound ligand, the increased concentration of TCR molecules in a high density zone would allow low-affinity receptors to initiate and maintain signals; (2) by increasing the concentration of cytosolic signaling molecules and second messengers at regionally organized focal points in the proximity of the TCRs; and (3) by excluding negative regulatory molecules such as phosphatases from the zone of antigen receptor signaling.

T cells display ~30,000–40,000 TCR on the cell surface but only 50–100 peptide/MHC complexes on APCs are required for T-cell activation [147 ]. Moreover, it appears that survival of naive peripheral CD4 and CD8 T cells is dependent on continuous recognition of self-peptide/MHC complexes by the TCR [148 ]. Why are these T cells not constantly activated? Ligand-induced formation of Caps and SMACs could introduce an additional level that regulates lymphocyte effector functions. Thus, TCR-mediated MAPK, SAPK/JNK, or NF-{kappa}B induction in the absence of Caps and SMACs might regulate cell survival. However, formation of higher-order clusters appears necessary to induce immune responses such as proliferation and expression of regulatory cytokines. In this scenario, recruitment and activation of a Cap- and SMAC-dependent signaling cascade is probably the crucial and limiting step required for the induction of lymphocyte effector functions.

A role for an actin-dependent clustering of signaling molecules is in line with fundamental regulatory mechanisms in signaling that might be stated in chemical terms as effective molarity, encompassing concepts of both proximity and orientation. The widespread use of dimerization domains, localization domains, and protein-protein interaction domains, as well as scaffolding proteins probably reflects the importance of effective molarity as a regulatory mechanism in intracellular signaling. Most steps in intracellular signaling can be regulated by induced proximity in specific architectures and actin-dependent clustering of signaling molecules could well serve the purpose of controlling effective molarity. Future understanding is likely to depend on the identification of the biochemical pathway(s) that are defective in the absence of Caps and SMACs and novel strategies to test the function of the molecules involved in these signaling clusters.

The clustering of receptors appears to be important for assembling signaling molecules at focal sites, an event that may promote sustained signaling necessary for lymphocyte activation. However, if true, why do the kinetics of tyrosine phosphorylation and the activation of SAPK, MAPK, and NF-{kappa}B appear normal in cells that cannot form Caps? One possible explanation is that the induction of physiological responses may require that distinct signaling pathways be activated in a temporally and spatially coordinated fashion. For example, oscillations of calcium flux within a focal site regulated by receptor clustering might be a mechanism by which multiple signaling pathways could be coordinated. Without SMACs or Caps, individual signaling pathways may fire but the synergy is lost. Alternatively, the formation of SMACs or Caps may lead to the recruitment and activation of members of a unique signaling cascade. The possibility that actin-dependent events activate a novel signaling pathway is suggested by findings that the downstream effects of Rac1 and CDC42 can be dissociated using different mutant alleles [61 ]. Thus, Rac1 and CDC42 mutants have been identified that fail to activate PAK and JNK/SAPK but still lead to cytoskeletal changes and cell cycle progression. The pathways downstream of these Rac1/CDC42 alleles leading to proliferation and actin changes remain to be discovered.

Signals that induce T-cell activation facilitate the formation of SMACs and lipid raft aggregation at the site of receptor contact. Negative regulators of T-cell activation could presumably play a crucial role in the regulation of cytoskeletal reorganization that results in immune synapse formation. We have identified Cbl-b, a member of the Cbl family of E3-ubiquitin ligases [149 , 150 ], as a key negative regulator of lipid raft aggregation, immune synapse formation, proliferation, and cytokine production in T cells [32 , 151 , 152 ]. T cells deficient in Cbl-b cluster their T cell receptors and lipid rafts in the absence of costimulatory signals. In addition, cbl-b-/- T cells exhibit sustained protein tyrosine phosphorylation after TCR stimulation alone, a phenomenon that has been shown to require costimulation [7 ]. These findings uncover a suppressive mechanism, mediated at least in part by Cbl-b, that is in place that forces a T cell to rely on additional signals, namely costimulatory signals, for immune synapse formation and full T-cell activation. How costimulatory signals bypass the negative regulation by Cbl-b is unknown. However, Vav1 is phosphorylated in response to TCR stimulation and this phosphorylation is enhanced when a costimulatory signal is delivered. In cbl-b-/- T cells, Vav1 is hyperphosphorylated in response to TCR stimulation alone at levels seen in cbl-b+/- cells when a costimulatory signal is delivered [151 , 152 ]. As discussed, Vav1 is a crucial regulator of cytoskeletal reorganization and perhaps once a level of Vav1 phosphorylation (or other signaling proteins) is achieved, induction of cytoskeletal reorganization is initiated, followed by T-cell activation. Therefore, phosphatases and molecules that facilitate protein degradation could play a crucial role in regulating immune synapse formation and T-cell activation by regulating levels of activated TCR proximal signaling proteins.

Activation of PKC can bypass the functional defects in T cells deficient for Vav1, Rac1, or WASP function [35 36 37 ]. Moreover, PKC activation restores the susceptibility to apoptosis of vav1-/- thymocytes, and inhibition of a PKC isoform inhibits TCR-mediated thymocyte apoptosis [54 ]. Of the several PKC isoforms, Vav1 was found to associate only with the Ca2+-independent PKC{theta} molecule [54 ] and Vav1 has been placed upstream of PKC{theta} activation [153 ]. PKC{theta} is highly expressed in the hematopoietic system, particularly in T cells, and has been shown to cooperate with calcineurin to induce transcription of the T cell growth factor IL-2 [154 ]. This observation is reminiscent of the coordinated transactivation of the IL-2 gene by calcineurin and Vav1/Rac1 [35 , 114 ]. In addition, PKC{theta} translocates to the central areas of SMACs, whereas the PKC{alpha}, ß1, {delta}, {eta}, and {zeta} isoforms are excluded from the contact site [46 , 155 ]. Thus, PKC{theta} is a candidate for an effector kinase that links Cap and SMAC formation to downstream signaling pathways involved in the development, proliferation, and activation of T cells. PKC{theta} knock-out mice have been recently described [156 ]. These mice display normal T and B cell development. However, peripheral T cells have a severe block in antigen receptor-induced proliferation and IL-2 production. Biochemically, loss of PKC{theta} results in abolished NF-{kappa}B activation but normal activation of other signaling pathways. It remains to be tested whether PKC{theta}-deficient T cells can form SMACs and/or whether the stability and segregation of SMACs is impaired.

In addition to the Vav1-regulated PKC{theta}-NF-{kappa}B pathway, overexpression of Vav1 activates NF-AT-dependent transcription [114 ], suggesting that NF-AT is an additional nuclear terminus of Cap- and actin-dependent signaling. Because NF-AT and NF-{kappa}B transcription complexes contribute to the transactivation of the IL-2 promoter, a biochemical pathway can be proposed that relays TCR engagement to Vav1 activation, actin-polymerization, NF-AT, and NF-{kappa}B activation and expression of immune response genes (Fig. 1) . In Vav1- and Rac1-mutant as well as cytochalasin D-treated T cells, NFATc1 is still able to translocate from the cytoplasm to the nucleus after TCR engagement, but the NFAT transcriptional complex is inactive [35 ]. Thus, Vav1- and Cap-dependent signaling activates either a cascade that induces the expression of an as yet unknown nuclear subunit of NF-AT complexes, or a unique pathway that mediates direct NF-AT activation.


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CONCLUSIONS
 
Antigen-specific stimulation of T cells leads to the formation of highly organized supramolecular activation clusters required for full T-cell activation. Antigen receptor activation may lead to the organization of focal actin-scaffolded signaling highways whose function is to sustain TCR signaling and coordinate downstream signaling events such that complete activation is achieved and late events such as proliferation and cytokine secretion can occur. The analyses of genetically engineered mouse mutants offer helpful insights into the molecules mediating SMAC and Cap formation and downstream transduction of antigen receptor signaling.

Received September 7, 2000; revised October 22, 2000; accepted October 25, 2000.


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