Integrin-mediated Activation of Focal Adhesion Kinase Is Independent of Focal Adhesion Formation or Integrin Activation

Abstract

Integrin α_IIbβ₃functions as the fibrinogen receptor on platelets and mediates platelet aggregation and clot retraction. Among the events that occur during either “inside-out” or “outside-in” signaling through α_IIbβ₃ is the phosphorylation of focal adhesion kinase (pp125^FAK) and the association of pp125^FAK with cytoskeletal components. To examine the role of pp125^FAK in these integrin-mediated events, pp125^FAK phosphorylation and association with the cytoskeleton was determined in cells expressing two mutant forms of α_IIbβ₃: α_IIbβ₃(D723A/E726A), a constitutively active integrin in which the putative binding site for pp125^FAK is altered, and α_IIbβ₃(F727A/K729E/F730A), in which the putative binding site for α-actinin is altered. Both mutants were expressed on the cell surface and were able to bind ligand, either spontaneously or upon activation. Whereas cells expressing α_IIbβ₃(D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing α_IIbβ₃(F727A/K729E/F730A) adhere to fibrinogen, but had reduced focal adhesions and stress fibers. pp125^FAK is recruited to focal adhesions in adherent cells expressing α_IIbβ₃(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing α_IIbβ₃(F727A/K729E/F730A), pp125^FAK was phosphorylated despite reduced formation of focal adhesions and stress fibers. We conclude that activation of pp125^FAK can be dissociated from two important events in integrin signaling, the assembly of focal adhesions in adherent cells and integrin activation following ligand occupation.