Department of Immunology, Mayo Graduate and Medical Schools, Mayo Clinic, Rochester, Minnesota
Correspondence: Paul J. Leibson, Department of Immunology, Mayo Clinic, 200 First Street, S.W., Rochester, MN 55905. E-mail: leibson.paul{at}mayo.edu
 TOP ABSTRACT INTRODUCTIONLYMPHOCYTE-MEDIATED CYTOTOXICITYREGULATION OF LYMPHOCYTE...SUMMARYREFERENCES |
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Key Words: natural killer cells • cytotoxic T lymphocytes • Rho GTPase family • second messengers • signal transduction
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TOPABSTRACTINTRODUCTIONLYMPHOCYTE-MEDIATED CYTOTOXICITYREGULATION OF LYMPHOCYTE...SUMMARYREFERENCES |
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TOPABSTRACTINTRODUCTIONLYMPHOCYTE-MEDIATED CYTOTOXICITYREGULATION OF LYMPHOCYTE...SUMMARYREFERENCES |
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RIII; CD16) and may also bind targets through adhesion molecules [2
, 3
]. Although natural cytotoxicity and ADCC trigger common intracellular signaling events and share downstream targets, they are also coupled to distinct biochemical pathways [3
]. Exocytosis of granules containing apoptosis-inducing proteins is one mechanism of target-cell killing by CTLs and NK cells. Such granules, which contain perforin and granzymes, are preformed in NK cells. In CTLs, cytotoxic granules are generated following peptide-MHC recognition by the T cell receptor (TCR)-CD3 complex and subsequent functional maturation of the CTL into an effector cell [4 ]. Redistribution of cytotoxic granules to an area of target-cell–effector-cell contact is initiated by specific interactions between surface ligands on the target cell and receptors on the effector lymphocytes. Subsequent effector-cell degranulation triggers apoptosis in the target cell [4 5 6 ]. It should be emphasized that the formation of a stable conjugate between a cytotoxic cell and its potential target cell, followed by directed delivery of granule components to the target cell are prerequisites of cell-mediated cytotoxicity [7 ]. Further, the process of effector-cell degranulation is controlled by actin polymerization and formation of a microtubule organizing center [8 ]. Critical to understanding the cytotoxic function of NK cells and CTLs is the delineation of the second messenger pathways specifically required for the directional release of cytoplasmic granule-derived proteins from these effectors toward target cells [9 ]. The low molecular weight GTP-binding proteins of the Rho family of GTPases have been identified as critical transducers of signals downstream of activating receptors on lymphocytes [10 ] and are required for many actin cytoskeleton-dependent cellular processes that may be essential for the formation of conjugates between killer cells and their targets and for the polarized release of granules [11 , 12 ].
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TOPABSTRACTINTRODUCTIONLYMPHOCYTE-MEDIATED CYTOTOXICITYREGULATION OF LYMPHOCYTE...SUMMARYREFERENCES |
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Low molecular weight GTP-binding proteins serve as molecular switches that transduce an upstream signal to downstream effector molecules (Fig. 1 ). The switch involves cycling between an inactive, GDP-bound state and an active, GTP-bound form [13 , 14 ]. An upstream signal stimulates the dissociation of GDP from the inactive form of the protein, which is followed by the binding of GTP and a conformational change in the molecule, allowing it to interact with downstream effectors. The GTP-bound form is converted by the intrinsic GTPase activity of the protein to the GDP-bound form, which then releases the bound, downstream effectors.
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Figure 1. Coupling of Rho family GTPases to upstream signals by Vav and subsequent activation of downstream effectors. Vav is recruited to activated immune-recognition receptors and requires tyrosine phosphorylation and the binding of phosphatidylinositol-3 kinase (PI-3K) products for localized full catalytic activity. Vav contains multiple domains including a Dbl-homology (DH) domain that mediates Vav guanine-nucleotide exchange activity for the Rho GTPases. In their GTP-bound form, Rho family GTPases interact with specific effectors to initiate downstream signal transduction pathways that regulate rearrangements of the actin cytoskeleton, which are critical for cytotoxicity. PTK, Protein tyrosine kinase; GDP, guanosine diphosphate; PAK, p21-activated kinase; ROCK, Rho-associated coiled-coil forming protein kinase; CH, calponin homology; AR, acidic region; PH, pleckstrin homology; CR, cysteine-rich region; PR, proline-rich region; SH, Src homology.
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The Rho family of small GTPases consists of several proteins including Rac, Rho, and Cdc42, which interact with downstream effectors upon binding to GTP [17 , 18 ]. The activated GTP-bound protein can interact with different signaling molecules, allowing several diverging pathways to be driven by a single family member [19 , 20 ]. The Rho GTPases also influence each other’s activity [17 ], and there is significant cross-talk between GTPases of the Ras and Rho subfamilies [21 ]. Rho family proteins can, therefore, coordinate multiple signaling pathways through their ability to regulate the cytoskeleton and gene expression [17 ]. Although these small GTPases control a diverse range of cellular functions, one general role is in the establishment of structural polarity through dynamic regulation of the actin cytoskeleton [19 , 21 ].
Regulators of the Rho Family
Regulators of Rho GTPases have emerged as important components of signal transduction pathways used by antigen receptors and costimulatory, cytokine, and chemokine receptors to regulate the immune response [18
]. Regulatory proteins that act on Rho family proteins include the guanine nucleotide dissociation inhibitors (GDIs), guanine nucleotide exchange factors (GEFs), and GTPase-activating proteins (GAPs) [13
, 15
, 20
].
GDIs play an important role in inhibiting some Rho family proteins by holding them in an inactive complex in the cytoplasm, thereby preventing their binding to membranes and induction of downstream signals. Activation of Rho family proteins by GTP is facilitated by GEFs, and inactivation is driven by GAPs [22 ]. GEFs are multidomain proteins that can activate Rho family proteins by catalyzing GDP-GTP exchange through their DH domain [13 , 15 , 20 ]. DH domains are invariably located next to a PH domain. In some GEFs (Sos and Vav), the PH domain acts as a negative regulator of the DH domain, and binding of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to the PH domain relieves this inhibition [20 ]. The lifetime of the active GTP-bound state is determined by the combination of slow, intrinsic GTPase activity and the action of GAPs, which accelerate GTP hydrolysis, thereby converting GTPases to their inactive, GDP-bound form [2 ]. GAP activity is subject to regulation by tyrosine phosphorylation [20 ].
The Vav Family of GEFs
The Vav family has three known members in mammalian cells (Vav, Vav2, and Vav3), each of which is structurally similar [23
]. Vav is a well-characterized GEF that activates Rho family GTP-binding proteins. It is primarily expressed in hematopoietic cells and acts as a GEF for Rho family GTPases, converting them from an inactive GDP-bound state to an active GTP-bound form [24
, 25
]. Vav is recruited to activated antigen receptors and requires tyrosine phosphorylation and the presence of activating phospholipids for catalytic activity [26
]. Vav contains an array of structural motifs including a DH domain, a single SH2 domain, two SH3 domains, a PH domain, and a CH domain, enabling it to interact with different proteins involved in multiple signal transduction pathways [24
, 27
, 28
]. The presence of a protein domain within Vav that confers GEF activity toward Rho family GTPases, along with protein-protein- and protein-lipid-interacting domains, suggests that Vav may interact with and influence multiple intracellular biochemical signaling cascades [24
].
Vav has the remarkable ability to couple PTK-dependent regulation to a G-protein-mediated control. The GEF activity of Vav is greatly increased upon its phosphorylation by tyrosine kinases after the cross-linking of many immune-recognition receptors [7 ] and upon binding of Vav’s PH domain to the PI-3K major lipid product, PIP3 [29 , 30 ]. Activation of PI-3K is a common response to triggering of antigen receptors and costimulatory and cytokine receptors. Phosphoinositide products of PI-3K activity may therefore couple immune receptors to Rac and Rho signaling pathways in lymphocytes via regulation of their subcellular localization [10 ]. Control of the subcellular localization of regulatory proteins for Ras, Rac, and Rho GTPases is a fundamental mechanism for regulating the activity of these molecules. The binding of PI-3K lipid products to PH domains of Rac and Rho GEFs may allosterically modify GEF activity or induce translocation of the protein to specific subcellular locations or to defined areas of the plasma membrane where activation can occur [10 , 18 ]. It is increasingly recognized that the plasma membrane is not uniform but divided into signaling subcompartments, which may contain different repertoires of PH domain-containing proteins. In this manner, Rho family GTPases may be organized into discrete subpopulations and may be linked by adaptor proteins to different effector pathways [31 ].
It has been shown that Vav undergoes tyrosine phosphorylation after the cross-linking of many activating receptors on hematopoietic cells, including the TCR, B cell receptor, and FcR, and during the generation of natural cytotoxicity [7 , 32 ]. In cytotoxic lymphocytes, Vav is a critical regulator of the intracellular polarization of cytolytic granules toward sensitive target cells required for cell-mediated killing [6 , 7 , 24 , 27 , 33 ]. Vav and Vav2 regulate distinct Rac and Rho family GTPases. Vav acts as a GEF toward Rac-1, Rac-2, and RhoG, and Vav2 displays GEF activity toward RhoA subfamily members and RhoG [32 ]. Vav2 is expressed in cytotoxic lymphocytes and undergoes receptor-mediated tyrosine phosphorylation after the cross-linking of activating receptors on CTLs and NK cells. A functional DH domain is required for Vav2 enhancement of cellular cytotoxicity, and an intact SH2 domain is required for Vav2 coupling to activating receptors and subsequent receptor-initiated tyrosine phosphorylation [32 ].
Vav and Rac1 in NK cells regulate the development of cell-mediated killing. There is a rapid increase in Vav tyrosine phosphorylation during the development of antibody-dependent cellular cytotoxicity and natural killing [7 ]. For Vav to enhance killing, it must have GEF activity and retain structural determinants in its N terminus to be correctly localized during its activation. It is also likely that Vav regulates, at least in part, cell-mediated cytotoxicity by enhancing Rac1 activation. Indirect evidence for this includes the finding that overexpression of dominant-negative Rac1 has been found to inhibit NK cell-mediated cytotoxicity [7 ]. Further, NK cells expressing dominant-negative Rac1 have a decreased ability to form conjugates with targets, and those that do form conjugates have a decreased ability to polarize their cytolytic granules toward the target cell.
The presence of multiple domains within Vav suggests that it may participate in many signal transduction pathways. For example, the CH domain, PH domain, and acidic domain of Vav can differentially affect the regulation of nuclear factor of activated T cells (NF-AT)/activated protein-1 (AP-1)-mediated gene transcription and the development of cell-mediated killing [24 ]. In this context, deletion or specific mutation of the CH domain results in a protein that lacks the ability to enhance NF-AT/AP-1-mediated gene transcription compared with wild-type Vav. It is interesting that these CH mutants are not defective in their ability to regulate cell-mediated killing, implying a differential role for this domain in Vav function during these two separate biological responses. Although the CH domain of Vav is required for Vav-dependent regulation of NF-AT/AP-1-mediated gene transcription, it is dispensable when it comes to the regulation of cellular cytotoxicity [24 ]. The PH domain has also been suggested to regulate Vav activation. However, whereas Vav mutants lacking their PH domain are defective in their ability to enhance NF-AT/AP-1 reporter activity and TCR- or FcR-mediated cellular cytotoxicity, enhancement of natural cytotoxicity by the PH deletion mutants is unaffected, indicating a differential requirement for the PH domain in the regulation of these two distinct forms of cell-mediated killing.
Regulation of Actin Polymerization
Extracellular signals regulate actin dynamics through small GTPases of the Rho family. In lymphocytes, cytoskeletal rearrangements and granule exocytosis are controlled in part through the activation of multiple Rho family members including Rac1, RhoA, and Cdc42. However, it remains unclear whether these proteins serve overlapping functions during granule exocytosis or if they regulate separate portions of the involved signaling pathways [7
]. Unlike Ras GTPases, which are constitutively membrane-bound, Rho GTPases are thought to be at least partially cytosolic (associated with GDIs) and therefore must translocate to the plasma membrane (where they would presumably bind a GEF). Precisely how they do so is not known [21
].
Rac1.
Many intracellular signaling pathways are activated during the development of cell-mediated killing. Rac1, a member of the Rho family of GTPases, plays a central role in cytotoxic cell granule redistribution and cytoskeletal reorganization. This GTPase has been shown to be a key early component of a pathway involved in the polarization of cytolytic granules in NK cells during spontaneous cytotoxicity. Activation of specific Src and Syk family protein kinases occurs in NK cells upon engagement of receptors involved in target-cell recognition [4
, 7
, 34
]. This leads to activation of PI-3K and sequential recruitment and activation of Rac1 
PAK1 mitogen-activated protein kinase (MAPK) kinase extracellular regulated kinase (ERK), in a Ras-independent manner [4
, 35
, 36
]. It is not known whether granule redistribution depends only on activation of this predominant pathway or whether it represents the net result of activation of several distinct pathways [4
].
In T cells, the pathways responsible for cytoskeletal reorganization similarly depend on Rac. A Vav-dependent phosphorylation event may be induced upon interaction of the TCR with peptide-MHC on antigen presenting cells (APCs). This leads to activation of Rac1 and the cytoskeletal reorganization that allows APC-T cell conjugation and formation of an "immunological synapse" (see below) [4 , 37 ]. In addition, Rac1 has been found to act downstream and upstream of PI-3K, thereby critically regulating the pathways leading to cytoskeleton rearrangements upon TCR engagement [4 , 38 ].
RhoA.
RhoA plays an important role in regulation of the actin cytoskeleton in cytotoxic cells. Actin polymerization is required for the assembly of proteins involved in TCR signaling into a specialized signaling domain, the immunological synapse, at the APC-T cell contact site [39
]. Plasma membrane microdomains containing sphingolipids and cholesterol (lipid rafts) are enriched with signaling molecules [40
]. The cross-linking of certain types of cell-surface receptors initiates the redistribution of these lipid rafts, resulting in the formation of signaling complexes. Signaling molecules cluster in a multimolecular complex around surface receptors. This clustering enables compartmentalization of key second messengers and results in the amplification of signal transduction cascades. Rafts can act as platforms holding signaling molecules together [40
]. Repositioning cell-surface molecules at the T cell-APC interface and the polarization of lipid rafts to the site of contact between the NK and target cell involve the regulation of the actin cytoskeleton [33
]. Regulation of the actin cytoskeleton by RhoA/p160ROCK/LIM-domain containing kinase (LIMK1) in cytotoxic lymphocytes is an important event in the development of cell-mediated killing and the polarization of lipid rafts to the effector-target interface [33
]. ROCK directly phosphorylates LIMK, which in turn is activated to phosphorylate cofilin. Cofilin exhibits actin-depolymerizing activity that is inhibited as a result of its phosphorylation by LIMK [13
, 41
]. Inhibition of actin depolymerization represents an essential control point in the overall cycle of actin polymerization and depolymerization, resulting in cytoskeletal changes. By inducing the phosphorylation and consequent inactivation of cofilin, Rho-ROCK signaling may stabilize actin filaments leading to their accumulation [41
, 42
].
Cdc42.
Cdc42 has been shown to regulate T cell polarization toward APCs [10
, 18
, 26
, 39
, 43
]. Within minutes of contacting an APC, a T cell undergoes a dramatic polymerization of actin filaments at the site of cell-cell contact [39
]. Cell-surface molecules on lymphocytes rapidly cluster into a Cap shortly after contact with activating molecules [44
]. This process involves rearrangements of the T cell cytoskeleton as well as redistribution of cell-surface molecules. After antigen stimulation, Vav is recruited to the plasma membrane where it becomes activated by tyrosine phosphorylation and by binding to products of activated PI-3K. Vav can then activate the Rho GTPases, which may be accomplished by two different mechanisms or by the integration of both as follows: Activated Cdc42 and PIP2 recruit Wiscott-Aldrich syndrome protein (WASP) to the membrane and stimulate the actin nucleation activity of the WASP-associated Arp2/3 complex; and activated Rac promotes actin polymerization by a yet-unknown mechanism involving PIP5-K activation or LIMK activation. It is not known whether a similar organization exists between CTLs and their targets [44
].
Downstream Targets—From Rho Proteins to the Actin Cytoskeleton
Rho GTPases control a variety of cellular responses, including regulation of gene expression and cell-cycle progression. The role of Rho GTPases during these responses is likely to be distinct from their involvement in maintenance of the actin cytoskeleton. It is therefore important to note that multiple effectors exist for activated Rac, Rho, and Cdc42. Some of these effectors regulate the actin cytoskeleton, and others have quite different functions [18
]. To understand the biochemical mechanisms through which Rho GTPases regulate the organization of the actin cytoskeleton and other associated activities, identification of their molecular targets (effectors) has been the focus of considerable investigation [21
].
Several Rho family targets are protein kinases, although little is known about which proteins these kinases phosphorylate [20 ]. Rac and Cdc42 targets include the serine/threonine kinase PAK and the tyrosine kinase activated-cdc42 kinase (ACK), and RhoA-binding kinases include ROCK (also known as ROK/Rho-kinase) and PKN (PKC-related serine/threonine kinases) [15, 20 ]. ROCK was among the first targets shown to affect actin organization. Rac, Rho, and Cdc42 do not simply regulate the formation of actin-containing structures but rather, appear to coordinate specific cellular responses by interacting with a number of downstream targets [20 ]. A fundamental feature of the biology of Rho GTPases is that receptors do not uniformly activate all cellular pathways mediated by a certain GTPase [18 ]. As mentioned earlier, this suggests that there must be subcellular compartments containing discrete pools of these proteins and that some spatial organization of Rac, Rho, and Cdc42 effector molecules exists by multivalent adaptor, anchoring, or scaffold proteins [18 ]. The importance of each Rho target is likely to differ depending on the circumstances and cell type, and undoubtedly, further links between Rho proteins and actin polymerization will be elucidated in the future [18 , 20 ].
Other GTPase families
Rab GTP-binding Proteins and Vesicular Transport
As stated above, CTLs and NK cells lyse target cells by exocytosing specialized secretory granules containing perforin and granzymes into a synapse-like junction that forms between the lymphocyte and the bound target cell [45
]. Rab GTPases reside on the surface of vesicles and organelles in the endocytic and secretory pathways, where they play critical roles in the targeting and fusion of these vesicles with their appropriate acceptor membrane by participating in the formation and/or function of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes [45
].
The intracellular movement of vesicles to the plasma membrane during activation-induced lysis has been recently shown to be dependent on the function of a small GTPase, Rab27a [8 , 45 ]. Rab27a localizes to granzyme-containing granules in CTLs and functions at a late step in the fusion of granules with the plasma membrane at the immunological synapse of CTLs, compatible with current models of Rab protein function in vesicle docking and fusion [45 ]. In this context, individuals diagnosed with Griscelli syndrome, caused by a mutation in Rab27a, and corresponding ashen mice with severely diminished Rab27a expression are impaired in their ability to secrete lytic granules from CTLs, resulting in a reduced ability to kill their targets [8 , 46 ].
Ras GTPases and NK Cell Function
NK cell cytotoxicity is critically dependent on MAPK, which drives perforin and granzyme B polarization following engagement of a target cell [5
]. MAPK activation in NK cells can occur via Ras-dependent and -independent signaling pathways. Whereas a Ras-dependent MAPK pathway results in NK cell-gene expression required for cytokine induction and cellular proliferation and differentiation, a Ras-independent MAPK pathway is involved in NK-cell engagement and lysis of target cells. Notably, a Ras-independent MAPK pathway critically controls the rapid mobilization of perforin and granzyme B in NK cells during target lysis without the need for new gene expression [5
].
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TOPABSTRACTINTRODUCTIONLYMPHOCYTE-MEDIATED CYTOTOXICITYREGULATION OF LYMPHOCYTE...SUMMARYREFERENCES |
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Received August 5, 2002; accepted October 4, 2002.
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TOPABSTRACTINTRODUCTIONLYMPHOCYTE-MEDIATED CYTOTOXICITYREGULATION OF LYMPHOCYTE...SUMMARYREFERENCES |
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and its lipid products Trends Cell Biol. 10,466-473[CrossRef][Medline]This article has been cited by other articles:
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