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 the 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.
Weekes
J
J
Department of Biochemistry, University of Leicester, U.K.
Barry
S T
ST
Critchley
D R
DR
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000096:biosynthesis; D007527:Isoenzymes / Q000494:pharmacology; D007700:Kinetics; D051419:Paxillin; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D016596:Vinculin / Q000737:chemistry* / Q000378:metabolism*
D000199:Actins; D003598:Cytoskeletal Proteins; D007527:Isoenzymes; D051419:Paxillin; D010446:Peptide Fragments; D010743:Phospholipids; D010750:Phosphoproteins; C496063:Pxn protein, rat; D011993:Recombinant Fusion Proteins; D016596:Vinculin; D005982:Glutathione Transferase; D011493:Protein Kinase C