INTEGRIN-DEPENDENT TYROSINE PHOSPHORYLATION IN CORNEAL FIBROBLASTS

Purpose. A major pathway for intracellular signaling from cell surface receptors, such as integrins, involves intracellular phosphorylation. In corneal fibroblasts, the authors have investigated the role of tyr osine phosphorylation in integrin-dependent cell adhesion to extracell ular matrix. Methods. Antibodies were used to detect phosphotyrosine-c ontaining proteins, including focal adhesion kinase in lysates and imm unoprecipitates of corneal fibroblasts. The authors used anti-phosphot yrosine antibodies to localize phosphotyrosines in fixed cultured corn eal fibroblasts. Similarly, immunocytochemical detection of vinculin w as used to identify focal adhesions, the subcellular structures in whi ch integrins organize attachment to matrix extracellularly and to cyto skeletal components intracellularly. Results. Suspension of corneal fi broblasts produced a dramatic decrease in detectable phosphotyrosines. During integrin-dependent fibroblast attachment to exogenously suppli ed fibronectin, the cytoplasmic phosphotyrosine kinase, focal adhesion kinase (FAK), pp125FAK, became tyrosine phosphorylated. However, FAK was not phosphorylated during fibroblast attachment to vitronectin or polylysine or when cells were kept in suspension. In addition, the tre atment of suspended cells with antibody to the extracellular domain of fibronectin receptor caused FAK phosphorylation. Phosphotyrosine was colocalized with vinculin in newly formed focal adhesions. Focal adhes ion formation was prevented by herbimycin A, an inhibitor of tyrosine kinases. Conclusions. In corneal fibroblasts, fibronectin receptor-spe cific signal transduction from extracellular matrix during the formati on of focal adhesions requires tyrosine kinase activation, including p hosphorylation of FAK. This underscores a role for the fibronectin rec eptor in signaling from the extracellular matrix in corneal fibroblast s.