Integrin-mediated activation of focal adhesion kinase is independent of focal adhesion formation or integrin activation - Studies with activated and inhibitory beta(3) cytoplasmic domain mutants

Integrin alpha(IIb)beta(3) functions 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 alpha(IIb)beta(3) 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 alpha(IIb)beta(3): alpha(IIb)beta(3)(D723A/E726A), a constitutively active integrin in which the putative binding site for pp125(FAK) in altered, and alpha(IIb)beta(3)(F727A/K729E/F730A), in which the putative binding site for a-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 alpha(IIb)beta(3)(D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing alpha(IIb)beta(3)(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 alpha(IIb)beta(3)(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing alpha(IIb)beta(3)(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.