Acidic phospholipids inhibit the intramolecular association between the N- and C-terminal regions of vinculin, exposing actin-binding and protein kinase C phosphorylation sites

Chick vinculin polypeptides expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins have been used to identify the sites involved in the intramolecular association between the 90 kDa N-terminal head and the 30 kDa C-terminal tail region of the vinculin molecule. Fusion proteins spanning vinculin residues 1–258 and 1–398, immobilized on glutathione–agarose beads, were shown to bind a C-terminal vinculin polypeptide spanning residues 881–1066 (liberated from GST by thrombin cleavage). However, the C-terminal polypeptide did not bind to a fusion protein spanning residues 399–881 or to itself. Binding was dependent on residues 167–207 within the N-terminal polypeptide, a sequence also essential for talin binding. Conversely, the 90 kDa head polypeptide was shown to bind to residues 1029–1036 in the tail region of vinculin. The association of head and tail was inhibited by acidic, but not neutral, phospholipids. Pre-incubation of vinculin with acidic phospholipids exposed the binding site for F-actin and a phosphorylation site for protein kinase C. The phosphorylation site was located in the tail region of the vinculin molecule. These results raise the possibility that acidic phospholipids play a role in regulating the activity of vinculin and therefore the assembly of both cell–cell and cell–matrix adherens-type junctions.