Antigen Receptor Stimulation Research Articles

Inositol (1,4,5) trisphosphate 3 kinase B controls positive selection of T cells and modulates Erk activity.

The mechanisms governing positive selection of T cells in the thymus are still incompletely understood. Here, we describe a N-ethyl-N-nitrosourea induced recessive mouse mutant, Ms. T-less, which lacks T cells in the peripheral blood because of a complete block of thymocyte development at the CD4(+)CD8(+) stage. Single nucleotide polymorphism mapping and candidate gene sequencing revealed a nonsense mutation in the inositol (1,4,5) trisphosphate 3 kinase B (Itpkb) gene in Ms. T-less mice. Accordingly, Ms. T-less thymocytes do not show detectable expression of Itpkb protein and have drastically reduced basal inositol (1,4,5) trisphosphate kinase activity. Itpkb converts inositol (1,4,5) trisphosphate to inositol (1,3,4,5) tetrakisphosphate, soluble second messengers that have been implicated in Ca(2+) signaling. Surprisingly, Ca(2+) responses show no significant differences between wild type (WT) and mutant thymocytes. However, extracellular signal-regulated kinase (Erk) activation in response to suboptimal antigen receptor stimulation is attenuated in Ms. T-less thymocytes, suggesting a role for Itpkb in linking T cell receptor signaling to efficient and sustained Erk activation.

Ca2+ Signaling in Identified T-lymphocytes from Human Intestinal Mucosa: RELATION TO HYPOREACTIVITY, PROLIFERATION, AND INFLAMMATORY BOWEL DISEASE

Ca2+ entry across the plasma membrane is necessary for the activation and proliferation of T-lymphocytes. Human intestinal lamina propria lymphocytes physiologically exhibit minimal proliferation in response to antigen receptor stimulation when compared with peripheral blood T-lymphocytes. This hyporeactivity is partially abolished in inflammatory bowel disease. We hypothesized that differences in Ca2+ signaling could be related to the disease. To test this possibility, we measured Ca2+ signals in identified lymphocytes from human blood and human intestinal mucosa. Ca2+ signals in lamina propria T-lymphocytes from non-inflamed tissue were drastically reduced when compared with Ca2+ signals of blood T-lymphocytes from the same persons. However, Ca2+ signals in T-lymphocytes from inflamed intestinal mucosa were much higher than the ones from non-inflamed mucosa and almost reached levels of Ca2+ signals in peripheral blood T-cells. Furthermore, Ca2+ influx was closely linked to cell proliferation in both peripheral blood T-lymphocytes and lamina propria lymphocytes cells. We conclude that differences in Ca2+ signaling can explain the differences of T-lymphocyte reactivity in blood versus lamina propria and, importantly, also between T-lymphocytes from inflamed and non-inflamed intestinal mucosa. Ca2+ channels in the plasma membrane of T-lymphocytes might thus prove an excellent target to screen for immunosuppressiva to potentially treat the symptoms of inflammatory bowel disease.

Bcl10 activates the NF-kappaB pathway through ubiquitination of NEMO.

The NF-kappaB family of transcription factors is activated in response to many stimuli, including pro-inflammatory cytokines, environmental stresses and, in the case of B and T lymphocytes, by antigenic stimulation. Bcl10 is essential for NF-kappaB activation by T- and B-cell receptors. T and B lymphocytes from Bcl10-deficient mice fail to activate NF-kappaB in response to antigen-receptor stimulation and, as a consequence, are unable to proliferate. Bcl10 overexpression is sufficient to activate NF-kappaB, a process that requires the NF-kappaB essential modulator NEMO (also known as IKK-gamma), which is the regulatory subunit of the IkappaB kinase complex. However, the cellular mechanism by which Bcl10 activates the NF-kappaB pathway remains unclear. Here we show that Bcl10 targets NEMO for lysine-63-linked ubiquitination. Notably, a mutant form of NEMO that cannot be ubiquitinated inhibited Bcl10-induced NF-kappaB activation. Paracaspase and a ubiquitin-conjugating enzyme (UBC13) were both required for Bcl10-induced NEMO ubiquitination and subsequent NF-kappaB activation. Furthermore, short interfering RNAs that reduced the expression of paracaspase and UBC13 abrogated the effects of Bcl10. Thus, the adaptor protein Bcl10 promotes activation of NF-kappaB transcription factors through paracaspase- and UBC13-dependent ubiquitination of NEMO.

Defective development and function of Bcl10-deficient follicular, marginal zone and B1 B cells.

Bcl10 is an intracellular protein essential for nuclear factor (NF)-kappaB activation after lymphocyte antigen receptor stimulation. Using knockout mice, we show that absence of Bcl10 impeded conversion from transitional type 2 to mature follicular B cells and caused substantial decreases in marginal zone and B1 B cells. Bcl10-deficient B cells showed no excessive apoptosis. However, both Bcl10-deficient follicular and marginal zone B cells failed to proliferate normally, although Bcl10-deficient marginal zone B cells uniquely failed to activate NF-kappaB efficiently after stimulation with lipopolysaccharide. Bcl10-deficient marginal zone B cells did not capture antigens, and Bcl10-deficient (Bcl10-/-) mice failed to initiate humoral responses, leading to an inability to clear blood-borne bacteria. Thus, Bcl10 is essential for the development of all mature B cell subsets.

T cell receptor-stimulated generation of hydrogen peroxide inhibits MEK-ERK activation and lck serine phosphorylation.

Previous studies indicated that antigen receptor (TcR) stimulation of mature T cells induced rapid generation of reactive oxygen species (ROS). The goal of the current study was to examine the role(s) of ROS in TcR signal transduction, with a focus upon the redox-sensitive MAPK family. TcR cross-linking of primary human T blasts and Jurkat human T cells rapidly activated the ERK, JNK, p38 and Akt kinases within minutes, and was temporally associated with TcR-stimulated production of hydrogen peroxide (H(2)O(2)). TcR-induced activation of ERK was selectively augmented and sustained in the presence of pharmacologic antioxidants that can quench or inhibit H(2)O(2) production (NAC, MnTBAP and Ebselen, but not DPI), while activation of JNK and Akt were largely unaffected. This was paralleled by concurrent changes in MEK1/2 phosphorylation, suggesting that ROS acted upstream of MEK-ERK activation. Molecular targeting of H(2)O(2) by overexpression of peroxiredoxin II, a thioredoxin dependent peroxidase, also increased and sustained ERK and MEK activation upon TcR cross-linking. Enhancement of ERK phosphorylation by antioxidants correlated with increased and sustained serine phosphorylation of the src-family kinase lck, a known ERK substrate. Thus, the data suggest that TcR-stimulated production of hydrogen peroxide negatively feeds back to dampen antigen-stimulated ERK activation and this redox-dependent regulation may serve to modulate key steps in TcR signaling.

Vav1: a key signal transducer downstream of the TCR.

Vav1 is a 95-kDa protein expressed in all hemopoietic cells that becomes rapidly tyrosine phosphorylated following T cell antigen receptor (TCR) stimulation. Vav1 contains multiple domains characteristic of signal transducing proteins, including a Dbl homology domain, a hallmark of a guanine nucleotide exchange factor (GEF) for Rho-family GTPases. Indeed Vav1 is a GEF for Rac1, Rac2 and RhoG, and it is activated following tyrosine phosphorylation. Generation of mice deficient in Vav1 has shown that it plays an important role in selection events within the thymus, including both positive and negative selection, consistent with Vav1 transducing TCR signals required to drive these processes. Furthermore, Vav1-deficient T cells are defective in TCR-induced proliferation and cytokine synthesis. Analysis of TCR signaling pathways in Vav1-deficient T cells and thymocytes has shown that Vav1 is required to transduce signals to the activation of a calcium flux, extracellular signal-regulated kinase (ERK) and the nuclear factor kappaB (NF-kappaB) transcription factor. Vav1 has also been shown to control the activation of phospholipase Cgamma1 (PLCgamma1) via both phosphoinositide-3-kinase (PI3K)-dependent and -independent pathways. Finally, Vav1 has been shown to transduce TCR signals to some but not all cytoskeleton-dependent pathways. In particular, Vav1 is required for efficient TCR-induced conjugate formation with antigen presenting cells (APCs), activation of the integrin leukocyte function-associated antigen-1 (LFA-1) and cell polarization.

Lethal T cell immunodeficiency induced by chronic costimulation via CD27-CD70 interactions.

It has been proposed that HIV-1, in addition to directly infecting and killing CD4+ T cells, causes T cell dysfunction and T cell loss by chronic immune activation. We analyzed the effects of chronic immune activation in mice that constitutively expressed CD70, the ligand for the tumor necrosis factor receptor family member CD27, on B cells. CD70 transgenic (CD70 Tg) mice showed a progressive conversion of naive T cells into effector-memory cells, which culminated in the depletion of naive T cells from lymph nodes and spleen. T cell changes depended on continuous CD27-CD70 interactions and T cell antigen receptor stimulation. Despite this hyperactive immune system, CD70 Tg mice died aged 6-8 months from Pneumocystis carinii infection, a hallmark of T cell immunodeficiency. Thus, persistent delivery of costimulatory signals via CD27-CD70 interactions, as may occur during chronic active viral infections, can exhaust the T cell pool and is sufficient to induce lethal immunodeficiency.

Characterization of the human ephrin-A4 promoter

Expression of the ephrin-A4 ligand, a family member of ligands binding the Eph receptor tyrosine kinases, is induced after an antigen-receptor stimulation of lymphocytes. To understand the transcription regulation of the ephrin-A4 gene, its promoter was identified and regulating elements were characterized. The ephrin-A4 promoter contains cis elements directing the cell-specific expression. By deletion studies, three specific regions, which were contributing to the transcription activity in lymphoid cells, were localized. In one of these regions, an inverted CCAAT box was identified and shown to bind the transcription activator nuclear factor-Y (NF-Y). The importance of NF-Y binding for the ephrin-A4 promoter activity is shown by a total abrogation of promoter activity after destruction of its binding site. NF-Y binding and activity are also crucially dependent on the integrity of the surrounding sequence. In addition, electrophoretic mobility-shift assay and serial-mutation analysis of the two remaining regulating regions revealed cis regulatory elements contributing to the transcription activity of the ephrin-A4 promoter.

Expression of human protein tyrosine phosphatase epsilon in leucocytes: a potential ERK pathway-regulating phosphatase.

The expression of protein tyrosine phosphatase epsilon (PTPepsilon) was studied in human tissues and blood cells. High mRNA expression was observed in peripheral blood leucocytes, particularly in monocytes and granulocytes which revealed at least four distinct transcripts. In lymphocytes, PTPepsilon expression was induced after 12-O-tetradecanoylphorbol-13-acetate (TPA) or antigen-receptor stimulation, indicating that PTPepsilon plays a role in the events taking place after antigen engagement. Previously, PTPepsilon has been shown to be involved in regulating voltage-gated potassium channel activity, insulin-receptor signalling and Janus kinase-signal transducers and activators of transcription (STAT) signalling. Transfection of cells with different PTPepsilon constructs and activator protein-1 reporter gene indicates that the catalytic activity of PTPepsilon is involved in the regulation of the mitogen-activated protein kinase cascade. In particular, the extracellular signal-regulated kinases (ERK1/2) were shown to be inhibited in both phosphorylation status and enzymatic activity after overexpression of PTPepsilon. Thus, PTPepsilon emerges as a phosphatase with a potential to regulate the ERK1/2 pathway either directly or indirectly through its catalytic activity.

Vav1 Couples T Cell Receptor to Serum Response Factor-dependent Transcription via a MEK-dependent Pathway

The Vav family of guanine nucleotide exchange factors for Rho family GTPases plays a critical role in lymphocyte proliferation, gene transcription, and cytoskeleton reorganization following immunoreceptor stimulation. However, its role in immediate early gene activation is unclear. In this study, we have investigated the mechanisms by which Vav1 can regulate c-fos serum response element transcriptional activity. We show that T cell antigen receptor (TCR) stimulation induces the phosphorylation of serum response factor (SRF) on serine 103 and increases the binding of SRF complexes on serum response element in a MEK- and p38-dependent pathway. The physiological relevance of our findings is supported by the inhibition of the interleukin-2 gene transcriptional activity by a dominant negative SRF mutant. Overexpression of Vav1, which partially mimics TCR stimulation, promotes SRF-dependent transcription, and dominant negative Vav1 mutants block SRF activation by TCR. SRF activation by Vav1 occurs through a signaling cascade consisting of Rac1/Cdc42 and the serine/threonine kinases Pak1 and MEK, but independently of the phosphatidylinositol 3-kinase pathway. Interestingly, Vav2 also enhances SRF through Rho GTPases, suggesting that Vav proteins are general regulators of SRF activation in lymphocytes. This report establishes Vav proteins as a direct link between antigen receptors and SRF-dependent early gene expression.

BANK regulates BCR-induced calcium mobilization by promoting tyrosine phosphorylation of IP(3) receptor.

B-cell activation mediated through the antigen receptor is dependent on activation of protein tyrosine kinases (PTKs) such as Lyn and Syk and subsequent phosphorylation of various signaling proteins. Here we report on the identification and characterization of the B-cell scaffold protein with ankyrin repeats (BANK), a novel substrate of tyrosine kinases. BANK is expressed in B cells and is tyrosine phosphorylated upon B-cell antigen receptor (BCR) stimulation, which is mediated predominantly by Syk. Overexpres sion of BANK in B cells leads to enhancement of BCR-induced calcium mobilization. We found that both Lyn and inositol 1,4,5-trisphosphate receptor (IP(3)R) associate with the distinct regions of BANK and that BANK promotes Lyn-mediated tyrosine phosphorylation of IP(3)R. Given that IP(3)R channel activity is up-regulated by its tyrosine phosphorylation, BANK appears to be a novel scaffold protein regulating BCR-induced calcium mobilization by connecting PTKs to IP(3)R. Because BANK expression is confined to functional BCR-expressing B cells, BANK-mediated calcium mobilization may be specific to foreign antigen-induced immune responses rather than to signaling required for B-cell development.

Differential Association of CD45 Isoforms with CD4 and CD8 Regulates the Actions of Specific Pools of p56lck Tyrosine Kinase in T Cell Antigen Receptor Signal Transduction

An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction was carried out by transfecting CD45-negative CD4(+)CD8(+) HPB-ALL T cells with the CD45R0, CD45RBC, and CD45RABC isoforms. Fluorescence resonance energy transfer analysis showed that the CD45R0 isoform, but not the CD45RBC or CD45RABC isoforms, was found as homodimers and also preferentially associated with CD4 and CD8 at the cell-surface. A comparison was therefore made of T cell antigen receptor signaling between sub-clones expressing either CD45R0 or CD45RBC. Under basal conditions CD4-associated p56(lck) tyrosine kinase activity and cellular protein tyrosine phosphorylation levels were higher in the CD45R0(+) than in the CD45RBC(+) sub-clones. Upon CD3-CD4 ligation, TCR-zeta phosphorylation, ZAP-70 recruitment to the p21/p23 TCR-zeta phosphoisomers, ZAP-70 phosphorylation, as well as p56(lck), c-Cbl and Slp-76 phosphorylation, were all markedly increased in CD45R0(+) compared with CD45RBC(+) cells. T cell antigen receptor (TCR) stimulation alone also promoted c-Cbl phosphorylation in CD45R0(+) but not in CD45RBC(+) cells. Our results are consistent with a model in which association of CD45R0 with CD4 generates a more active pool of CD4-associated p56(lck) kinase molecules. Upon CD3-CD4 co-ligation, the active p56(lck) increases the intensity of T cell antigen receptor signal transduction coupling by promoting TCR-zeta chain phosphorylation and ZAP-70 recruitment.

Identification of novel targets of immunosuppressive agents by cDNA-based microarray analysis.

The immunosuppressive agents cyclosporin A (CsA) and tacrolimus (FK506) bind to unrelated intracellular immunophilin receptors, cyclophilin (CyP) and FK506-binding protein (FKBP), respectively. The complexes of CsA-CyP and of FK506-FKBP both bind to and inhibit the activity of the calcium/calmodulin-dependent serine/threonine phosphatase calcineurin. We used cDNA microarray analysis to characterize early human peripheral blood T cell transcriptional responses following antigen receptor stimulation in the absence or presence of CsA or FK506, hoping to identify novel targets dependent upon calcineurin or immunophilins or, perhaps, specific targets of either CyP or FKBP inhibitable by one drug alone. The array data failed to identify genes uniquely sensitive to only one drug, suggesting that transcriptionally regulated, immunophilin-dependent but calcineurin-independent targets fell below the limits of detection in this system. In contrast, transcript profiling identified and mRNA and protein analysis confirmed novel as well as known genes reproducibly induced or inhibited by both immunosuppressive agents. In this context, we show that transcriptional activation of Stat5a and repression of the cytokine interleukin-16 are regulated by T cell receptor engagement and dependent upon drug-immunophilin complexes and, presumably, calcineurin activity.

Hematopoietic Progenitor Kinase 1 Associates Physically and Functionally with the Adaptor Proteins B Cell Linker Protein and SLP-76 in Lymphocytes

B cell linker protein (BLNK) is a SLP-76-related adaptor protein essential for signal transduction from the BCR. To identify components of BLNK-associated signaling pathways, we performed a phosphorylation-dependent yeast two-hybrid analysis using BLNK probes. Here we report that the serine/threonine kinase hematopoietic progenitor kinase 1 (HPK1), which is activated upon antigen-receptor stimulation and which has been implicated in the regulation of MAP kinase pathways, interacts physically and functionally with BLNK in B cells and with SLP-76 in T cells. This interaction requires Tyr(379) of HPK1 and the Src homology 2 (SH2) domain of BLNK/SLP-76. Via homology modeling, we defined a consensus binding site within ligands for SLP family SH2 domains. We further demonstrate that the SH2 domain of SLP-76 participates in the regulation of AP-1 and NFAT activation in response to T cell receptor (TCR) stimulation and that HPK1 inhibits AP-1 activation in a manner partially dependent on its interaction with SLP-76. Our data are consistent with a model in which full activation of HPK1 requires its own phosphorylation on tyrosine and subsequent interaction with adaptors of the SLP family, providing a mechanistic basis for the integration of this kinase into antigen receptor signaling cascades.

Zap-70-independent Ca(2+) mobilization and Erk activation in Jurkat T cells in response to T-cell antigen receptor ligation.

The tyrosine kinase ZAP-70 has been implicated as a critical intermediary between T-cell antigen receptor (TCR) stimulation and Erk activation on the basis of the ability of dominant negative ZAP-70 to inhibit TCR-stimulated Erk activation, and the reported inability of anti-CD3 antibodies to activate Erk in ZAP-70-negative Jurkat cells. However, Erk is activated in T cells receiving a partial agonist signal, despite failing to activate ZAP-70. This discrepancy led us to reanalyze the ZAP-70-negative Jurkat T-cell line P116 for its ability to support Erk activation in response to TCR/CD3 stimulation. Erk was activated by CD3 cross-linking in P116 cells. However, this response required a higher concentration of anti-CD3 antibody and was delayed and transient compared to that in Jurkat T cells. Activation of Raf-1 and MEK-1 was coincident with Erk activation. Remarkably, the time course of Ras activation was comparable in the two cell lines, despite proceeding in the absence of LAT tyrosine phosphorylation in the P116 cells. CD3 stimulation of P116 cells also induced tyrosine phosphorylation of phospholipase C-gamma1 (PLCgamma1) and increased the intracellular Ca(2+) concentration. Protein kinase C (PKC) inhibitors blocked CD3-stimulated Erk activation in P116 cells, while parental Jurkat cells were refractory to PKC inhibition. The physiologic relevance of these signaling events is further supported by the finding of PLCgamma1 tyrosine phosphorylation, Erk activation, and CD69 upregulation in P116 cells on stimulation with superantigen and antigen-presenting cells. These results demonstrate the existence of two pathways leading to TCR-stimulated Erk activation in Jurkat T cells: a ZAP-70-independent pathway requiring PKC and a ZAP-70-dependent pathway that is PKC independent.

The stress kinase mitogen-activated protein kinase kinase (MKK)7 is a negative regulator of antigen receptor and growth factor receptor-induced proliferation in hematopoietic cells.

The dual specificity kinases mitogen-activated protein kinase (MAPK) kinase (MKK)7 and MKK4 are the only molecules known to directly activate the stress kinases stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) in response to environmental or mitogenic stimuli. To examine the physiological role of MKK7 in hematopoietic cells, we used a gene targeting strategy to mutate MKK7 in murine T and B cells and non-lymphoid mast cells. Loss of MKK7 in thymocytes and mature B cells results in hyperproliferation in response to growth factor and antigen receptor stimulation and increased thymic cellularity. Mutation of mkk7 in mast cells resulted in hyperproliferation in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF). SAPK/JNK activation was completely abolished in the absence of MKK7, even though expression of MKK4 was strongly upregulated in mkk7(-/-) mast cell lines, and phosphorylation of MKK4 occurred normally in response to multiple stress stimuli. Loss of MKK7 did not affect activation of extracellular signal-regulated kinase (ERK)1/2 or p38 MAPK. mkk7(-/-) mast cells display reduced expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1. Reexpression of p16INK4a in mkk7(-/-) mast cells abrogates the hyperproliferative response. Apoptotic responses to a variety of stimuli were not affected. Thus, MKK7 is an essential and specific regulator of stress-induced SAPK/JNK activation in mast cells and MKK7 negatively regulates growth factor and antigen receptor-driven proliferation in hematopoietic cells. These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.

Bimp1, a MAGUK Family Member Linking Protein Kinase C Activation to Bcl10-mediated NF-κB Induction

Bcl10 and MALT1, products of distinct chromosomal translocations in mucosa-associated lymphoid tissue lymphoma, cooperate in activating NF-kappaB. Mice lacking Bcl10 demonstrate severe immunodeficiency associated with failure of lymphocytes to activate nuclear factor kappaB (NF-kappaB) in response to antigen receptor stimulation and protein kinase C activation. We characterize Bimp1, a new signaling protein that binds Bcl10 and activates NF-kappaB. Bimp1-mediated NF-kappaB activation requires Bcl10 and IkappaB kinases, indicating that Bimp1 acts upstream of these mediators. Bimp1, Bcl10, and MALT1 form a ternary complex, with Bcl10 bridging the Bimp1/MALT1 interaction. A dominant negative Bimp1 mutant inhibits NF-kappaB activation by anti-CD3 ligation, phorbol ester, and protein kinase C expression. These results suggest that Bimp1 links surface receptor stimulation and protein kinase C activation to Bcl10/MALT1, thus leading to NF-kappaB induction.

PTP-PEST, a scaffold protein tyrosine phosphatase, negatively regulates lymphocyte activation by targeting a unique set of substrates.

There is increasing interest in elucidating the mechanisms involved in the negative regulation of lymphocyte activation. Herein, we show that the cytosolic protein tyrosine phosphatase PTP-PEST is expressed abundantly in a wide variety of haemopoietic cell types, including B cells and T cells. In a model B-cell line, PTP-PEST was found to be constitutively associated with several signalling molecules, including Shc, paxillin, Csk and Cas. The interaction between Shc and PTP-PEST was augmented further by antigen receptor stimulation. Overexpression studies, antisense experiments and structure-function analyses provided evidence that PTP-PEST is an efficient negative regulator of lymphocyte activation. This function correlated with the ability of PTP-PEST to induce dephosphorylation of Shc, Pyk2, Fak and Cas, and inactivate the Ras pathway. Taken together, these data suggest that PTP-PEST is a novel and unique component of the inhibitory signalling machinery in lymphocytes.

Molecular motors involved in T cell receptor clusterings

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.

Vav-2 controls NFAT-dependent transcription inB- but not T-lymphocytes

We show here that Vav-2 is tyrosine phosphorylated following antigen receptor engagement in both B- and T-cells, but potentiates nuclear factor of activated T cells (NFAT)-dependent transcription only in B cells. Vav-2 function requires the N-terminus, as well as functional Dbl homology and SH2 domains. More over, the enhancement of NFAT-dependent transcription by Vav-2 can be inhibited by a number of dominant-negative GTPases. The ability of Vav-2 to potentiate NFAT-dependent transcription correlates with its ability to promote a sustained calcium flux. Thus, Vav-2 augments the calcium signal in B cells but not T cells, and a truncated form of Vav-2 can neither activate NFAT nor augment calcium signaling. The CD19 co-receptor physically interacts with Vav-2 and synergistically enhances Vav-2 phosphorylation induced by the B-cell receptor (BCR). In addition, we found that Vav-2 augments CD19-stimulated NFAT- dependent transcription, as well as transcription from the CD5 enhancer. These data suggest a role for Vav-2 in transducing BCR signals to the transcription factor NFAT and implicate Vav-2 in the integration of BCR and CD19 signaling.