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

Abstract

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.

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

“…Therefore, we tested in vitro PKC␣ binding to FLN domain constructs after preincubation with phospholipid vesicles, using a protocol that has been applied previously in our laboratory to analyze PKC binding to vinculin (44). To map filamin binding sites in dot overlay assays, deletion constructs of the N and C terminus were generated from human FLNa cDNA by PCR and cloned into expression vectors.…”

“…Characterization of PKC Binding Properties-PKC ligand binding can involve a complex interplay of protein-protein and protein-lipid interactions, as exemplified by its ligands syndecan 4 (45) or vinculin (44). To identify the main characteristics of the interaction with filamin, domain constructs of the lipid and Ca 2ϩ -binding PKC regulatory domain (rd), the variable hinge region (V3), that is involved in intercellular targeting (46), and ligand binding (9), and the kinase domain (kd) were employed in overlay assays.…”

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

“…In vitro studies as well as in vivo studies in several cell types have described both tyrosine and serine phosphorylation sites on the vinculin molecule (37,38). There is evidence that phosphorylation at the Tyr 1065 site near the C terminus of the tail domain affects cell traction and spreading and the dynamics of the incorporation of vinculin into focal adhesions (37,39,40).…”

A novel 2D‐based approach to the discovery of candidate substrates for the metalloendopeptidase meprin

“…It thus serves a regulatory, dynamic linkage between the ECM and intracellular actin cytoskeleton. It was demonstrated that acidic phospholipids inhibit intramolecular association between the N-and C-terminal regions of vinculin, exposing actin-binding and protein kinase C phosphorylation sites on serines 1033 and 1045 [34,35]. Upon activation of hmeprina ⁄ b in stably transfected MDCK cells, the 116 kDa full-length form of vinculin and truncated forms (75 and 85 kDa) were detected in cell culture supernatants (Fig.…”

“…Those half-cleaved products are most probably related to in-gel digestion artefacts because cleavage within this protein sequence stretch by meprin must be excluded due to an overall amino acid sequence coverage of this protein that exceeded amino acid 182. In addition, the two half-cleaved peptides DQAVSDTELQEMSTEGSK (residues [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] and DTELQEMSTEGSK (residues 28-40) in SSP 502 and 1502 were chromatographically separated and were not in-source fragmentation products generated during the ionization process. The former peptide represented the mature N-terminus of clusterin (aspartate at position 23) and hence was not generated by meprin activity.…”