The Vinculin Binding Sites of Talin and α-Actinin Are Sufficient to Activate Vinculin

Philippe R.J Bois,Brendan P O'Hara,Daniel Nietlispach,John Kirkpatrick,Tina Izard

doi:10.1074/jbc.m510397200

Philippe R.J Bois, Brendan P O'Hara + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m510397200

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Abstract

Vinculin regulates both cell-cell and cell-matrix junctions and anchors adhesion complexes to the actin cytoskeleton through its interactions with the vinculin binding sites of alpha-actinin or talin. Activation of vinculin requires a severing of the intramolecular interactions between its N- and C-terminal domains, which is necessary for vinculin to bind to F-actin; yet how this occurs in cells is not resolved. We tested the hypothesis that talin and alpha-actinin activate vinculin through their vinculin binding sites. Indeed, we show that these vinculin binding sites have a high affinity for full-length vinculin, are sufficient to sever the head-tail interactions of vinculin, and they induce conformational changes that allow vinculin to bind to F-actin. Finally, microinjection of these vinculin binding sites specifically targets vinculin in cells, disrupting its interactions with talin and alpha-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and alpha-actinin are buried within helical bundles present in their central rod domains. Collectively, these results support a model where the engagement of adhesion receptors first activates talin or alpha-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin.

Highlights

Cancer Center Support (CORE) Grant CA21765, and by the American Lebanese Syrian Associated Charities (ALSAC)
Vinculin binding to talin or ␣-actinin provides essential links for adhesion receptors with the actin cytoskeleton and, as underscored by the studies presented here, vinculin plays essential roles in stabilizing adhesion complexes
PIP2 impairs the associations of vinculin with actin [29], and the structures of inactive vinculin revealed that the PIP2 binding site is occluded in its inactive state [12, 15]

Summary

Introduction

Cancer Center Support (CORE) Grant CA21765, and by the American Lebanese Syrian Associated Charities (ALSAC). Talin interacts with vinculin through several high-affinity vinculin binding sites (VBS) present in its central rod domain (16, 34 –36), which could allow talin to bind to multiple vinculin molecules and amplify outside-in integrin signaling [35]. These interactions are likely essential, as targeted deletion of talin abolishes the formation of focal adhesions [37]. ␣-actinin plays an important role in the maturation of adhesion complexes [38, 39] and binds to vinculin through a single high affinity VBS (␣VBS) present in the R4 spectrin repeat at the end of its rod domain [19, 20, 40]. The structures of talin-VBS- and ␣VBS-bound to Vh1 have demonstrated that these amphipathic ␣-helices disrupt the Vh1-Vt interaction from a distance, by provoking conformational changes in the N-terminal helical bundle of vinculin, by a process coined helical bundle conversion [24, 35, 40]

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