Abstract 3854: Identification of crucial residues at the rim of the binding cleft of the C1 domain of Vav1 that determine its ligand sensitivity

Abstract

Vav1 is a guanine exchange factor (GEF) for the Rho family of GTPases. It plays a pivotal role in T-cell maturation and development, cytoskeleton organization, and oncogenic transformation. The GEF activity of Vav1 is regulated by several factors, including interaction between its catalytic DH domain and its C1 domain. Its C1 domain shows homology with “typical” C1 domains that are sensitive to the second messenger diacylglycerol (DAG) and phorbol esters (PEs), but it is classified as “atypical” based on its unresponsiveness to these ligands. However, crystallographic analysis has shown that, unlike atypical C1 domains (e.g. Raf1) which possess a distorted structure, the Vav1 C1 retains the geometry of the binding cavity. We hypothesized that residues in the vicinity of the binding pocket might interfere with ligand binding. Sequence alignment with typical C1 domains revealed six unique residues situated along the rim of the putative binding cleft in Vav1 C1: Glu9, Glu10, Pro11, Trp22, Thr24, Tyr26.To probe the role of these residues on DAG/PE sensitivity, we first mutated these sites in the potent PE-sensitive C1b domain of PKCΔ to that of the corresponding sites of Vav1 C1, and analyzed the potency of the mutants for PEs. In vitro binding assays showed that 5 of 6 single-site-mutations (except Trp22) caused significant but limited (10-15 fold) reduction in the binding affinity to phorbol 12,13-dibutyrate (PDBU). Introduction of multiple mutations further decreased the affinity, in a cumulative fashion, leading to no detectable binding in the quintuple mutant. Correspondingly, in vivo confocal microscopy revealed that double and triple GFP-tagged mutants showed much slower and weaker plasma membrane translocation in response to PE than did WT C1bΔ, whereas the quintuple mutation was completely unresponsive. Thus, the ligand-insensitivity of Vav1 C1 reflects the combined effects of Glu9, Glu10, Pro11, Thr24, Tyr26 rather than the effect of a single specific residue. Conversely, introducing “reverse” mutations (corresponding to the residues of PKCΔ C1b) into Vav1 C1 generate binding activity. The quintuple (PKCΔ-like) mutation restored the phorbol-ester-sensitivity of Vav1 C1 to the level of the potent PKCΔ C1b both in vitro and in vivo. In addition, the quintuple mutation conferred PE-sensitivity to the full length Vav1, as revealed by translocation studies. Computer modeling suggests that the presence of these residues confers on Vav1 C1 a hydrophilic surface at the tip of the binding cavity (as opposed to the rather lipophilic surface of PKCΔ C1b), thus impeding interactions with the membrane bilayer and hindering the formation of the ternary binding complex of ligand, receptor (C1) and membrane lipid. We speculate that targeting those unique hydrophilic residues with specific DAG/PE analogs may provide a rationale for selectively manipulating Vav1 function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3854. doi:10.1158/1538-7445.AM2011-3854