ACIDIC PHOSPHOLIPIDS INHIBIT THE INTRAMOLECULAR ASSOCIATION BETWEEN THE N-TERMINAL AND C-TERMINAL REGIONS OF VINCULIN, EXPOSING ACTIN-BINDING AND PROTEIN-KINASE-C PHOSPHORYLATION SITES

Chick vinculin polypeptides expressed in Escherichia coli as glutathio ne 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 mo lecule. Fusion proteins spanning vinculin residues 1-258 and 1-398, im mobilized on glutathione-agarose beads, were shown to bind a C-termina l vinculin polypeptide spanning residues 881-1066 (liberated from GST by thrombin cleavage). However, the C-terminal polypeptide did not bin d 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 hea d polypeptide was shown to bind to residues 1029-1036 in the tail regi on of vinculin. The association of head and tail was inhibited by acid ic, but not neutral, phospholipids. Pre-incubation of vinculin with ac idic phospholipids exposed the binding site for F-actin and a phosphor ylation site for protein kinase C. The phosphorylation site was locate d in the tail region of the vinculin molecule. These results raise the possibility that acidic phospholipids play a role in regulating the a ctivity of vinculin and therefore the assembly of both cell-cell and c ell-matrix adherens-type junctions.