Structure And Function Of The Human Vav2and Arap3

We have found and confirmed that Arap3is a novel interaction partner for Vav2. While Vav2is a guanine nucleotide exchange factor (GEF) for Rac, Arap3is a GTPase activating protein (GAP) for both Arf6and RhoA. Vav2SH2domain can interact directly with the two tyrosine-phosphorylated sites pY1403and pY1408in Arap3. Further, we have solved the solution structure of Vav2-SH2domain in complex with a phosphoryted peptide derived from the tyrosine-phorylated site pY1408in Arap3. In addition, we also have performed a preliminary study for the solution structure of Arap3ArfGAP domain.There is an overview of Vav2, Arap3and the related small GTPases in chapter1. Vav2and Arap3are important celluar signaling proteins, and both have been found to play key roles in the lamellipodia formation and cell migration.Chapter2focuses on our finding that Arap3is a novel interaction partner for Vav2. This interaction is regulated by the phosphorylation level of Arap3, as mutation the two phosphorylated sites pY1403and pY1408in Arap3decreased the interaction sharply. Tyrosine residue Y140(?) of Arap3is embedded within a YEEP motif, such a motif is consistent with the consensus sequence reported to be preferred by Vav2SH2domain. Tyrosine residue Y1408of Arap3is embedded within a YEEV motif, with a Val at pY+3position of ligand, which has never been shown to be suitable for Vav2SH2domain binding. However, synthesized peptides derived from these two tyrosine-phosphorylated sites pY1403and pY1408in Arap3both showed high affinites to Vav2SH2domain according to our ITC and NMR perturbation assays, with dissociation constants (Kd) of-0.27μM and-1.40μM, respectively. The complex structure of Vav2SH2/peptide pY1408was determined. The hydrophobic pY+3binding pocket of Vav2SH2domain is shallow, and the pY+3residue Val just patchs on the surface of this pocket through a lot of hydrophobic interactions.Chapter3is about our preliminary work on the determination of solution structure of Arap3ArfGAP domain. We selected a protein fragment containing the entire ArfGAP domain and a-30residues extension in its C-terminal, basing on the method of limited proteolysis. The work of chemical shifts assignment has been done and the solution structure is under determination.