Volume 272, Number 36, Issue of September 5, 1997 pp. 22538-22547
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Integrin-mediated Activation of Focal Adhesion Kinase Is Independent of Focal Adhesion Formation or Integrin Activation
STUDIES WITH ACTIVATED AND INHIBITORY ₃ CYTOPLASMIC DOMAIN MUTANTS

(Received for publication, April 4, 1997, and in revised form, June 6, 1997)

Suzanne Lyman ^§ , Andrew Gilmore ^¶ , Keith Burridge ^¶ , Susan Gidwitz and Gilbert C. White II^§

From the Departments of  Medicine, ^§ Pharmacology, and ^¶ Cell Biology and Anatomy,  The Center for Thrombosis and Hemostasis, University of North Carolina, Chapel Hill, North Carolina 27599

Integrin _IIb3 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 _IIb3 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 _IIb3: _IIb3(D723A/E726A), a constitutively active integrin in which the putative binding site for pp125^FAK is altered, and _IIb3(F727A/K729E/F730A), in which the putative binding site for -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 _IIb3(D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing _IIb3(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 _IIb3(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing _IIb3(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.

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