Integrin signaling: Roles for the cytoplasmic tails of αIIbβ3 in the tyrosine phosphorylation of pp125FAK

Abstract

pp125FAK (focal adhesion kinase) a protein tyrosine kinase that may mediate cellular responses to adhesion, is activated and tyrosine-phosphorylated when platelets adhere to fibrinogen via the integrin, αIIbβ3. To determine whether either of the cytoplasmic tails of αIIbβ3 regulates FAK phosphorylation, CHO cells were stably transfected with αIIbβ3 or various cytoplasmic tail truncation mutants. Cells expressing wild-type αIIbβ3 or αIIbβ3 that lacked the COOH-terminal 13 or 18 residues of the 20 residue αIIb tail adhered to and spread on fibrinogen or on an anti-αIIb antibody, and FAK became tyrosine-phosphorylated. FAK also became phosphorylated in adherent cells lacking the COOH-terminal 35 or 39 residues of the 47 residue β3 tail, although the extent of phosphorylation was reduced by about 50% in the latter mutant. Little or no FAK phosphorylation was observed if 46 residues were deleted from the β3 tail. None of these β3 truncation mutants spread on the anti-αIIb antibody. When cells with wild-type αIIbβ3 or truncated β3 were detached from a surface, FAK became rapidly dephosphorylated. In contrast, FAK remained phosphorylated in the two αIIb truncation mutants for up to 90 minutes in suspension. This persistent phosphorylation was not due to occupancy of αIIbβ3 by adhesive ligands because it was also observed with an αIIb tail truncation mutant that contained an additional mutation in the extracellular portion of the receptor that prevents ligand binding. These studies demonstrate that: (1) the β3 cytoplasmic tail, including the membrane-proximal portion, is involved in initiation of FAK phosphorylation; (2) FAK phosphorylation can be initiated by cell adhesion in the absence of cell spreading; and (3) the membrane-distal portion of the αIIb cytoplasmic tail may normally function to dampen FAK phosphorylation in non-anchored cells.