The Phosphorylation of Vinculin on Tyrosine Residues 100 and 1065, Mediated by Src Kinases, Affects Cell Spreading

Abstract

Vinculin is a conserved actin binding protein localized in focal adhesions and cell-cell junctions. Here, we report that vinculin is tyrosine phosphorylated in platelets spread on fibrinogen and that the phosphorylation is Src kinases dependent. The phosphorylation of vinculin on tyrosine was reconstituted in vanadate treated COS-7 cells coexpressing c-Src. The tyrosine phosphorylation sites in vinculin were mapped to residues 100 and 1065. A phosphorylation-specific antibody directed against tyrosine residue 1065 reacted with phosphorylated platelet vinculin but failed to react with vinculin from unstimulated platelet lysates. Tyrosine residue 1065 located in the vinculin tail domain was phosphorylated by c-Src in vitro. When phosphorylated, the vinculin tail exhibited significantly less binding to the vinculin head domain than the unphosphorylated tail. In contrast, the phosphorylation did not affect the binding of vinculin to actin in vitro. A double vinculin mutant protein Y100F/Y1065F localized to focal adhesion plaques. Wild-type vinculin and single tyrosine phosphorylation mutant proteins Y100F and Y1065F were significantly more effective at rescuing the spreading defect of vinculin null cells than the double mutant Y100F/Y1065F. The phosphorylation of vinculin by Src kinases may be one mechanism by which these kinases regulate actin filament assembly and cell spreading.

Structural Determinants of the Mechanical Stability of α-Catenin

“…Vinculin activity is controlled by a high affinity interaction between the head and tail domains (34). Its activation and recruitment to force-activated talin (and likely to ␣-catenin) involve phosphorylation and allosteric interactions with cytosolic binding partners (34,61,63,64). Conversely, recent experimental data demonstrated that force directly activates ␣-catenin (30,42), and the present simulations suggest that this occurs by a process involving salt-bridge disruption within the M region.…”

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

“…Previousinvestigatorshavesuggestedthatvinculinphosphorylation at Tyr 1065 may contribute to the regulation of its conformation (37,48). Although vinculin has long been recognized as a tyrosine phosphorylation substrate (49), Zhang et al (37) were the first to map one of the phosphorylation sites to Tyr 1065 near the C terminus of the molecule within the tail domain and to demonstrate that phosphorylation at this site, as well as at Tyr 100 , affects cell spreading in activated platelets.…”

“…Although vinculin has long been recognized as a tyrosine phosphorylation substrate (49), Zhang et al (37) were the first to map one of the phosphorylation sites to Tyr 1065 near the C terminus of the molecule within the tail domain and to demonstrate that phosphorylation at this site, as well as at Tyr 100 , affects cell spreading in activated platelets. They also used pull-down assays to analyze the effect of Tyr 1065 phosphorylation on the affinity of vinculin tail domain fragments for the head domain and found that the phosphorylation markedly reduced the affinity of the tail domain for the head domain.…”

“…In vitro studies as well as in vivo studies in several cell types have described both tyrosine and serine phosphorylation sites on the vinculin molecule (37,38). There is evidence that phosphorylation at the Tyr 1065 site near the C terminus of the tail domain affects cell traction and spreading and the dynamics of the incorporation of vinculin into focal adhesions (37,39,40).…”

“…In vitro studies as well as in vivo studies in several cell types have described both tyrosine and serine phosphorylation sites on the vinculin molecule (37,38). There is evidence that phosphorylation at the Tyr 1065 site near the C terminus of the tail domain affects cell traction and spreading and the dynamics of the incorporation of vinculin into focal adhesions (37,39,40). We previously found that vinculin undergoes phosphorylation at tyrosine 1065 concurrently with force development during the stimulation of tracheal smooth muscle tissues with acetylcholine and that the phosphorylation of vinculin at this site remains elevated for the duration of the contraction (10).…”