Lh. Romer et al., TYROSINE KINASE-ACTIVITY, CYTOSKELETAL ORGANIZATION AND MOTILITY IN HUMAN VASCULAR ENDOTHELIAL-CELLS, Molecular biology of the cell, 5(3), 1994, pp. 349-361
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.