Integrin-dependent tyrosine phosphorylation in corneal fibroblasts.
OBJECTIVE
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 tyrosine phosphorylation in integrin-dependent cell adhesion to extracellular matrix.


METHODS
Antibodies were used to detect phosphotyrosine-containing proteins, including focal adhesion kinase in lysates and immunoprecipitates of corneal fibroblasts. The authors used anti-phosphotyrosine antibodies to localize phosphotyrosines in fixed cultured corneal fibroblasts. Similarly, immunocytochemical detection of vinculin was used to identify focal adhesions, the subcellular structures in which integrins organize attachment to matrix extracellularly and to cytoskeletal components intracellularly.


RESULTS
Suspension of corneal fibroblasts produced a dramatic decrease in detectable phosphotyrosines. During integrin-dependent fibroblast attachment to exogenously supplied 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 treatment 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 adhesion formation was prevented by herbimycin A, an inhibitor of tyrosine kinases.


CONCLUSIONS
In corneal fibroblasts, fibronectin receptor-specific signal transduction from extracellular matrix during the formation of focal adhesions requires tyrosine kinase activation, including phosphorylation of FAK. This underscores a role for the fibronectin receptor in signaling from the extracellular matrix in corneal fibroblasts.
Masur
S K
SK
Department of Ophthalmology, Mount Sinai School of Medicine, City University of New York, NY 10029-6574, USA.
Idris
A
A
Michelson
K
K
Antohi
S
S
Zhu
L X
LX
Weissberg
J
J
D000818:Animals; D002448:Cell Adhesion; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D016023:Integrins / Q000502:physiology*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011817:Rabbits; D017469:Receptors, Fibronectin / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism*
D005353:Fibronectins; D005456:Fluorescent Dyes; D016023:Integrins; D017469:Receptors, Fibronectin; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases