title: Injury of stromal fibroblasts induces phosphorylation of focal adhesion proteins.
abstract: OBJECTIVE
  The extracellular matrix serves as a structural support for the corneal stroma and mediates signaling events that regulate the intracellular environment of stromal keratocytes. We hypothesize that adhesion and injury mediate signal transduction events causing the phosphorylation of tyrosine residues of specific adhesion proteins and that phosphorylation is required for cellular adhesion and migration.
  
  
  METHODS
  For the adhesion experiments; primary rabbit stromal fibroblasts were seeded and phosphorylation of tyrosine residues was followed from 1 min to 24 h. For the injury experiments, confluent primary cultures were rendered quiescent, wounded, and tyrosine phosphorylation was followed from 30 s to 6 h. The antibody (py-20) was used to detect proteins phosphorylated on tyrosine residues. We examined changes in the phosphorylation of focal adhesion kinase (FAK), paxillin and cortactin, using immunoprecipitation and Western blot analysis.
  
  
  RESULTS
  In the adhesion experiments, the phosphorylation of a 68-kDa protein was detected after 1 min, and the phosphorylation of a 125-kDa protein was not detected until 15 min. These proteins were identified in re-probed blots as paxillin and FAK. In the injury experiments, FAK phosphorylation was detected within 30 s and remained elevated for 6 h when cells were cultured on fibronectin. Both FAK and paxillin phosphorylation were prominent after injury, but unlike FAK phosphorylation, paxillin phosphorylation decreased over time. Phosphorylation was prominent at the wound margin. After wound closure, it returned to background levels. Tyrosine kinase inhibitors, genistein and herbimycin, decreased the number of adherent cells and altered the rate of cell migration after injury, compared to control (DMSO alone).
  
  
  CONCLUSIONS
  The results indicate that injury and cell-matrix interaction mediate the phosphorylation of specific adhesion proteins and that phosphorylation is required for wound repair.
authors.affiliation: Department of Ophthalmology and Biochemistry, Boston University School of Medicine, MA 02118, USA.
authors.forename: F
authors.initials: F
authors.lastname: Haq
authors.forename: V
authors.initials: V
authors.lastname: Trinkaus-Randall
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology / Q000293:injuries*; D051356:Cortactin; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D051416:Focal Adhesion Protein-Tyrosine Kinases; D008840:Microfilament Proteins / Q000378:metabolism*; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D014443:Tyrosine / Q000378:metabolism; D014945:Wound Healing
chemical_list: D015815:Cell Adhesion Molecules; D051356:Cortactin; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D051419:Paxillin; D010750:Phosphoproteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases