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

Abstract

Integrin ␣ IIb ␤ 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 ␣ IIb ␤ 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, pp125FAK phosphorylation and association with the cytoskeleton was determined in cells expressing two mutant forms of ␣ IIb ␤ 3 : ␣ IIb ␤ 3 (D723A/ E726A), a constitutively active integrin in which the putative binding site for pp125FAK is altered, and ␣ IIb ␤ 3 (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 ␣ IIb ␤ 3 (D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing ␣ IIb ␤ 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 ␣ IIb ␤ 3 (D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing ␣ IIb ␤ 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.The cytoplasmic domains of integrins constitute an essential link between the extracellular, ligand-binding domain of the receptor and both signaling and structural mechanisms inside the cell. Ranging in length from 15 amino acids for the ␣ 1 subunit to over 1000 amino acids for the ␤ 4 subunit, the cytoplasmic domains extend inward from the plasma membrane toward various cytosolic components known to be involved in integrin-mediated signaling such as heterotrimeric G proteins, proteins involved in phospholipid metabolism, serine-threonine kinases, tyrosine kinases, calcium transport systems, and cytoskeleton components (1-7). Although structurally distinct, the different ␣ and ␤ cytoplasmic domains show areas of regional homology, suggesting a conserved tertiary structure. The nine known ␤ subunits contain a highly conserved membrane-proximal polar region with the consensus sequence HDRREFAKFEKEK, which is separated by a stretch of nine amino acids from a central NPXY motif and a stretch of 16 -25 amino acids from a distal NPXY motif. The NPXY sequence prescribes a signal for clathrin-coated pit-mediated internalization of integral membrane proteins (8) and is a recognition site for Shc, an SH2-containing adaptor protein (9). The spacing of the tyrosine residues in the ␣ 1 , ␤ 3 , and ␤ 6 cytoplasmic tails conforms to the spacing of tyrosine residues in immu...

Dynamic Modulation of Cytoskeletal Proteins Linking Integrins to Signaling Complexes in Spreading Cells

“…Thus, although talin may be able to link ␤ 3 -integrin to the cytoskeleton in focal adhesions (in which the integrin is intact), it would not be able to link the calpain-cleaved integrin to the cytoskeleton in the newly identified integrin clusters. In contrast, a putative binding site for ␣-actinin has been identified in the membrane proximal region (34,37) that would remain in calpain-cleaved integrin (Fig. 1).…”

Integrin-independent Tyrosine Phosphorylation of p125fak in Human Platelets Stimulated by Collagen

“…FAK phosphorylation in platelets has been dissociated from both ␣ IIb ␤ 3 occupancy and focal adhesion formation centering on ␣ IIb ␤ 3 in the absence of fibrinogen binding (49,50).…”

Expression and Function of Calcium Binding Domain Chimeras of the Integrins αIIb and α5

“…Cell Culture and Transfections-Chinese hamster ovary K1 (CHO) cells were maintained and transfected as described previously (50). COS-7 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin.…”

“…Flow Cytometry-Flow cytometry was performed as described previously (50) except that for PAC1 studies, cells were incubated with a 1:100 dilution of LIBS 6 ascites with or without 1 mM GRGDSP peptide for 15 min at 37°C. Cells were washed and stained with FITC-conjugated rabbit anti-mouse IgM.…”

“…Immunoprecipitation-Surface labeling and immunoprecipitation were performed essentially as described previously (50).…”