A guanine nucleotide exchange factor-independent function of Vav1 in transcriptional activation.

T cell antigen receptor (TCR) stimulation induces the tyrosine phosphorylation of several intracellular proteins including the protooncogene Vav1. Vav1 expression is necessary for normal T cell development and activation. We previously showed that overexpression of Vav1 in Jurkat T cells potentiates the activity of the transcription factor nuclear factor of activated T cells (NF-AT). The mechanism by which Vav1 participates in TCR signaling events is not clear. Vav1 contains a guanine nucleotide exchange factor (GEF) domain that has specificity for Rac and other Rho GTPases that have been recently implicated in T cell activation events. Significantly, in vitro tyrosine phosphoryation of Vav1 by Lck activates its exchange activity. This Lck-mediated phosphorylation of Vav1 has been reported to depend upon Tyr-174 in Vav1, a site implicated in Vav1 function by other studies as well. In this report, we demonstrated that Tyr-174 is not required for the TCR-induced phosphorylation of Vav1 in vivo. Moreover, mutation of Tyr-174 augmented the ability of Vav1 to up-regulate NF-AT activation as well as the Vav1 GEF function leading to Rac activation. However, we also showed that the GEF activity of Vav1 was neither sufficient nor necessary for potentiation of NF-AT, and thereby we identify a GEF-independent role of Vav1 in potentiating NF-AT-driven transcription. Oncogenic Vav1 in which the amino-terminal 67 amino acids were deleted had elevated GEF activity but did not potentiate NF-AT when overexpressed in Jurkat cells. We also showed that a GEF mutant form of Vav1 that had impaired GEF function could still potentiate NF-AT. These studies reveal a previously unrecognized negative regulatory function of Tyr-174 in Vav1 and suggest that domains other than the Vav1 GEF domain contribute to TCR signals leading to NF-AT activation.