The Calponin Homology Domain of Vav1 Associates with Calmodulin and Is Prerequisite to T Cell Antigen Receptor-induced Calcium Release in Jurkat T Lymphocytes

Abstract

Vav1 is a guanine nucleotide exchange factor that is expressed specifically in hematopoietic cells and plays important roles in T cell development and activation. Vav1 consists of multiple structural domains so as to facilitate both its guanine nucleotide exchange activity and scaffold function following T cell antigen receptor ( Stimulation of the T cell antigen receptor (TCR)2 initiates a cascade of signaling events that lead to T cell activation. Calcium plays a central role in this process and has been studied intensively (1-4). Engagement of TCR triggers the activation and accumulation of enzymes and adapter molecules to the proximal membrane (5, 6), such as tyrosine phosphorylation and activation of phospholipase-C␥1 (PLC-␥1), thereby increasing the production of inositol 1,4,5-trisphosphate (IP 3 ). IP 3 binds to and activates the inositol 1,4,5-trisphosphate receptor (IP 3 R), which results in Ca 2ϩ release from the endoplasmic reticulum (ER) and the subsequent calcium influx from Ca 2ϩ release-activated Ca 2ϩ channel (CRAC) (1, 7). The elevated cytoplasmic [Ca 2ϩ ] i evokes a multitude of cellular responses, such as the NFAT-mediated gene expressions and the cell proliferation (8, 9).Vav1 is expressed specifically in hematopoietic cells as a 95-kDa protein, which plays pivotal roles as a guanine exchange factor (GEF) for small GTPases as well as a scaffold protein in the activation of hematopoietic cells (10 -12). The importance of Vav1 is because of its multiple structural elements, including a calponin homology (CH) domain, an acidic motif, a Dbl homology domain, a pleckstrin homology (PH) domain, a cysteine-rich motif, and one single SH2 domain flanked by two SH3 domains responsible for signaling protein assembly (12, 13). Upon TCR engagement, Vav1 is phosphorylated on the key tyrosine residues in the acidic motif, leading to the exposure of active Dbl homology domain for GDP/GTP exchange activity (14). Studies on vav1 Ϫ/Ϫ T cells isolated from knock-out mice demonstrated that Vav1 is essential for normal T cell activation and proliferation (15)(16)(17). In addition, the vav1-null cell line, J.Vav1, derived from Jurkat cells by somatic gene targeting approach, also exhibits pleiotropic defects in TCR-mediated signaling pathways (18).T cell stimulation evokes a biphasic calcium flux as follows: calcium release from intracellular stores followed by calcium influx across the plasma membrane (7,19). IP 3 Rs dominantly control the initiation of IP 3 -induced calcium release, demonstrated by using antisense knockdown of IP 3 R to block calcium release from the ER (20). Jurkat T cells express three IP 3 R isoforms, IP 3 R-1, IP 3 R-2, and IP 3 R-3 (21), which differ significantly in their sensitivity to IP 3 (22,23). A tyrosine kinase, Fyn, was suggested to modulate IP 3 R channel activities (24,25). Interactions between IP 3 R and other proteins, such as calmodulin (CaM), were reported to control the channel opening. Although some observations viewed CaM as an inhibitory protein of IP 3 R (26 -28), more...

Vav1 Oncogenic Mutation Inhibits T Cell Receptor-induced Calcium Mobilization through Inhibition of Phospholipase Cγ1 Activation

“…1C). These results are consistent with previously reported findings (52). It has also been reported that PLC␥1 phosphorylation is not significantly altered by oncoVav1 expression (52).…”

“…These results are consistent with previously reported findings (52). It has also been reported that PLC␥1 phosphorylation is not significantly altered by oncoVav1 expression (52). However, our results indicate that the TCR-induced phosphorylation of the critical Tyr-783 residue of PLC␥1 is diminished in cells expressing oncoVav1 compared with cells expressing WT Vav1 (Fig.…”

“…The calcium releaseactivated calcium influx also seems to be unimpaired by these defects in Vav1 expression because following the initial rise, [Ca 2ϩ ] i stabilized at virtually identical elevated plateaus in all four cell types. These data are in agreement with previously reported evidence (52).…”

The N-terminal 20-Amino Acid Region of Guanine Nucleotide Exchange Factor Vav1 Plays a Distinguished Role in T Cell Receptor-mediated Calcium Signaling

“…We previously reported that the CH domain was indispensable for Vav1-CaM interaction, and that the complex potentiated calcium release in a GEF-independent manner (11). However, the precise role of Vav1 that is different from other Vav proteins still remains to be investigated.…”

“…Plasmid DNA Constructs-The plasmids encoding Vav1 and the CH-truncated mutation, Vav1⌬CH, were prepared in our laboratory as described (11). Other constructs for the expression of N-terminal-truncated mutants of Vav1 were derived from plasmid encoding wild type Vav1 by polymerase chain reactions (PCR) with the corresponding oligonucleotides, and the mutant fragments were subcloned into pcDNA4/HisMax.C vector at BamHI and XhoI restriction sites.…”