TYROSINE KINASE-ACTIVITY, CYTOSKELETAL ORGANIZATION AND MOTILITY IN HUMAN VASCULAR ENDOTHELIAL-CELLS

Tyrosine phosphorylation of cytoskeletal proteins occurs during integr in-mediated cell adhesion to extracellular matrix proteins. We have in vestigated the role of tyrosine phosphorylation in the migration and i nitial spreading of human umbilical vein endothelial cells (HUVEC). El evated phosphotyrosine concentrations were noted in the focal adhesion s of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots o f extracts of wounded HUVEC monolayers demonstrated increased phosphor ylation at 120-130 kDa when compared with extracts of intact monolayer s. The pp125(FAK) immunoprecipitated from wounded monolayers exhibited increased kinase activity as compared to pp125(FAK) from intact monol ayers. The time to wound closure in HUVEC monolayers was doubled by ty rphostin AG 213 treatment. The same concentration of AG 213 interfered with HUVEC focal adhesion and stress fiber formation. AG 213 inhibite d adhesion-associated tyrosine phosphorylation of pp125(FAK) in HUVEC. Tyrphostins AG 213 and AG 808 inhibited pp125(FAK) activity in in vit ro kinase assays. pp125(FAK) immunoprecipitates from HUVEC treated wit h both of these inhibitors also had kinase activity in vitro that was below levels seen in untreated HUVEC. These findings suggest that tyro sine phosphorylation of cytoskeletal proteins may be important in HUVE C spreading and migration and that pp125(FAK) may mediate phosphotyros ine formation during these processes.