Isolation and characterization of a vinculin cDNA from chick-embryo fibroblasts

Abstract

A chick-embryo fibroblast lambda gt11 cDNA library was screened with affinity-purified antibodies to chick gizzard vinculin. One recombinant was purified to homogeneity and the fusion protein expressed in Escherichia coli strain C600. The fusion protein was unstable, but polypeptides that reacted with vinculin antibodies, but not non-immune immunoglobulin, were detected by Western blotting. The recombinant contained a single EcoRI fragment of 2891 bp with a single open reading frame. The deduced protein sequence could be aligned with that of six CNBr-cleavage peptides and two tryptic peptides derived from chicken gizzard vinculin. AUG-247 has tentatively been identified as the initiation codon, as it is contained within the consensus sequence for initiation sites of higher eukaryotes. The cDNA lacks 3' sequence and encodes 74% of the vinculin sequence, presuming the molecular mass of vinculin to be 130,000 Da. Analysis of the deduced sequence showed no homologies with other protein sequences, but it does display a triple internal repeat of 112 amino acid residues covering residues 259-589. The sequences surrounding the seven tyrosine residues in the available sequence were aligned with the tyrosine autophosphorylation consensus sequence found in protein tyrosine kinases. Tyr-822 showed a good match to this consensus, and may represent one of the two major sites of tyrosine phosphorylation by pp60v-sre. Northern blots showed that the 2.89 kb vinculin cDNA hybridized to one size of mRNA (approx. 7 kb) in chick-embryo fibroblasts, chick smooth muscle and chick skeletal muscle. Southern blots revealed multiple hybridizing bands in genomic DNA.

Identification of a talin binding site in the cytoskeletal protein vinculin.

“…We have previously analyzed a 2.89-kb chick vinculin eDNA which contained 246 bp of 5' untranslated sequence, the initiation codon, and 83% of the total coding sequence, but lacked 3' coding and noncoding sequence (Price et al, 1987). Comparison of the restriction map of this clone with that of a 5-kb chick vinculin eDNA isolated by Bendori et al (1987) showed the latter to contain the additional 3' sequence missing from the 2.89-kb eDNA (Fig.…”

“…An analysis of the complete sequence of chick vinculin (Price et al, 1987(Price et al, , 1989Coutu and Craig, 1988) shows that the molecule contains 1,066 amino acids, with a deduced molecular mass of'x,117 kD. The most notable feature of the molecule is that it contains a proline-rich region spanning residues 837-879, with two VS-proteinase cleavage sites (Price et al, 1989).…”

“…The two chick vinculin cDNAs used in this study (referred to as the 2.89-kb eDNA and cVinS) were both isolated from a chick embryo fibroblast Lgtll eDNA I~rary, and have been described elsewhere (Price et al, 1987(Price et al, , 1989Bendori et al, 1987).…”

“…A restriction map of two vineulin cDNAs: (a) a 2.89-kb eDNA (Price et al, 1987); (b) a 5-kb eDNA referred to as eVin5 (Bendori et al, 1987). Each eDNA is contained on a single Eco RI fragment.…”

Complete sequence of human vinculin and assignment of the gene to chromosome 10.

“…3). To obtain a more complete understanding of the role of vinculin in adherens junctions, the sequence of chicken embryo vinculin has been determined (14)(15)(16). The sequence of nematode Caenorhabditis elegans vinculin also has been published (17).…”