The protooncoprotein Vav, a key regulator of lymphocyte signal transduction pathways, is a guanine nucleotide exchange factor (GEF) for Rho GTPases. The catalytic DH domain of Vav is autoinhibited by an adjacent N-terminal peptide, which binds the GEF active site. Phosphorylation of a buried tyrosine in this arm by Lck dissociates the arm from the DH domain, activating Vav. This proposal integrates biochemical and NMR spectroscopic investigations to discover the pathway to Vav activation. We hypothesize that A) additional tyrosine motifs (Y-motifs) in the Vav N-terminus may bind the DH domain and contribute to autoinhibition, and B) initial phosphorylation of exposed Y-motifs recruits Lck through its SH2 domain, enabling the kinetically disfavored phosphorylation event to occur in intramolecular fashion. Affinities of phosphorylated Vav proteins for the Lck SH2 domain, and of Vav N-terminal peptides for the DH domain will be determined, revealing which Y-motifs in the Vav N-terminus can bind the DH domain, and, when phosphorylated, recruit Lck. Effects of phosphorylation and SH2 binding on DH-inhibitory peptide affinity and binding kinetics will be determined, revealing whether these initial steps could contribute to relief of autoinhibition. Kinetic parameters for Lck kinase domain-catalyzed phosphorylation of Vav peptides, both free and bound intramolecularly to the DH domain, will be determined. Parameters will also be determined for full-length Lck, revealing the extent to which SH2-mediated docking to phosphorylated sites can overcome the sequestering effect of the DH domain toward the buried tyrosine. Amide and methyl group [mu]s-ms timescale dynamics in autoinhibited Vav will be measured by NMR relaxation dispersion analysis at multiple fields. The data will quantitate the rates of these motions, as well as the populations of the ground and excited states and the chemical shift differences between them. The chemical shift and rate data, combined with biochemically-measured helix dissociation rates, will reveal if the NMR dynamics represent excursions to a helix-dissociated state. Comparison of NMR dynamics rates with kinetics of phosphorylation will reveal if NMR dynamics could represent motions that govern the rate of Vav activation by Lck. These results will test our ideas that autoinhibited proteins often possess exposed """"""""access points"""""""" to overcome kinetic barriers to activation inherent in their design.
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