Vav-family proteins in T-cell signalling

Vav-family proteins in T-cell signalling Victor LJ Tybulewicz The Vav family proteins (Vav1, Vav2, Vav3) are cytoplasmic guanine nucleotide exchange factors (GEFs) for Rho-family GTPases. T-cell antigen receptor (TCR) signalling results in the tyrosine phosphorylation of Vav proteins and hence their activation. Results from mice deficient in one or more Vav proteins has shown that they play critical roles in T-cell development and activation. Vav1 is required for TCR-induced calcium flux, activation of the ERK MAP kinase pathway, activation of the NF-kB transcription factor, inside-out activation of the integrin LFA-1, TCR clustering, and polarisation of the T cell. Although many of these processes may require the GEF activity of Vav1, it is possible that Vav1 also has adaptor-like functions. Recent evidence suggests that Vav1 might also function in the nucleus, where it undergoes arginine methylation. An emerging theme is that Vav proteins may have important functions downstream of receptors other than the TCR, such as integrins and chemokine receptors. Addresses Division of Immune Cell Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK Corresponding author: Tybulewicz, Victor LJ ([email protected])

Current Opinion in Immunology 2005, 17:267–274 This review comes from a themed issue on Lymphocyte activation Edited by Gail A Bishop and Jonathan R Lamb Available online 13th April 2005 0952-7915/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2005.04.003

Introduction In mammals, the Vav family of proteins consists of three known members – Vav1, Vav2 and Vav3. The first member of the Vav family of proteins to be described, initially termed Vav, now known as Vav1, was found in a screen for novel oncogenes capable of transforming 3T3 fibroblasts [1]. Analysis of its sequence showed several domains characteristic of signal transducing proteins (Figure 1). Subsequently, two further members of the family, Vav2 and Vav3 were isolated, and sequence analysis showed an identical arrangement of domains [2–4]. Whereas the expression of Vav1 appears to be predominantly limited to the haemopoietic system, Vav2 and Vav3 are expressed more ubiquitously [1,3,4]. A role for Vav1 in T-cell signalling was first suggested by the observation that Vav1 is tyrosine phosphorylated following engagement www.sciencedirect.com

of the T-cell antigen receptor (TCR) [5,6]. More recently, Vav2 and Vav3 have also been shown to undergo TCRinduced phosphorylation [7–10,11]. In addition, phosphorylation of Vav proteins has been reported in a wide range of cell types and downstream of many different receptors, including the B-cell antigen receptor, FceRI, FcgRI/II/III, growth factor receptors, integrins, cytokine receptors and chemokine receptors [12]. In this review I will not attempt to comprehensively cover all aspects of the function of Vav-family proteins, as these have been covered in several recent reviews [12–15]. Instead I will focus on more recent advances in our understanding of the function of Vav-family proteins in transducing signals within T cells. First I will discuss how Vav proteins are regulated, then their role downstream of the TCR and finally the emerging theme of their role in signalling from other receptors.

Regulation of Vav proteins Sequence analysis of Vav proteins showed that they contain a Dbl homology (DH) region, typical of the GDP/GTP exchange factors (GEFs), which activate Rho-family GTPases (Figure 1) [16,17]. This domain is flanked by several domains thought to regulate the GEF activity. Amino-terminal to the DH domain is a calponin homology (CH) domain and an acidic domain, whereas to the carboxy-terminal side is a pleckstrin homology (PH) and a C1 domain (Figure 1). Finally, at the carboxyterminal end of the protein there are two SH3 domains and one SH2 domain, which are most likely to be involved in protein–protein interactions. Many potential binding partners have been identified for these domains, and some of these are listed in Figure 1. Biochemical analysis confirmed that the DH domain of Vav proteins confers GEF activity, although there has been disagreement with respect to the specific GTPases these proteins can activate. In some reports it has been demonstrated that Vav1 is a GEF for Rac1, Rac2 and RhoG, that Vav2 is a GEF for RhoA, RhoB and RhoG, whereas Vav3 preferentially activates RhoA, RhoG and, to a lesser extent, Rac1 [4,18,19]. In other reports it has been shown that Vav1 may also act on RhoA and Cdc42, and that Vav2 may activate Rac1 and Cdc42 [20,21]. Most interestingly, tyrosine phosphorylation of Vav1 was shown to result in the activation of its GEF activity [18,20]. Similarly, Vav2 and Vav3 are activated by tyrosine phosphorylation leading to their activation [4,19]. Vav proteins contain at least three tyrosine phosphorylation sites, which are located within the acidic domain Current Opinion in Immunology 2005, 17:267–274

268 Lymphocyte activation

Figure 1

Inhibition of GEF GEF

Regulation of GEF

YYY N

C CH

Ac

DH

LyGDI RhoGDI Enx1 APS

PH

C1

PIP2 PIP3

SH3A SH2 SH3B Grb2 SLP-76 Nef ZAP70 Zyxin Syk Ku-70 hnRNP-K hnRNP-C Dynamin2 VIK-1

Btk, Itk, Tec, p85, Cbl-b, Nek3 Current Opinion in Immunology

Domain structure of Vav-family proteins. The calponin homology (CH) domain inhibits GEF activity, perhaps by binding the cysteine-rich C1 domain. The acidic (Ac) domain contains at least three sites of tyrosine phosphorylation. Tyrosine 174 inhibits GEF activity of Vav1, and this inhibition is relieved by phosphorylation. The Dbl homology (DH) domain has catalytic GEF activity towards Rho-family GTPases. The pleckstrin homology (PH) domain may regulate GEF activity following binding of the phospholipids PIP2 and PIP3. The C1 (zinc finger) domain may contribute to GEF activity by binding to the GTPases. The SH3A, SH2 and SH3B domains mediate several protein–protein interactions. Potential binding partners for each domain are listed below the Vav structure [25,50,70–72].

(Figure 1) [22]. Structural analysis has shown that, in Vav1, one of these residues, Tyr174 binds to the DH domain and inhibits its GEF activity [23]. Phosphorylation of Tyr174 causes the tyrosine to move away from the DH domain, thereby relieving the inhibition (Figure 2). The activity of the DH domain is also inhibited by the CH domain, as deletion of this domain results in a constitutively active GEF [12]. Recent work has shown that the CH domain of Vav1 can bind to the C1 region, suggesting that this inhibition occurs through an intramolecular interaction that occludes the DH domain and blocks access to its substrate GTPases (Figure 2) [24]. Such a model is further supported by electron microscopic analysis of Vav3, showing that deletion of the CH domain alters Vav3 from a closed to an open conformation (X Bustelo, personal communication). If the CH domain mediates auto-inhibition of the DH domain of Vav proteins, activation might require a ligand to bind to the CH domain, displacing it from the DH and C1 domains. Such a ligand could be the adaptor protein APS (adaptor molecules containing PH and SH2 domains), which has been shown to bind to the CH domains of Vav1 and Vav3 and augment their ability to transform fibroblasts [25]. An invariant feature of all DH-containing proteins is an adjacent PH domain, which may regulate GEF activity. It has been proposed that the PH domain of Vav1 binds to the phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate Current Opinion in Immunology 2005, 17:267–274

(PIP3) and that the binding of these two lipids either inhibits or activates the in vitro GEF activity of Vav1, respectively [26]. Consistent with this, mutation of Arg422 to glycine (R422G) within the PH domain, which abolishes binding of both PIP2 and PIP3, resulted in a mutant form of Vav1 which could not be inhibited by PIP2 or activated by PIP3 [27]. To test this hypothesis in vivo, we generated a mouse strain expressing Vav1R422G mutant protein. In CD4+ T cells from these mice, TCRinduced Rac1 activation was reduced, although it was still higher than in Vav1-deficient T cells (A Prisco et al., unpublished data). Our results are consistent with a positive role for the PH domain in regulating GEF activity, potentially following binding of PIP3 (Figure 2). However, the results also show that binding of phospholipid to the PH domain is not absolutely required for GEF activity.

T-cell development The observation that TCR signalling leads to the phosphorylation and hence activation of Vav proteins suggested that Vav proteins might play important roles in T-cell development and activation. Initial studies with chimeric mice generated using Vav1-deficient embryonic stem (ES) cells showed reduced numbers of thymocytes and peripheral CD4+ and CD8+ T cells [28–30]. The establishment of Vav1-deficient mice enabled a more detailed analysis which showed that in the absence of Vav1 there was a partial block in thymic development at www.sciencedirect.com

Vav-family proteins Tybulewicz

269

Figure 2

CH ligand?

CH

Ac Y Y Y

Ac

DH CH

Tyrosine phosphorylation CH ligand? PIP3

PH

C1

SH3A SH2 SH3B Inactive

YP YP YP

DH GTPase PH PIP3

C1

SH3A SH2 SH3B Active

Current Opinion in Immunology

Hypothetical mechanism of activation of Vav-family proteins. In the inactive state, Vav is folded in such a way as to inhibit the GEF activity of the DH domain. This is achieved through binding of tyrosines in the acidic domain to the DH domain and through binding of the CH domain to the C1 region. Activation of Vav may involve at least three different events to relieve this auto-inhibition. Phosphorylation of the tyrosines causes them to be displaced from the DH domain, binding of a ligand to the CH domain may cause it to release the C1 domain and PIP3 may bind to the PH domain, altering its conformation. The result of this is an open active configuration of Vav which can productively interact with the GTPase through its DH domain and possibly also through the PH and C1 regions.

the CD4 CD8 (double negative; DN) to CD4+CD8+ (double positive; DP) transition, consistent with a role for Vav1 in transducing pre-TCR signals [31], although it has never been shown directly that pre-TCR signalling activates Vav1. Analysis of TCR transgenic mice deficient in Vav1 showed a further block in development between DP and CD4+CD8 and CD4 CD8+ single positive (SP) cells, indicating less efficient positive selection of DP thymocytes bearing either MHC class I- or class IIrestricted ab TCRs [31,32]. Furthermore, these studies also showed less efficient negative selection of DP cells, consistent with Vav1 transducing the TCR signals required for the selection of DPs. By contrast, the development of T cells bearing a gd TCR was unaffected by the absence of Vav1, demonstrating the existence of distinct signalling pathways mediating the selection of ab versus gd T cells [33]. In contrast to the effect of the Vav1 mutation, mice deficient in either Vav2 or Vav3 have normal T-cell development [10,11,34]. Mice deficient in both Vav1 and Vav3, however, show an enhanced block at the DN/ DP and DP/SP transitions compared to Vav1-deficient mice, which is further exacerbated in mice deficient in all three Vav proteins [11]. In Vav1/2/3-deficient mice, although the number of DN cells is unaffected, there is 100-fold reduction in the number of DP and SP thymocytes, and peripheral T cells. Thus, in thymic development, the Vav proteins have overlapping redundant functions. Whereas Vav1 seems to play the most www.sciencedirect.com

critical role in these developmental transitions, Vav3 can compensate, at least in part, for the lack of Vav1, whereas a compensatory function of Vav2 in T cells becomes apparent only in the absence of both Vav1 and Vav3.

Role of Vav proteins in T-cell activation and TCR signalling In addition to defects in T-cell development, the activation of mature T cells was also found to be defective in the absence of Vav1. TCR-induced proliferation, upregulation of activation markers and the secretion of cytokines was greatly reduced in Vav1-deficient T cells [28,29,35,36]. Analysis of immune responses in Vav1deficient mice showed defective T-cell help during antibody responses to T-dependent antigens, and also a greatly impaired primary cytotoxic T cell response to viral infection [36,37]. Studies of Vav1-deficient gd T cells has shown that, although their development is normal, their TCR-induced activation is not; in the absence of Vav1, TCR-induced proliferation and cytokine secretion were greatly reduced [33]. Biochemical analysis of TCR signalling pathways has revealed several Vav-dependent events. Vav1-deficient T cells have defects in TCR-induced intracellular [Ca2+] flux, activation of the ERK MAP kinases and activation of the transcription factor NF-kB [35,38,39]. In all cases, these defects are only partial, suggesting that there might be redundancy of function between Vav1 and the other family members. Analysis of Vav1/2/3-deficient T cells Current Opinion in Immunology 2005, 17:267–274

270 Lymphocyte activation

confirms this, showing that this triple mutation results in a complete block in TCR-induced calcium flux and ERK activation [11]. Calcium flux

Analysis of the defective TCR-induced calcium flux in Vav1-deficient DP thymocytes has shown it is probably due to a failure to activate phospholipase Cg1 (PLCg1) [40]. Vav1 controls two pathways leading to PLCg1 activation. In the first pathway, Vav1 is required for the TCR-induced activation of the Tec-family kinases Itk and Tec, which may in turn directly phosphorylate PLCg1. This defect in Tec activation might be secondary to the observed defective activation of phosphoinositide 3-kinase (PI3K), as Itk and Tec contain PH domains capable of binding PIP3, the second messenger generated by PI3K [41]. In the second pathway, in the absence of Vav1 there is defective LAT (linker for activation of T cells) phosphorylation and assembly of a LAT–SLP76–PLCg1 complex [40,42]. This pathway is PI3K-independent, as it is unaffected by PI3K inhibitors. It remains unclear how Vav1 controls these two pathways. Vav1 might activate PI3K through Rac1, as this GTPase has been proposed to activate the lipid kinase [43–45]. By contrast, Vav1 might control the formation of the LAT– SLP-76–PLCg1 complex through allosteric means. Vav1 has been shown to bind through its SH2 domain to a phosphotyrosine on SLP-76, and thus may be required to stabilise the LAT–SLP-76–PLCg1 complex. Although the TCR-induced phosphorylation and activation of Itk was diminished in the absence of Vav1 [40], a recent study has shown that, in the absence of Itk, the recruitment of Vav1 to the immunological synapse was reduced [46]. Itk might be required for the phosphorylation of either LAT or SLP-76 adaptor proteins, thereby generating phosphotyrosines to which Vav1 is recruited through its SH2 domain. Taken together, these observations suggest a functional interdependence between Vav1 and Itk. Vav1 may transduce TCR signals to Itk via Rac1, PI3K and PIP3. Subsequently, Itk may phosphorylate adaptor proteins that recruit Vav1 to the immunological synapse resulting in a localised activation of Rho-family GTPases. This might be critical for the generation of a polarised remodelling of the actin cytoskeleton (see below). ERK MAP kinase

Further studies in Vav1-deficient DP thymocytes have delineated two pathways by which Vav1 controls ERK. Firstly, in the absence of Vav1, the decreased TCRinduced activation of PLCg1 leads to a reduction in the second messenger diacylglycerol, reduced activation of RasGRP1, a Ras GEF and, hence, defective activation of Ras, B-Raf, MEK and ERK [42]. Secondly, the TCRinduced recruitment of the Sos1 and Sos2 Ras GEFs to LAT is decreased in Vav1-deficient cells, presumably Current Opinion in Immunology 2005, 17:267–274

because of decreased LAT phosphorylation. Similar conclusions were reached in studies in the Jurkat human T cell leukaemic cell line. Overexpression of Vav1 and RasGRP1 in these cells leads to hyperactivation of Ras in a PLCg1-dependent fashion [47]. Vav1-deficient Jurkat T cells

In a recent study, the generation of a Vav1-deficient Jurkat T cell line was reported [48]. This recapitulated some but not all of the phenotypes described in Vav1-deficient primary murine T cells. In particular, in Vav1-deficient Jurkat cells, TCR-induced calcium flux, IL-2 transcription and activation of the NF-kB transcription factor were all reduced, whereas ERK activation remained normal [48]. It is unclear why there is a difference in the effect of Vav1 deficiency on ERK activation, but it might reflect differences in the wiring of the TCR-ERK pathway between Jurkat cells and primary murine T cells. Further analysis of the Vav1-deficient Jurkat cells has shown decreased TCR-induced activation of the JNK MAP kinase pathway and of the NFAT (nuclear factor of activated T cells), AP-1 and REAP (CD28 response element) transcription factors. Experiments using RNA interference to knockdown expression of Vav1 or Vav3 in Jurkat T cells has shown distinct roles for the two GEFs [49]. Although knockdown of Vav1 reduced TCR-induced activation of the IL-2 promoter, and the NFAT and NF-kB transcription factors, it had no effect on TCR-induced activation of the serum response factor (SRF). Conversely, Vav3 was required for TCR-induced SRF activation, but not for activation of NFAT, NF-kB or the IL-2 promoter. The availability of Vav1-deficient Jurkat cells has enabled complementation studies. Vav1 bearing a mutation in the DH domain was found to be unable to rescue TCRinduced activation of JNK, NFAT, AP-1, NF-kB or the IL-2 promoter [48]. This result emphasises the critical role of the DH domain and hence the GEF activity of Vav1 in transducing TCR signals. However, it remains unknown how the activation of Rho-family GTPases transduces signals to these numerous signalling pathways. Dynamin2

Dynamin2 is a large GTPase of the dynamin superfamily which is implicated in controlling protein–protein interactions as well in reshaping the cortical actin cytoskeleton. Dynamin2 binds through a proline-rich domain to the carboxy-terminal SH3B domain of Vav1 [50]. Knockdown of dynamin2 in Jurkat T cells replicated many of the phenotypes seen in Vav1-deficient cells, causing reduced TCR-induced calcium flux, phosphorylation of PLCg1, activation of Rac1, and activation of ERK and JNK MAP kinases [50]. By contrast, knockdown of dynamin2 did not affect assembly of the LAT–SLP76–PLCg1 complex. Taken together these results suggest that dynamin2 plays a critical role in transducing Vav1 signals to the activation of Rac1 and to numerous www.sciencedirect.com

Vav-family proteins Tybulewicz

downstream signalling pathways. The mechanism by which dynamin2 carries out this function is unknown, but is clearly an important area for future research.

Vav proteins and the regulation of the cytoskeleton In view of the well-established role for Rho-family GTPases in controlling the actin cytoskeleton [51], it has been proposed that by virtue of being Rho-family GEFs, Vav proteins might transduce TCR signals leading to the reorganisation of the cytoskeleton [52]. When a T cell interacts with an antigen-presenting cell (APC), several events occur that require cytoskeletal rearrangements. TCR triggering by peptide–MHC complexes on the APC results in the activation of the integrin lymphocyte function-associated antigen-1 (LFA-1) which then binds to ICAM-1 on the APC leading to the formation of a stable T cell–APC conjugate [53]. This activation of LFA-1 is caused in part by clustering of the integrin leading to high avidity binding of its ligand; cytoskeletal changes are required for this clustering. When a conjugate forms, an ordered array of proteins termed an immunological synapse (IS) is assembled at the interface, characterised by a central accumulation of TCR molecules surrounded by a ring of LFA-1 [54]. Again, this movement of proteins requires actin rearrangements. Finally, the T cell becomes polarised, as exemplified by movement of the microtubule organising centre (MTOC) to face the APC [55]. In support of the proposal that Vav1 can regulate the cytoskeleton, several of the above processes have been found to be Vav1-dependent. Vav1 transduces TCR inside-out signals, leading to the activation of LFA-1 such that, in the absence of Vav1, T cells and thymocytes are inefficient at forming antigenspecific conjugates with APCs [56,57]. Furthermore, this defect is probably due to a failure to cluster LFA-1, possibly caused by a failure to rearrange the actin cytoskeleton, although there is no direct evidence for this. Antigen-induced clustering of the TCR and of lipid rafts at the centre of the synapse is also defective in Vav1deficient T cells [58,59]. However, another study found no defect in TCR clustering in Vav1-deficient T cells [57]. The difference in these results remains unclear, but probably relates to technical differences in the assays used. Finally, antigen-induced polarisation of the MTOC was defective in Vav1-deficient DP thymocytes [57]. In contrast to the requirement for Vav1 for inside-out activation of LFA-1, TCR clustering and cell polarisation, the characteristic cell shape changes that occur following conjugate formation are unaffected by Vav1 deficiency, demonstrating that Vav1 transduces signals selectively to some but not all cytoskeletal rearrangements [57]. Although Vav1 transduces TCR signals to several cell biological processes known to require rearrangement of the cytoskeleton, the precise pathways by which it does so www.sciencedirect.com

271

are not known. Just because Vav1 is a Rho-family GEF, it does not follow that Vav1 necessarily contributes to these pathways by controlling remodelling of the actin cytoskeleton. For example, the inside-out activation of LFA-1 has been shown to require a calcium-dependent protease [60], and thus the defective LFA-1 activation in Vav1deficient T cells might be secondary to the defect in calcium flux. Nonetheless, several recent studies have suggested mechanisms by which Vav1 might control the cytoskeleton. WASP

When activated, Wiskott-Aldrich syndrome protein (WASP) binds to and activates the actin nucleation complex Arp2/3. Antigen stimulation of T cells leads to the recruitment of WASP to the IS through binding to the adapters Nck and SLP-76, where it is then activated by Cdc42-GTP [61]. Vav1 is also recruited to SLP-76 adaptor following TCR stimulation, and so is co-localised with WASP. In Vav1-deficient T cells, the recruitment of WASP to the immunological synapse is unaffected, but its activation is greatly reduced, as is the accumulation of active Cdc42-GTP [61]. Taken together, this suggests that Vav1 might transduce TCR signals via Cdc42 and WASP to Arp2/3 and, hence, actin polymerisation. ERM

Ezrin/Radixin/Moesin (ERM) proteins form crosslinks between the cortical actin cytoskeleton and the plasma membrane and thereby contribute to cell rigidity. TCR stimulation leads to the dephosphorylation and hence inactivation of ERM proteins, resulting in less rigidity [62]. This may be important in enabling the T cell to deform while forming a conjugate with an APC. In Vav1deficient T cells, or in T cells expressing dominant negative Rac1, TCR-induced dephosphorylation of ERM is greatly reduced, suggesting the existence of a Vav1/Rac1 pathway which transduces TCR signals to the inactivation of ERM proteins [63].

Vav proteins in the nucleus Vav proteins are found in the cytoplasm of resting lymphocytes, and TCR stimulation has been shown to cause translocation to the plasma membrane, presumably because of recruitment to either ZAP-70 or to the LAT–SLP-76 complex. However, a few reports using several cell types, including Jurkat T cells, have shown Vav1 in the nucleus. In particular, it has been shown that stimulation of mast cells through FceRI, a receptor related to the TCR, results in accumulation of Vav1 in the nucleus after 30 minutes, where it is found associated with the NFAT and NF-kB transcription factors [64]. This nuclear entry depends on a nuclear localisation sequence within the PH domain of Vav1. In addition, the same study showed that the SH3B domain is required to maintain Vav1 in the cytoplasm of unstimulated cells. Interestingly, the SH3B domain has been shown to bind Current Opinion in Immunology 2005, 17:267–274

272 Lymphocyte activation

to several nuclear proteins such as Ku-70, hnRNPC and VIK-1 (Figure 1); however, the significance of these interactions is unknown. In an unexpected finding, TCR signalling has been shown to induce methylation of Vav1 on arginine [65]. The significance of this modification is unclear; however, as all of the arginine methylated Vav1 was found in the nucleus, it might be an important modification for the putative nuclear function of Vav1.

Role of Vav proteins downstream of receptors other than the TCR Most of the research on Vav proteins in T cells has focused on their role in TCR signalling. However Vav proteins are phosphorylated following activation of a diverse number of receptors, including cytokine receptors, chemokine receptors, integrins and growth factor receptors [12]. Thus it is possible that Vav proteins play important roles in T cells downstream of receptors other than the TCR, although to date there is only limited evidence for this. Vav1-deficient T cells signal normally from both b1 and b2 integrins [57,66]. However this might be because there is redundancy between Vav proteins. Vav3-deficient osteoclasts and Vav1/3-deficient neutrophils have defects in avb3 and b2 integrin signalling, respectively [67,68], suggesting that T cells deficient in multiple Vav proteins may also demonstrate defects in integrin signalling. Treatment of T cells with the chemokine stromal cell-derived factor (SDF)-1a causes tyrosine phosphorylation of Vav1, and overexpression of a mutant form of Vav1 blocked SDF-1a-induced polarisation and migration [69]. Vav1-deficient T cells, however, exhibited normal SDF-1a-induced migration, suggesting that the Vav proteins may have redundant roles in this pathway. Studies with T cells deficient in one or more of Vav1/2/3 are needed to address this possibility.

Conclusions Vav proteins play important roles in T-cell development and activation. Vav1 transduces TCR signals to multiple biochemical pathways and to several cytoskeleton-dependent processes. Future research will need to elucidate the specific molecular mechanisms by which Vav proteins are activated and by which they transduce these signals. Is GEF activity required for some or all of these processes? How does the activation of Rho-family GTPases contribute to these signal transduction processes? How does the function of Vav2 and Vav3 differ from Vav1? Which other receptors on T cells transduce signals through Vav proteins? Answers to these questions will come from a combination of genetic, biochemical and structural biology approaches.

Update The work referred to in the text as (X Bustelo, personal communication) is now in press [73]. Current Opinion in Immunology 2005, 17:267–274

Acknowledgements I thank A Saveliev for critical reading of the manuscript.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Katzav S, Martin-Zanca D, Barbacid M: Vav, a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells. EMBO J 1989, 8:2283-2290.

2.

Henske EP, Short MP, Jozwiak S, Bovey CM, Ramlakhan S, Haines JL, Kwiatkowski DJ: Identification of VAV2 on 9q34 and its exclusion as the tuberous sclerosis gene TSC1. Ann Hum Genet 1995, 59:25-37.

3.

Schuebel KE, Bustelo XR, Nielsen DA, Song BJ, Barbacid M, Goldman D, Lee IJ: Isolation and characterization of murine vav2, a member of the vav family of proto-oncogenes. Oncogene 1996, 13:363-371.

4.

Movilla N, Bustelo XR: Biological and regulatory properties of Vav-3, a new member of the Vav family of oncoproteins. Mol Cell Biol 1999, 19:7870-7885.

5.

Bustelo XR, Ledbetter JA, Barbacid M: Product of vav protooncogene defines a new class of tyrosine protein kinase substrates. Nature 1992, 356:68-71.

6.

Margolis B, Hu P, Katzav S, Li W, Oliver JM, Ullrich A, Weiss A, Schlessinger J: Tyrosine phosphorylation of vav protooncogene product containing SH2 domain and transcription factor motifs. Nature 1992, 356:71-74.

7.

Moores SL, Selfors LM, Fredericks J, Breit T, Fujikawa K, Alt FW, Brugge JS, Swat W: Vav family proteins couple to diverse cell surface receptors. Mol Cell Biol 2000, 20:6364-6373.

8.

Billadeau DD, Mackie SM, Schoon RA, Leibson PJ: The Rho family guanine nucleotide exchange factor Vav-2 regulates the development of cell-mediated cytotoxicity. J Exp Med 2000, 192:381-392.

9.

Tartare-Deckert S, Monthouel MN, Charvet C, Foucault I, Van Obberghen E, Bernard A, Altman A, Deckert M: Vav2 activates c-fos serum response element and CD69 expression but negatively regulates nuclear factor of activated T cells and interleukin-2 gene activation in T lymphocyte. J Biol Chem 2001, 276:20849-20857.

10. Doody GM, Bell SE, Vigorito E, Clayton E, McAdam S, Tooze R, Fernandez C, Lee IJ, Turner M: Signal transduction through Vav-2 participates in humoral immune responses and B cell maturation. Nat Immunol 2001, 2:542-547. 11. Fujikawa K, Miletic AV, Alt FW, Faccio R, Brown T, Hoog J,  Fredericks J, Nishi S, Mildiner S, Moores SL et al.: Vav1/2/3-null Mice Define an Essential Role for Vav Family Proteins in Lymphocyte Development and Activation but a Differential Requirement in MAPK Signalling in T and B Cells. J Exp Med 2003, 198:1595-1608. This paper reports the generation of mice deficient in all three Vav proteins — Vav1, Vav2 and Vav3 — and shows functional redundancy between them for T-cell development and activation. 12. Bustelo XR: Regulatory and signalling properties of the Vav family. Mol Cell Biol 2000, 20:1461-1477. 13. Bustelo XR: Vav proteins, adaptors and cell signalling. Oncogene 2001, 20:6372-6381. 14. Turner M, Billadeau DD: VAV proteins as signal integrators for multi-subunit immune-recognition receptors. Nat Rev Immunol 2002, 2:476-486. 15. Katzav S: Vav1: an oncogene that regulates specific transcriptional activation of T cells. Blood 2004, 103:2443-2451. 16. Adams JM, Houston H, Allen J, Lints T, Harvey R: The hematopoietically expressed vav proto-oncogene shares homology with the dbl GDP-GTP exchange factor, the bcr www.sciencedirect.com

Vav-family proteins Tybulewicz

gene and a yeast gene (CDC24) involved in cytoskeletal organization. Oncogene 1992, 7:611-618. 17. Zheng Y: Dbl family guanine nucleotide exchange factors. Trends Biochem Sci 2001, 26:724-732. 18. Crespo P, Schuebel KE, Ostrom AA, Gutkind JS, Bustelo XR: Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product. Nature 1997, 385:169-172. 19. Schuebel KE, Movilla N, Rosa JL, Bustelo XR: Phosphorylationdependent and constitutive activation of Rho proteins by wildtype and oncogenic Vav-2. EMBO J 1998, 17:6608-6621. 20. Han J, Das B, Wei W, Van Aelst L, Mosteller RD, Khosravi-Far R, Westwick JK, Der CJ, Broek D: Lck regulates Vav activation of members of the Rho family of GTPases. Mol Cell Biol 1997, 17:1346-1353. 21. Abe K, Rossman KL, Liu B, Ritola KD, Chiang D, Campbell SL, Burridge K, Der CJ: Vav2 is an activator of Cdc42, Rac1, and RhoA. J Biol Chem 2000, 275:10141-10149. 22. Lopez-Lago M, Lee H, Cruz C, Movilla N, Bustelo XR: Tyrosine phosphorylation mediates both activation and downmodulation of the biological activity of Vav. Mol Cell Biol 2000, 20:1678-1691. 23. Aghazadeh B, Lowry WE, Huang XY, Rosen MK: Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation. Cell 2000, 102:625-633. 24. Zugaza JL, Lopez-Lago MA, Caloca MJ, Dosil M, Movilla N, Bustelo XR: Structural determinants for the biological activity of Vav proteins. J Biol Chem 2002, 277:45377-45392. 25. Yabana N, Shibuya M: Adaptor protein APS binds the NH2-terminal autoinhibitory domain of guanine nucleotide exchange factor Vav3 and augments its activity. Oncogene 2002, 21:7720-7729. 26. Han J, Luby-Phelps K, Das B, Shu X, Xia Y, Mosteller RD, Krishna UM, Falck JR, White MA, Broek D: Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science 1998, 279:558-560. 27. Das B, Shu X, Day GJ, Han J, Krishna UM, Falck JR, Broek D: Control of intramolecular interactions between the pleckstrin homology and Dbl homology domains of Vav and Sos1 regulates Rac binding. J Biol Chem 2000, 275:15074-15081. 28. Tarakhovsky A, Turner M, Schaal S, Mee PJ, Duddy LP, Rajewsky K, Tybulewicz VLJ: Defective antigen receptormediated proliferation of B and T cells in the absence of Vav. Nature 1995, 374:467-470. 29. Zhang R, Alt FW, Davidson L, Orkin SH, Swat W: Defective signalling through the T- and B-cell antigen receptors in lymphoid cells lacking the vav proto-oncogene. Nature 1995, 374:470-473. 30. Fischer KD, Zmuidzinas A, Gardner S, Barbacid M, Bernstein A, Guidos C: Defective T-cell receptor signalling and positive selection of Vav-deficient CD4+ CD8+ thymocytes. Nature 1995, 374:474-477. 31. Turner M, Mee PJ, Walters A, Quinn ME, Mellor AL, Zamoyska R, Tybulewicz VLJ: A requirement for the Rho-family GTP exchange factor Vav in positive and negative selection of thymocytes. Immunity 1997, 7:451-460. 32. Kong YY, Fischer KD, Bachmann MF, Mariathasan S, Kozieradzki I, Nghiem MP, Bouchard D, Bernstein A, Ohashi PS, Penninger JM: Vav regulates peptide-specific apoptosis in thymocytes. J Exp Med 1998, 188:2099-2111. 33. Swat W, Xavier R, Mizoguchi A, Mizoguchi E, Fredericks J,  Fujikawa K, Bhan AK, Alt FW: Essential role for Vav1 in activation, but not development, of gd T cells. Int Immunol 2003, 15:215-221. This paper shows a role for Vav 1 in the activation, but not development, of gd T cells. 34. Tedford K, Nitschke L, Girkontaite I, Charlesworth A, Chan G, Sakk V, Barbacid M, Fischer KD: Compensation between Vav-1 www.sciencedirect.com

273

and Vav-2 in B cell development and antigen receptor signalling. Nat Immunol 2001, 2:548-555. 35. Fischer K-D, Kong Y-Y, Nishina H, Tedford K, Marenge`re LEM, Kozieradzki I, Sasaki T, Starr M, Chan G, Gardener S et al.: Vav is a regulator of cytoskeletal reorganization mediated by the T-cell receptor. Curr Biol 1998, 8:554-562. 36. Penninger JM, Fischer KD, Sasaki T, Kozieradzki I, Le J, Tedford K, Bachmaier K, Ohashi PS, Bachmann MF: The oncogene product Vav is a crucial regulator of primary cytotoxic T cell responses but has no apparent role in CD28-mediated co-stimulation. Eur J Immunol 1999, 29:1709-1718. 37. Gulbranson-Judge A, Tybulewicz VL, Walters AE, Toellner KM, MacLennan IC, Turner M: Defective immunoglobulin class switching in Vav-deficient mice is attributable to compromised T cell help. Eur J Immunol 1999, 29:477-487. 38. Costello PS, Walters AE, Mee PJ, Turner M, Reynolds LF, Prisco A, Sarner N, Zamoyska R, Tybulewicz VLJ: The Rho-family GTP exchange factor Vav is a critical transducer of TCR signals to the calcium, ERK and NF-kB pathways. Proc Natl Acad Sci USA 1999, 96:3035-3040. 39. Holsinger LJ, Graef I, Swat W, Chi T, Bautista DM, Davidson L, Lewis RS, Alt FW, Crabtree GR: Defects in actin cap formation in Vav-deficient mice implicate an actin requirement for lymphocyte signal transduction. Curr Biol 1998, 8:563-572. 40. Reynolds LF, Smyth LA, Norton T, Freshney N, Downward J, Kioussis D, Tybulewicz VLJ: Vav1 transduces T cell receptor signals to the activation of phospholipase C-g1 via phosphoinositide 3-kinase-dependent and -independent pathways. J Exp Med 2002, 195:1103-1114. 41. Lewis CM, Broussard C, Czar MJ, Schwartzberg PL: Tec kinases: modulators of lymphocyte signalling and development. Curr Opin Immunol 2001, 13:317-325. 42. Reynolds LF, de Bettignies C, Norton T, Beeser A, Chernoff J,  Tybulewicz VLJ: Vav1 transduces T cell receptor signals to the activation of the Ras/ERK pathway via LAT, Sos and RasGRP1. J Biol Chem 2004, 279:18239-18246. This paper extends studies reported in [40] showing that Vav1 transduces TCR signals to the activation of PLCg1 via PI3K and the Tec-family kinases, as well as controlling the assembly of a LAT–SLP-76–PLCg1 complex. In this paper, Vav1 is shown to transduce TCR signals to a Ras– B-Raf–MEK–ERK pathway via PLCg1 and RasGRP1. See also annotation to [47]. 43. Tolias KF, Cantley LC, Carpenter CL: Rho family GTPases bind to phosphoinositide kinases. J Biol Chem 1995, 270:17656-17659. 44. Zheng Y, Bagrodia S, Cerione RA: Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85. J Biol Chem 1994, 269:18727-18730. 45. Bokoch GM, Vlahos CJ, Wang Y, Knaus UG, Traynor-Kaplan AE: Rac GTPase interacts specifically with phosphatidylinositol 3-kinase. Biochem J 1996, 315:775-779. 46. Labno CM, Lewis CM, You D, Leung DW, Takesono A,  Kamberos N, Seth A, Finkelstein LD, Rosen MK, Schwartzberg PL et al.: Itk functions to control actin polymerization at the immune synapse through localized activation of Cdc42 and WASP. Curr Biol 2003, 13:1619-1624. This paper shows that Itk is required for the recruitment of Vav1 to the immunological synapse. 47. Zugaza JL, Caloca MJ, Bustelo XR: Inverted signalling hierarchy  between RAS and RAC in T-lymphocytes. Oncogene 2004, 23:5823-5833. This paper reports a study in Jurkat T cells showing that Vav1 activates RasGRP1 and Ras via PLCg1. See also annotation to [42]. 48. Cao Y, Janssen EM, Duncan AW, Altman A, Billadeau DD, Abraham RT: Pleiotropic defects in TCR signalling in a Vav-1-null Jurkat T-cell line. EMBO J 2002, 21:4809-4819. 49. Zakaria S, Gomez TS, Savoy DN, McAdam S, Turner M,  Abraham RT, Billadeau DD: Differential regulation of TCRmediated gene transcription by Vav family members. J Exp Med 2004, 199:429-434. Current Opinion in Immunology 2005, 17:267–274

274 Lymphocyte activation

RNAi-mediated knockdown of Vav1 and Vav3 in Jurkat T cells shows distinct functions for the two proteins.

This paper shows that WASP is recruited to the adaptor SLP-76 via Nck where it is co-localised with Vav1 and activated by Cdc42.

50. Gomez TS, Hamann MJ, McCarney S, Savoy DN, Lubking CM,  Heldebrant MP, Labno CM, McKean DJ, McNiven MA, Burkhardt JK et al.: The Large GTPase Dynamin2 Interacts with Vav1 and Regulates T Cell Activation by Controlling Actin Polymerization at the T Cell-APC Contact Site. Nat Immunol 2005, 6:261-270. Demonstrates an interaction between dynamin2 and the SH3B domain of Vav1, and, using RNAi, shows that dynamin2 is critical for TCR signal transduction.

62. Delon J, Kaibuchi K, Germain RN: Exclusion of CD43 from the immunological synapse is mediated by phosphorylationregulated relocation of the cytoskeletal adaptor moesin. Immunity 2001, 15:691-701.

51. Etienne-Manneville S, Hall A: Rho GTPases in cell biology. Nature 2002, 420:629-635. 52. Fischer KD, Tedford K, Penninger JM: Vav links antigen-receptor signalling to the actin cytoskeleton. Semin Immunol 1998, 10:317-327. 53. Dustin ML, Bromley SK, Kan Z, Peterson DA, Unanue ER: Antigen receptor engagement delivers a stop signal to migrating T lymphocytes. Proc Natl Acad Sci USA 1997, 94:3909-3913. 54. Monks CRF, Freiberg BA, Kupfer H, Sciaky N, Kupfer A: Threedimensional segregation of supramolecular activation clusters in T cells. Nature 1998, 395:82-86. 55. Kupfer A, Singer SJ: The specific interaction of helper T cells and antigen-presenting B cells. IV. Membrane and cytoskeletal reorganizations in the bound T cell as a function of antigen dose. J Exp Med 1989, 170:1697-1713. 56. Krawczyk C, Oliveira-dos-Santos A, Sasaki T, Griffiths E, Ohashi PS, Snapper S, Alt F, Penninger JM: Vav1 controls integrin clustering and MHC/peptide-specific cell adhesion to antigen-presenting cells. Immunity 2002, 16:331-343. 57. Ardouin L, Bracke M, Mathiot A, Pagakis SN, Norton T, Hogg N,  Tybulewicz VLJ: Vav1 transduces TCR signals required for LFA-1 function and cell polarization at the immunological synapse. Eur J Immunol 2003, 33:790-797. Vav1 transduces an inside-out signal from the TCR to LFA-1, and is required for cell polarisation. See also [56]. 58. Wu¨ lfing C, Bauch A, Crabtree GR, Davis MM: The vav exchange factor is an essential regulator in actin-dependent receptor translocation to the lymphocyte-antigen-presenting cell interface. Proc Natl Acad Sci USA 2000, 97:10150-10155. 59. Villalba M, Bi K, Rodriguez F, Tanaka Y, Schoenberger S, Altman A: Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells. J Cell Biol 2001, 155:331-338. 60. Stewart MP, McDowall A, Hogg N: LFA-1-mediated adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease, calpain. J Cell Biol 1998, 140:699-707. 61. Zeng R, Cannon JL, Abraham RT, Way M, Billadeau DD,  Bubeck-Wardenberg J, Burkhardt JK: SLP-76 coordinates Nckdependent Wiskott-Aldrich syndrome protein recruitment with Vav-1/Cdc42-dependent Wiskott-Aldrich syndrome protein activation at the T cell-APC contact site. J Immunol 2003, 171:1360-1368.

Current Opinion in Immunology 2005, 17:267–274

63. Faure S, Salazar-Fontana LI, Semichon M, Tybulewicz VL, Bismuth  G, Trautmann A, Germain RN, Delon J: ERM proteins regulate cytoskeleton relaxation promoting T cell-APC conjugation. Nat Immunol 2004, 5:272-279. TCR-induced dephosphorylation of ERM proteins is mediated by Vav1 and Rac1. 64. Houlard M, Arudchandran R, Regnier-Ricard F, Germani A, Gisselbrecht S, Blank U, Rivera J, Varin-Blank N: Vav1 is a component of transcriptionally active complexes. J Exp Med 2002, 195:1115-1127. 65. Baldari CT, Koretzky G, Telford JL, Acuto O: Antigen receptor signalling: the Tuscan chronicles. Nat Immunol 2005, 6:3-5. 66. del Pozo MA, Schwartz MA, Hu J, Kiosses WB, Altman A, Villalba M: Guanine exchange-dependent and -independent effects of Vav1 on integrin-induced T cell spreading. J Immunol 2003, 170:41-47. 67. Faccio R, Teitelbaum SL, Fujikawa K, Chappel J, Zallone A, Tybulewicz VL, Ross FP, Swat W: Vav3 regulates osteoclast function and bone mass. Nat Med 2005, 11 in press. 68. Gakidis MA, Cullere X, Olson T, Wilsbacher JL, Zhang B, Moores SL, Ley K, Swat W, Mayadas T, Brugge JS: Vav GEFs are required for beta2 integrin-dependent functions of neutrophils. J Cell Biol 2004, 166:273-282. 69. Vicente-Manzanares M, Cruz-Adalia A, Martin-Cofreces NB,  Cabrero JR, Dosil M, Alvarado-Sanchez B, Bustelo XR, Sanchez-Madrid F: Control of lymphocyte shape and the chemotactic response by the GTP exchange factor Vav. Blood 2005, 105:3026-3034. Overexpression of a mutant form of Vav1 blocks SDF-1a-induced chemotaxis, although no defect is seen in Vav1-deficient T cells. 70. Tybulewicz V, Ardouin L, Prisco A, Reynolds LF: Vav1: a key signal transducer downstream of the TCR. Immunol Rev 2003, 192:42-52. 71. Houlard M, Romero-Portillo F, Germani A, Depaux A, Regnier-Ricard F, Gisselbrecht S, Varin-Blank N: Characterization of VIK-1: a new Vav-interacting Kruppel-like protein. Oncogene 2005, 24:28-38. 72. Miller SL, Demaria JE, Freier DO, Riegel AM, Clevenger CV: Novel association of Vav2 and Nek3 modulates signalling through the human prolactin receptor. Mol Endocrinol 2004. 73. Llorca O, Arias-Palomo E, Zugaza JL, Bustelo XR: Global  conformational rearrangements during the activation of the GDP/GTP exchange factor Vav3. EMBO J 2005, DOI: 10.1038/ sj.emboj.7600617 (Published online March 10 2005). This paper reports the electron microscopic analysis of Vav3, supporting the model that the CH domain binds the C1 domain, thereby holding the protein in a closed auto-inhibited state. See also [24]

www.sciencedirect.com