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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.
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