Vav-Rac1-Mediated Activation of the c-Jun N-Terminal Kinase/c-Jun/AP-1 Pathway Plays a Major Role in Stimulation of the Distal NFAT Site in the Interleukin-2 Gene Promoter

Abstract

The proto-oncogene product Vav, which is expressed specifically in hematopoietic and trophoblast cells, plays crucial roles in the development and activation of T cells triggered through the antigen-specific T-cell receptor (TCR) (7, 48). Vav enhances basal and TCR-activated transcription of the interleukin-2 (IL-2) gene, and this enhancement is largely mediated by activation of the distal NFAT element in the IL-2 gene promoter (NFAT-IL-2) (12, 24, 63). As in the case of other NFAT-binding sites (44), this element represents a binding site for a cooperative complex of the transcription factors NFAT and AP-1 (25, 40). This fact confounds an accurate assessment of the relative importance of NFAT versus AP-1 in NFAT-IL-2 activation, as measured by standard reporter assays. Consistent with the ability of Vav to upregulate the activity of NFAT, several studies demonstrated reduced Ca 2ϩ mobilization in T cells from Vav-deficient mice (9,16,17,23,60). However, this issue remains controversial in view of apparently contradictory findings that documented intact nuclear translocation and DNA-binding activities of NFAT (23) (10,42,59) suggests that Vav may also enhance AP-1 activation, since JNK is one of the upstream kinases involved in AP-1 activation via the phosphorylation of c-Jun (13,22,55). Consistent with this notion, we recently found that transient overexpression of Vav greatly increases AP-1 activity in T cells (61), although another recent study reported that Vav does not play a role in AP-1 activation (15).Here, we further analyzed the mechanism of Vav-mediated NFAT-IL-2 activation, with particular emphasis on the contribution of AP-1 and its potential importance as a Vav target in T cells. We also assessed the effects of Vav on the nuclear translocation and DNA-binding activities of NFAT proteins. Our findings indicate that Vav-induced activation of c-Jun/ AP-1, which depends on an intact Rac or JNK pathway, plays a major role in NFAT-IL-2 activation and, furthermore, that Vav may have a relatively minor role in direct NFAT activation.

Cytohesin Binder and Regulator Augments T Cell Receptor-induced Nuclear Factor of Activated T Cells·AP-1 Activation through Regulation of the JNK Pathway

“…We hypothesized that NFAT activation by Cybr was mediated by AP-1 rather than increased binding of NFAT proteins. Therefore, we utilized an NFAT reporter construct containing the distal IFN-␥ high affinity NFAT site, which does not include AP-1 binding sites (25). As shown in Fig.…”

“…Vav mediates NFAT activation but does not influence TCR-induced proximal PTK activation or the cytoplasmic calcium levels (29). Instead, Vav is more important for augmenting the TCR-induced JNK/AP-1 cascade upstream of NFAT activation (25). We have shown that Cybr expression enhances TCR-dependent phosphorylation of Vav, therefore linking these molecules Jurkat T cells were transfected with either X-press-EV or X-press-Cybr plasmids.…”

“…The NFAT luciferase reporter construct pGL3-NFAT 3 -luciferase (pNFAT-Luc), consisting of three direct copies of the murine IL-4 high affinity NFAT site (region Ϫ82 to Ϫ64 of the murine IL-4 promoter) (23,24) was a generous gift of Dr. James A. Goodrich (University of Colorado, Boulder, CO). The NFAT-IFN luciferase reporter construct was a generous gift of Dr. A. Altman (La Jolla Institute for Allergy and Immunology, La Jolla, CA) and was described previously (25). pAP-1-Luc (AP-1, 7ϫ) luciferase reporter gene construct was kindly provided by Dr. M. Chiariello (CNR, Napoli, Italy).…”

“…Moreover, Vav can regulate the JNK-MAPK pathway downstream of TCR (25,29). We therefore explored the possibility that Cybr could influence Vav activation.…”

LIME Mediates Immunological Synapse Formation through Activation of VAV

“…The expression plasmids encoding Lck wild-type (wt) or mutant (F505) have been described previously (Choi et al, 1999). The pEFneo plasmid encoding human Vav-myc was kindly provided by Dr. Yun-Cai Liu (Kaminuma et al, 2001). The E. coli expression construct for the GST-Rho binding domain (RBD) of PAK1 was kindly provided by J.H.…”