Two Distinct Head-Tail Interfaces Cooperate to Suppress Activation of Vinculin by Talin

Department of Biological Chemistry, Johns Hopkins University, Baltimore, Maryland, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2005; 280(17):17109-17. DOI: 10.1074/jbc.M414704200
Source: PubMed


Vinculin is autoinhibited by an intramolecular interaction that masks binding sites for talin and F-actin. Although a recent
structural model explains autoinhibition solely in terms of the interaction between vinculin tail (Vt) and residues 1–258 (D1), we find an absolute requirement for an interface involving the D4 domain of head (Vh residues 710–836) and Vt. Charge-to-alanine mutations in Vt revealed a class of mutants, T12 and T19, distal to the V-(1–258) binding site, which showed increases in their Kd values for head binding of 100- and 42-fold, respectively. Reciprocal mutation of residues in the D4 domain that contact
Vt yielded a head-tail interaction mutant of comparable magnitude to T19. These findings account for the approximately 120-fold
difference in Kd values between Vt binding to V-(1–258), as opposed to full-length Vh-(1–851). The significance of a bipartite autoinhibitory site is evidenced by its effects on talin binding to Vh. Whereas Vt fails to compete with the talin rod domain for binding to V-(1–258), competition occurs readily with full-length Vh, and this requires the D4 interface. Moreover in intact vinculin, mutations in the D4-Vt interface stabilize association of vinculin and talin rod. In cells, these head-tail interaction mutants induce hypertrophy
and elongation of focal adhesions. Definition of a second autoinhibitory site, the D4-Vt interface, supports the competing model of vinculin activation that invokes cooperative action of ligands at two sites. Together
the D1-Vt and D4-Vt interfaces provide the high affinity (∼10–9) autoinhibition observed in full-length vinculin.

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Available from: Daniel M Cohen, Sep 22, 2015
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    • "Recently, Janssen et al. [3] proposed a model whereby F-actin binding to vinculin may weaken the interaction between the head and tail domain, which would increase the likelihood for talin (in cell–matrix interactions) or alpha-catenin (in cell–cell adhesions) to bind and fully activate vinculin [4]. According to these researchers , the D1 region of the vinculin head stericly clashes with F-actin that prevents the linkage between vinculin tail and F-actin. "
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    ABSTRACT: We investigated the effect of the point mutation E29R on vinculin under cell mechanical aspects. MEFvcl KO cells were transfected with intact eGFP-vinculin (rescue) or mutant E29R vinculin. Cellular stiffness and adhesion strength of mutant E29R vinculin were considerably higher compared to rescue and MEFvcl KO cells. 2D traction microscopy also indicated markedly higher strain energy in E29R mutant cells compared to rescue and MEFvcl KO cells. Fluorescence recovery after photobleaching showed that the recovery time for mutant E29R cells was drastically slower than for MEFvcl rescue cells and that the mobile fraction was larger for rescue compared to E29R mutant cells. These results indicate that E29R mutation might prime the vinculin head for F-actin binding, which results in higher cell stiffness and contractile force.
    Biochemical and Biophysical Research Communications 09/2014; 452(3). DOI:10.1016/j.bbrc.2014.08.133 · 2.30 Impact Factor
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    • "the tandem SH3 domains of CAP . This obser - vation is potentially interesting because the D4 domain has been previously observed to make contacts with the Vt residues R1049 and T1050 in the crystal structure ( Bakolitsa et al . , 2004 ) , and the disruption of these contacts is necessary to allow the release of the D1 - Vh interactions by talin ( Cohen et al . , 2005 ) . We there - fore employed co - sedimentation assays to examine whether the binding of the tandem SH3 domains of CAP to vinculin influences the interaction of F - actin to vinculin . However , our co - sedimenta - tion assays results ( Fig . S11 ) indicate that the increase in tandem SH3 domains causes almost no changes to the amount "
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    ABSTRACT: C-Cbl-associated protein (CAP) is an important cytoskeletal adaptor protein involved in the regulation of adhesion turnover. The interaction between CAP and vinculin is critical for the recruitment of CAP to focal adhesions. The tandem SH3 domains (herein termed SH3a and SH3b) of CAP are responsible for its interaction with vinculin. However, the structural mechanism underlying the interaction between CAP and vinculin is poorly understood. In this manuscript, we report the solution structure of the tandem SH3 domains of CAP. Our NMR and ITC data indicate that the SH3a and SH3b domains of CAP simultaneously bind to a long proline-rich region of vinculin with different binding specificities. Furthermore, the crystal structures of the individual SH3a and SH3b domains complexed with their substrate peptides indicate that Q807(SH3a) and D881(SH3b) are the critical residues determining the different binding specificities of the SH3 domains. Based on the obtained structural information, a model of the SH3ab-vinculin complex was generated using MD simulation and SAXS data.
    Journal of Structural Biology 05/2014; 187(2):194-205. DOI:10.1016/j.jsb.2014.05.009 · 3.23 Impact Factor
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    • "Many researchers have used various vinculin mutants to study the function of vinculin. Vinculin 8/19 (vin8/19) and vinculin T12 (vinT12) mutants interfere with the head–tail interaction characterizing constitutive activation [51]. The vinculin A50I (vinA50I) mutant inhibits the head/tail dissociation of vinculin [52] and increases the vinculin turnover rate in focal adhesions [53]. "
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    ABSTRACT: Vinculin, a 116-kDa membrane cytoskeletal protein, is an important molecule for cell adhesion; however, little is known about its other cellular functions. Here, we demonstrated that vinculin binds to Rab5 and is required for Staphylococcus aureus (S. aureus) uptake in cells. Viunculin directly bound to Rab5 and enhanced the activation of S. aureus uptake. Over-expression of active vinculin mutants enhanced S. aureus uptake, whereas over-expression of an inactive vinculin mutant decreased S. aureus uptake. Vinculin bound to Rab5 at the N-terminal region (1-258) of vinculin. Vinculin and Rab5 were involved in the S. aureus-induced phosphorylation of MAP kinases (p38, Erk, and JNK) and IL-6 expression. Finally, vinculin and Rab5 knockdown reduced infection of S. aureus, phosphorylation of MAPKs and IL-6 expression in murine lungs. Our results suggest that vinculin binds to Rab5 and that these two molecules cooperatively enhance bacterial infection and the inflammatory response.
    PLoS ONE 01/2014; 9(1):e87373. DOI:10.1371/journal.pone.0087373 · 3.23 Impact Factor
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