Characterization of Two F‐Actin‐Binding and Oligornerization Sites in the Cell‐Contact Protein Vinculin

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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.

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

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“…For this assay (Fig. 5B), we used full-length metavinculin and vinculin and probed their binding to actin filaments as an indicator of the open conformation, with the actin binding site in the tail (11,12,20,21) accessible. Cosedimentation with F-actin was performed in the presence of increasing amounts of the talin peptide.…”

Section: Metavinculin and Vinculin Are Both Regulated By Intramoleculmentioning

confidence: 99%

“…It is a multiligand protein, and binding of VH to talin or ␣-catenin targets vinculin to microfilament-associated cell adhesions (7)(8)(9)(10)). 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).…”

mentioning

confidence: 99%

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

Witt

Zieseniß

Fock

et al. 2004

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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.

“…Each discrete vinculin domain recognizes multiple binding partners: the vinculin head (Vh) 4 binds to talin, ␣-actinin, and IpaA (14 -16); the prolinerich linker interacts with vasodilator stimulated phosphoprotein (VASP), CAP/ponsin, vinexin, and the Arp2/3 complex (17)(18)(19)(20); the vinculin tail (Vt) associates with filamentous actin (F-actin), phosphatidylinositol (4,5)-bisphosphate, and paxillin (21)(22)(23). In the autoinhibitory form of vinculin, tight intramolecular interactions between Vh and Vt occlude binding to most of its ligands (13).…”

mentioning

confidence: 99%

The Vinculin C-terminal Hairpin Mediates F-actin Bundle Formation, Focal Adhesion, and Cell Mechanical Properties

Shen

Tolbert

Guilluy

et al. 2011

112

Vinculin is an essential and highly conserved cell adhesion protein, found at both focal adhesions and adherens junctions, where it couples integrins or cadherins to the actin cytoskeleton. Vinculin is involved in controlling cell shape, motility, and cell survival, and has more recently been shown to play a role in force transduction. The tail domain of vinculin (Vt) contains determinants necessary for binding and bundling of actin filaments. Actin binding to Vt has been proposed to induce formation of a Vt dimer that is necessary for cross-linking actin filaments. Results from this study provide additional support for actininduced Vt self-association. Moreover, the actin-induced Vt dimer appears distinct from the dimer formed in the absence of actin. To better characterize the role of the Vt strap and carboxyl terminus (CT) in actin binding, Vt self-association, and actin bundling, we employed smaller amino-terminal (NT) and CT deletions that do not perturb the structural integrity of Vt. Although both NT and CT deletions retain actin binding, removal of the CT hairpin (1061-1066) selectively impairs actin bundling in vitro. Moreover, expression of vinculin lacking the CT hairpin in vinculin knock-out murine embryonic fibroblasts affects the number of focal adhesions formed, cell spreading as well as cellular stiffening in response to mechanical force.The ability of cells to sense and respond to environmental cues such as mechanical forces is critical for multiple cellular processes including embryogenesis and wound healing (1-3). To transmit forces across the cell membrane in both directions, the actin cytoskeleton couples transmembrane receptors (integrin or cadherin), through points of cell adhesion consisting of multiple protein complexes that form cell-extracellular matrix (focal adhesions) and cell-cell (adherens junctions) contacts (3, 4). Vinculin is an abundant protein found in both focal adhesions and adherens junctions, and plays a key role in regulating cell morphology, cell motility, and force transduction (2, 5). Vinculin is essential during development as vinculin knockout (KO) mouse embryos fail to survive beyond day E10 with extensive defects in myocardial and endocardial structures (6). Consistent with a role in muscle structure, vinculin KO mice are predisposed to stress-induced cardiomyopathy (7). Moreover, mutations and deletions in the tail domain of metavinculin, a splice isoform of vinculin, are associated with dilated cardiomyopathy (8 -10). Vinculin also possesses tumor suppressor properties as vinculin KO cells are less adherent, have a rounded morphology, reduced lamellipodial stability, increased motility (6, 11), and are resistant to apoptosis and anoikis (12).Vinculin is a highly conserved cytoskeletal protein that has 1066 residues and contains a globular head, a flexible prolinerich linker, and a tail domain (13). Each discrete vinculin domain recognizes multiple binding partners: the vinculin head (Vh) 4 binds to talin, ␣-actinin, and IpaA (14 -16); the prolinerich linker inte...