The interaction of the cell-contact proteins VASP and vinculin is regulated by phosphatidylinositol-4,5-bisphosphate

140

Talin interactions with vinculin are essential for focal adhesions. Curiously, talin contains three noncontiguous vinculin binding sites (VBS) that can bind individually to the vinculin head (Vh) domain. Here we report the crystal structure of the human Vh⅐VBS1 complex, a validated model of the Vh⅐VBS2 structure, and biochemical studies that demonstrate that all of talin VBSs activate vinculin by provoking helical bundle conversion of the Vh domain, which displaces the vinculin tail (Vt) domain. Thus, helical bundle conversion is a structurally conserved response in talin-vinculin interactions. Furthermore, talin VBSs bind to Vh in a mutually exclusive manner but do differ in their affinity for Vh and in their ability to displace Vt, suggesting that the strengths of these interactions could lead to differences in signaling outcome. These findings support a model in which talin binds to and activates multiple vinculin molecules to provoke rapid reorganization of the actin cytoskeleton.

Section: Discussionmentioning

confidence: 99%

Structural Basis for Amplifying Vinculin Activation by Talin

Izard

Vonrhein

2004

140

“…In Vitro Chemical Cross-linking-Chemical cross-linking of recombinant VT, MVT, and VH was performed essentially as described (13). Purified (M)VT alone with VH or mixtures of both tail domains (100 pmol, 3.5 M) were incubated in the presence or absence of lipids at 37°C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

“…Anchorage of microfilaments is mediated by VT that harbors two actin binding regions (11,12). Through the recruitment of the vasodilator-stimulated phosphoprotein VASP and the Arp 2/3 complex to the hinge region, vinculin is also tightly linked to actin dynamics at cell adhesion sites (13)(14)(15). Ligand interactions are regulated by an intramolecular association of VH and VT, which masks the binding sites for most ligands (12,(15)(16)(17)(18)(19).…”

mentioning

confidence: 99%

Comparative Biochemical Analysis Suggests That Vinculin and Metavinculin Cooperate in Muscular Adhesion Sites

Witt

Zieseniß

Fock

et al. 2004

Self Cite

Metavinculin, the muscle-specific splice variant of the cell adhesion protein vinculin, is characterized by a 68-amino acid insert within the C-terminal tail domain. The findings that mutations within this region correlate with hereditary idiopathic dilated cardiomyopathy in man suggest a specific contribution of metavinculin to the molecular architecture of muscular actin-membrane attachment sites, the nature of which, however, is still unknown. In mice, metavinculin is expressed in smooth and skeletal muscle, where it co-localizes with vinculin in dense plaques and costameres, respectively, but is of conspicuously low abundance in the heart. Immunoprecipitates suggest that both isoforms are present in the same complex. On the molecular level, both vinculin isoforms are regulated via an intramolecular head-tail interaction, with the metavinculin tail domain having a lower affinity for the head as compared with the vinculin tail. In addition, metavinculin displays impaired binding to acidic phospholipids and reduced homodimerization. Only in the presence of phospholipidactivated vinculin tail, the metavinculin tail domain is readily incorporated into heterodimers. Mutational analysis revealed that the metavinculin insert significantly alters binding of the C-terminal hairpin loop to acidic phospholipids. In summary, our data lead to a model in which unfurling of the metavinculin tail domain is impaired by the negative charges of the 68-amino acid insert, thus requiring vinculin to fully activate the metavinculin molecule. As a consequence, microfilament anchorage may be modulated at muscular adhesion sites through heterodimer formation.

“…Phospholipids were prepared as described in Huttelmaier et al (39). Turkey gizzard FLN was incubated without or with a 5-fold molar excess of phosphatidylinositol 4,5-bisphosphate or phosphatidylserine and recombinant FLNa fragments were incubated without or with a 5-fold molar excess of phospholipids (phosphatidylcholine:phosphatidylserine: phosphatidylinositol 4,5-bisphosphate ϭ 9:9:2) for 30 min at 4°C and immobilized on nitrocellulose membrane (5-20 pmol of protein/spot) using a dot blotter with circular slots (Biometra, Göttingen, Germany).…”

Section: Methodsmentioning

confidence: 99%

The F-actin Cross-linking and Focal Adhesion Protein Filamin A Is a Ligand and in Vivo Substrate for Protein Kinase Cα

Tigges

Koch

Wissing

et al. 2003

Self Cite

Filamin A is an established structural component of cell-matrix adhesion sites. In addition, it serves as a scaffold for the subcellular targeting of different signaling molecules. Protein kinase C (PKC) has been found associated with filamin; however, details about this interaction and its significance for cell-matrix adhesiondependent signaling have remained elusive. We performed a yeast two-hybrid analysis using protein kinase C␣ as a bait and identified filamin as a direct binding partner. The interaction was confirmed in transfected HeLa cells, and serial truncation fragments of filamin A were employed to identify two binding sites on filamin. In vitro ligand binding assays revealed a Ca 2؉ and phospholipid-dependent association of the regulatory domain of protein kinase C with these sites. Phosphorylation of filamin was found to be isoform-restricted, leading to phosphate incorporation in the C termini of filamin A and C, but not B. PKC-dependent phosphorylation of filamin was also detected in cells. Our data suggest an intimate interaction between filamin and PKC in cell signaling.