Structural and Energetic Mechanisms of Cooperative Autoinhibition and Activation of Vav1

Department of Biochemistry, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8816, USA.
Cell (Impact Factor: 33.12). 01/2010; 140(2):246-56. DOI: 10.1016/j.cell.2009.12.033
Source: PubMed

ABSTRACT Vav proteins are guanine nucleotide exchange factors (GEFs) for Rho family GTPases. They control processes including T cell activation, phagocytosis, and migration of normal and transformed cells. We report the structure and biophysical and cellular analyses of the five-domain autoinhibitory element of Vav1. The catalytic Dbl homology (DH) domain of Vav1 is controlled by two energetically coupled processes. The DH active site is directly, but weakly, inhibited by a helix from the adjacent Acidic domain. This core interaction is strengthened 10-fold by contacts of the calponin homology (CH) domain with the Acidic, pleckstrin homology, and DH domains. This construction enables efficient, stepwise relief of autoinhibition: initial phosphorylation events disrupt the modulatory CH contacts, facilitating phosphorylation of the inhibitory helix and consequent GEF activation. Our findings illustrate how the opposing requirements of strong suppression of activity and rapid kinetics of activation can be achieved in multidomain systems.

  • Source
    • "However, this hypothesis is unlikely because we did not detect an increase in Vav2 tyrosine phosphorylation upon Wnt stimulation. Alternatively and more likely, it is possible that the interaction frees the Vav2 catalytic Dbl homology domain from the coordinated auto-inhibition by the Acidic and Calponin homology domains (Yu et al., 2010). Thus, p120-catenin interaction would mimic the effects of Vav2 phosphorylation in the Acidic domain. "
    [Show abstract] [Hide abstract]
    ABSTRACT: A role for Rac1 GTPase in canonical Wnt signalling has been recently demonstrated, being required for β-catenin translocation to the nucleus. In this article we have investigated the mechanism of Rac1 stimulation by Wnt. Up-regulation of Rac1activity by Wnt3a temporally correlates with enhanced p120-catenin binding to Rac1 and Vav2. Vav2 and Rac1 association with p120-catenin is modulated by phosphorylation of this protein: it is stimulated upon serine/threonine phosphorylation by CK1 and inhibited by tyrosine phosphorylation by Src or Fyn. Acting on these two post-translational modifications, Wnt3a induces the release of p120-catenin from E-cadherin, enables p120-catenin interaction with Vav2 and Rac1 and facilitates Rac1 activation by Vav2. Since p120-catenin depletion disrupts gastrulation in Xenopus, we analysed p120-catenin mutants for their ability to rescue this phenotype. In contrast to the wild-type protein or other controls, p120-catenin point mutants deficient in the release from E-cadherin or in Vav2- or Rac1-binding failed to rescue p120-catenin depletion. Collectively, these results indicate that p120-catenin binding to Vav2 and Rac1 is required for the activation of this GTPase upon Wnt signalling.
    Journal of Cell Science 09/2012; 125(22). DOI:10.1242/jcs.101030 · 5.33 Impact Factor
  • Source
    • "They all display a conserved tyrosine residue (Y174, Y172 and Y173 respectively) in the short N-terminal helix that gets phosphorylated by Src Family- Kinases (SFK), destructuring the helix to facilitate access to the DH domain. These phosphorylations are considered as signatures of the active state of those GEFs (Aghazadeh et al., 2000; Colomba et al., 2008; Han et al., 1997; Lopez-Lago et al., 2000; Yohe et al., 2007; Yu et al., 2010). Mutations or deletions of this N-terminal domain often give rise to constitutive activation (Colacios et al., 2011; Schuebel et al., 1998). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Rho GTPases act as molecular switches central in cellular processes such as cytoskeleton dynamics, migration, cell proliferation, growth or survival. Their activation is tightly regulated downstream of cell surface receptors by Guanine nucleotide Exchange Factors (GEFs), that are responsible for the specificity, the accuracy, and the spatial restriction of Rho GTPases response to extracellular cues. Because there is about four time more RhoGEFs that Rho GTPases, and GEFs do not always show a strict specificity for GTPases, it is clear that their regulation depends on specific interactions with the subcellular environment. RhoGEFs bear a peculiar structure, highly conserved though evolution, consisting of a DH-PH tandem, the DH (Dbl homology) domain being responsible for the exchange activity. The function of the PH (Pleckstrin homology) domain known to bind phosphoinositides, however, remains elusive, and reports are in many cases rather confusing. This review summarizes data on the regulation of RhoGEFs activity through interaction of the PH-associated DH domain with phosphoinositides which are considered as critical players in the spatial organization of major signaling pathways.
    05/2012; 52(2):303-14. DOI:10.1016/j.jbior.2012.04.001
  • Source
    • "M2GRED/LAND analysis suggests that incorporation of the information from the two-state system, indeed, dramatically reduces the roughness of TFS. Further NMR spectroscopic analysis of the different biochemical entities offer orthogonal chemical shift information to facilitate solution of four-state equilibria (Li et al. 2008; Yu et al. 2010). Fig. 1 "
    [Show abstract] [Hide abstract]
    ABSTRACT: Coupled equilibria play important roles in controlling information flow in biochemical systems, including allosteric molecules and multidomain proteins. In the simplest case, two equilibria are coupled to produce four interconverting states. In this study, we assessed the feasibility of determining the degree of coupling between two equilibria in a four-state system via relaxation dispersion measurements. A major bottleneck in this effort is the lack of efficient approaches to data analysis. To this end, we designed a strategy to efficiently evaluate the smoothness of the target function surface (TFS). Using this approach, we found that the TFS is very rough when fitting benchmark CPMG data to all adjustable variables of the four-state equilibria. After constraining a portion of the adjustable variables, which can often be achieved through independent biochemical manipulation of the system, the smoothness of TFS improves dramatically, although it is still insufficient to pinpoint the solution. The four-state equilibria can be finally solved with further incorporation of independent chemical shift information that is readily available. We also used Monte Carlo simulations to evaluate how well each adjustable parameter can be determined in a large kinetic and thermodynamic parameter space and how much improvement can be achieved in defining the parameters through additional measurements. The results show that in favorable conditions the combination of relaxation dispersion and biochemical manipulation allow the four-state equilibrium to be parameterized, and thus coupling strength between two processes to be determined.
    Journal of Biomolecular NMR 09/2011; 51(1-2):57-70. DOI:10.1007/s10858-011-9541-1 · 3.31 Impact Factor