Further characterization of the interaction between the cytoskeletal proteins talin and vinculin.
The cytoskeletal protein talin, which is thought to couple integrins to F-actin, contains three binding sites (VBS1-VBS3) for vinculin, a protein implicated in the negative regulation of cell motility and whose activity is modulated by an intramolecular interaction between the vinculin head (Vh) and vinculin tail (Vt) domains. In the present study we show that recombinant talin polypeptides containing the three VBSs (VBS1, residues 498-636; VBS2, residues 727-965; and VBS3, residues 1943-2157) each bind tightly to the same or overlapping sites within vinculin(1-258). A short synthetic talin VBS3 peptide (residues 1944-1969) was sufficient to inhibit binding of a (125)I-labelled talin VBS3 polypeptide to vinculin(1-258), and NMR spectroscopy confirmed that this peptide forms a 1:1 complex in slow exchange with vinculin(1-258). Binding of the (125)I-labelled VBS3 polypeptide was markedly temperature dependent, but was not inhibited by 1 M salt or 10% (v/v) 2-methyl-2-propanol. Attempts to further define the talin-binding site within vinculin(1-258) using a gel-blot assay were unsuccessful, but near maximal talin-binding activity was retained by a construct spanning vinculin residues 1-131 in a yeast two-hybrid assay. Interestingly, the talin VBS3 polypeptide was a potent inhibitor of the Vh-Vt interaction, and the VBS3 synthetic peptide was able to expose the actin-binding site in intact vinculin, which is otherwise masked by the Vh-Vt interaction. The results suggest that under certain conditions, talin may be an effective activator of vinculin.
Bass
Mark D
MD
Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, U.K.
Patel
Bipin
B
Barsukov
Igor G
IG
Fillingham
Ian J
IJ
Mason
Robert
R
Smith
Beverley J
BJ
Bagshaw
Clive R
CR
Critchley
David R
DR
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D002645:Chickens; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016608:Talin / Q000737:chemistry* / Q000378:metabolism*; D014422:Turkeys; D016596:Vinculin / Q000737:chemistry* / Q000378:metabolism*
D000199:Actins; D003598:Cytoskeletal Proteins; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016608:Talin; D016596:Vinculin