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

Abstract

Integrin alphaIIb beta3 functions as the fibrinogen receptor on platelets and mediates platelet aggregation and clot re- traction. Among the events that occur during either “in- side-out” or “outside-in” signaling through alphaIIb beta3 is the phosphorylation of focal adhesion kinase (pp125FAK) and the association of pp125FAK with cytoskeletal com- ponents. To examine the role of pp125FAK in these inte- grin-mediated events, pp125FAK phosphorylation and as- sociation with the cytoskeleton was determined in cells expressing two mutant forms of alphaIIb beta3: alphaIIb beta3(D723A/ E726A), a constitutively active integrin in which the putative binding site for pp125FAK is altered, and alphaIIb beta3(F727A/K729E/F730A), in which the putative binding site for alpha-actinin is altered. Both mutants were ex- pressed on the cell surface and were able to bind ligand, either spontaneously or upon activation. Whereas cells expressing alphaIIb beta3(D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrino- gen, cells expressing alphaIIb beta3(F727A/K729E/F730A) adhere to fibrinogen, but had reduced focal adhesions and stress fibers. pp125FAK is recruited to focal adhesions in adherent cells expressing alphaIIb beta3(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspen- sion in the presence of fibrinogen. In adherent cells expressing alphaIIb beta3(F727A/K729E/F730A), pp125FAK was phosphorylated despite reduced formation of focal ad- hesions and stress fibers. We conclude that activation of pp125FAK can be dissociated from two important events in integrin signaling, the assembly of focal adhesions in adherent cells and integrin activation following ligand occupation.