Intramolecular interactions regulate serine/threonine phosphorylation of vinculin

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During cell spreading, binding of actin-organizing proteins to acidic phospholipids and phosphorylation are important for localization and activity of these proteins at nascent cell-matrix adhesion sites. Here, we report on a transient interaction between the lipid-dependent protein kinase C␣ and vinculin, an early component of these sites, during spreading of HeLa cells on collagen. In vitro binding of protein kinase C␣ to vinculin tail was found dependent on free calcium and acidic phospholipids but independent of a functional kinase domain. The interaction was enhanced by conditions that favor the oligomerization of vinculin. Phosphorylation by protein kinase C␣ reached 1.5 mol of phosphate/mol of vinculin tail and required the C-terminal hydrophobic hairpin, a putative phosphatidylinositol 4,5-bisphosphate-binding site. Mass spectroscopy of peptides derived from in vitro phosphorylated vinculin tail identified phosphorylation of serines 1033 and 1045. Inhibition of C-terminal phospholipid binding at the vinculin tail by mutagenesis or deletion reduced the rate of phosphorylation to <50%. We suggest a possible mechanism whereby phospholipid-regulated conformational changes in vinculin may lead to exposure of a docking site for protein kinase C␣ and subsequent phosphorylation of vinculin and/or vinculin interaction partners, thereby affecting the formation of cell adhesion complexes.

Section: Pkc␣ and Vinculin Form Complexes In Vivo-supporting

confidence: 80%

Section: Pkc␣ and Vinculin Form Complexes In Vivo-mentioning

confidence: 99%

mentioning

confidence: 99%

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A Lipid-regulated Docking Site on Vinculin for Protein Kinase C

Ziegler

Tigges

Zieseniß

et al. 2002

Self Cite

“…Theoretically, there are other ligand combinations that might activate vinculin. Both phosphatidylinositol 4,5-bisphosphate binding to Vt (77) and phosphorylation of Vt (78,79) have been shown to block bimolecular interactions of Vh and Vt. Possibly, these effectors can also cooperate with talin to activate vinculin.…”

Section: ϫ9mentioning

confidence: 99%

Coincidence of Actin Filaments and Talin Is Required to Activate Vinculin

Chen¹,

Choudhury²,

Craig³

2006

132

182

Vinculin regulates cell adhesion by strengthening contacts between extracellular matrix and the cytoskeleton. Binding of the integrin ligand, talin, to the head domain of vinculin and F-actin to its tail domain is a potential mechanism for this function, but vinculin is autoinhibited by intramolecular interactions between its head and tail domain and must be activated to bind talin and actin. Because autoinhibition of vinculin occurs by synergism between two head and tail interfaces, one hypothesis is that activation could occur by two ligands that coordinately disrupt both interfaces. To test this idea we use a fluorescence resonance energy transfer probe that reports directly on activation of vinculin. Neither talin rod, VBS3 (a talin peptide that mimics a postulated activated state of talin), nor F-actin alone can activate vinculin. But in the presence of F-actin either talin rod or VBS3 induces dose-dependent activation of vinculin. The activation data are supported by solution phase binding studies, which show that talin rod or VBS3 fails to bind vinculin, whereas the same two ligands bind tightly to vinculin head domain (K d ϳ 100 nM). These data strongly support a combinatorial mechanism of vinculin activation; moreover, they are inconsistent with a model in which talin or activated talin is sufficient to activate vinculin. Combinatorial activation implies that at cell adhesion sites vinculin is a coincidence detector awaiting simultaneous signals from talin and actin polymerization to unleash its scaffolding activity. Vinculin (V),3 a 116-kDa soluble protein, is recruited to membrane-associated protein complexes that link the actin cytoskeleton to the extracellular matrix or neighboring cells (1). These adhesive sites have an organization visible in the light microscope and include skeletal and cardiac muscle costameres (2-4), cardiocyte intercalated discs, smooth muscle dense plaques, the zonula adherens between epithelial cells (5), myotendinous junctions (4), as well as focal adhesions and focal complexes in lamellipodia of migrating cells in culture (1). Cell adhesion structures consist of three domains; a transmembrane receptor such as an integrin, cadherin, or IgCam, extracellular matrix proteins or cell surface proteins that bind to the extracellular portion of these receptors, and a cytoplasmic plaque containing signaling and cytoskeletal proteins, such as focal adhesion kinase, Src, vinculin, talin, and actin, that assemble on the cytoplasmic domain of the receptors and regulate adhesion site turnover and strength. Cell adhesion is essential for motility in embryogenesis, wound healing, inflammation, and metastasis; therefore, it is important to discover the regulatory mechanisms.Previous work shows that vinculin both strengthens cell adhesions and regulates signaling. Nematodes that lack vinculin fail to develop connections between muscle myofibrils and the sarcolemma, arresting at the 2-fold stage of development with muscles that do not contract (6). Mice heterozygous at the vinculin locus are...

“…Vinculin probably functions by interacting with particular structural and regulatory proteins found at focal contacts and zonulae adherens (11). Biochemical experiments with purified proteins show that the binding sites on vinculin for talin (12), F-actin (13), acidic phospholipids (14), and vasodilator-stimulated phosphoprotein (15), and sites for protein kinase C-mediated phosphorylation (16,17), are blocked by the intramolecular association (K d ϳ 50 nM) of the head (V h ) and tail (V t ) regions (12). Therefore, regulation of the head-tail interaction to expose cryptic ligand binding and regulatory sites is hypothesized to be critical for recruitment of vinculin to sites of cell adhesion and/or for vinculin-dependent assembly of focal adhesion complexes (13).…”

mentioning

confidence: 99%

Polyphosphoinositides Inhibit the Interaction of Vinculin with Actin Filaments

Steimle

Hoffert

Adey

et al. 1999

Binding of vinculin to adhesion plaque proteins is restricted by an intramolecular association of vinculin's head and tail regions. Results of previous work suggest that polyphosphoinositides disrupt this interaction and thereby promote binding of vinculin to both talin and actin. However, data presented here show that phosphatidylinositol 4,5-bisphosphate (PI4,5P 2 ) inhibits the interaction of purified tail domain with F-actin. Upon re-examining the effect of PI4,5P 2 on the actin and talinbinding activities of intact vinculin, we find that when the experimental design controls for the effect of magnesium on aggregation of PI4,5P 2 micelles, polyphosphoinositides promote interactions with the talin-binding domain, but block interactions of the actin-binding domain. In contrast, if vinculin is trapped in an open confirmation by a peptide specific for the talin-binding domain of vinculin, actin binding is allowed. These results demonstrate that activation of the actin-binding activity of vinculin requires steps other than or in addition to the binding of PI4,5P 2 .Vinculin, a 117-kDa component of microfilament-associated cell junctions (1), is a modular protein composed of a 95-kDa N-terminal, globular head domain connected by a short prolinerich region to a 30-kDa tail domain (reviewed in Ref.2). Vinculin has an essential role in embryogenesis (3, 4) and regulatory roles in adhesion, spreading, and motility of cells in culture (5-10). Vinculin probably functions by interacting with particular structural and regulatory proteins found at focal contacts and zonulae adherens (11). Biochemical experiments with purified proteins show that the binding sites on vinculin for talin (12), F-actin (13), acidic phospholipids (14), and vasodilator-stimulated phosphoprotein (15), and sites for protein kinase C-mediated phosphorylation (16,17), are blocked by the intramolecular association (K d ϳ 50 nM) of the head (V h ) and tail (V t ) regions (12). Therefore, regulation of the head-tail interaction to expose cryptic ligand binding and regulatory sites is hypothesized to be critical for recruitment of vinculin to sites of cell adhesion and/or for vinculin-dependent assembly of focal adhesion complexes (13). Elucidation of the factors that regulate the head-tail interaction is central to understanding how events at the cell surface are expressed ultimately in the activities of molecules directly responsible for the functions of focal adhesion plaques.Evidence from in vitro experiments shows that acidic phospholipids in general (16), or specifically PI4,5P 2 1 (18) block the interaction of purified head and tail domains and act on intact vinculin to expose the binding sites for talin on V h (18) and for F-actin on V t (16,18). This observation provides a mechanistic link between cell surface receptors that modulate the synthesis of PI4,5P 2 , a signaling molecule (19) and vinculin, a structural molecule involved in assembling a focal adhesion plaque and mediating anchorage to the actin cytoskeleton.Here we provide new informat...