The phosphorylation of vinculin on tyrosine residues 100 and 1065, mediated by SRC kinases, affects cell spreading.
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.
Zhang
Zhiyong
Z
Department of Surgery and the Cancer Institute of New Jersey, Robert Wood Johnson Medical School-University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08903, USA.
Izaguirre
Gonzalo
G
Lin
Siang-Yo
SY
Lee
Hwa Young
HY
Schaefer
Erik
E
Haimovich
Beatrice
B
D000818:Animals; D030161:Avian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001792:Blood Platelets / Q000378:metabolism; D019556:COS Cells; D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D002999:Clone Cells; D022001:Focal Adhesions; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D010974:Platelet Aggregation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry*; D016596:Vinculin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
D030161:Avian Proteins; D011994:Recombinant Proteins; C035689:VCL protein, Gallus gallus; C488955:VCL protein, human; D016596:Vinculin; D014443:Tyrosine; D019061:src-Family Kinases