title: Acidic region tyrosines provide access points for allosteric activation of the autoinhibited Vav1 Dbl homology domain.
abstract: Autoinhibited proteins serve key roles in many signal transduction pathways, and therefore proper regulation of these proteins is critical for normal cellular function. Proto-oncogene Vav1 is an autoinhibited guanine nucleotide exchange factor (GEF) for Rho family GTPases. The core autoinhibitory module of Vav1 consists of the catalytic Dbl homology (DH) domain bound through its active site to an alpha helix centered about Tyr174 in the Acidic (Ac) region of the protein. Phosphorylation of Tyr174 and two other tyrosines in the Ac region, Tyr142 and Tyr160, relieves autoinhibition and activates the catalytic DH domain. In this study, we use biochemical and structural analyses of the Vav1 Ac and DH domains to examine the kinetic and thermodynamic properties of Vav1 activation by the Src family kinase, Lck, and the role of the Lck SH2 domain in this process. We find that in the Ac-DH fragment of Vav1, Tyr174, but not Tyr142 or Tyr160, is protected from phosphorylation by interactions with the DH domain. Binding of the Lck SH2 domain to phosphorylated Tyr142 increases kcat/KM for Tyr174 by 4-fold, likely because the kinase domain can act on the substrate effectively in an intramolecular fashion. These studies of the autoinhibited Ac-DH module provide the foundation for a quantitative structural and thermodynamic understanding of the regulation of full length Vav1. Moreover, kinetic pathways involving initial interactions with exposed sites or "access points", as observed here for Vav1, may be generally important in the regulation of many autoinhibited proteins.
authors.affiliation: Howard Hughes Medical Institute and Department of Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8816, USA.
authors.forename: Gaya K
authors.initials: GK
authors.lastname: Amarasinghe
authors.forename: Michael K
authors.initials: MK
authors.lastname: Rosen
mesh_terms: D000494:Allosteric Regulation; D020816:Amino Acid Motifs / Q000502:physiology; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry; D051567:Proto-Oncogene Proteins c-vav / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; D014443:Tyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)