Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells.
Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation at 120-130 kDa when compared with extracts of intact monolayers. The pp125FAK immunoprecipitated from wounded monolayers exhibited increased kinase activity as compared to pp125FAK from intact monolayers. The time to wound closure in HUVEC monolayers was doubled by tyrphostin AG 213 treatment. The same concentration of AG 213 interfered with HUVEC focal adhesion and stress fiber formation. AG 213 inhibited adhesion-associated tyrosine phosphorylation of pp125FAK in HUVEC. Tyrphostins AG 213 and AG 808 inhibited pp125FAK activity in in vitro kinase assays. pp125FAK immunoprecipitates from HUVEC treated with both of these inhibitors also had kinase activity in vitro that was below levels seen in untreated HUVEC. These findings suggest that tyrosine phosphorylation of cytoskeletal proteins may be important in HUVEC spreading and migration and that pp125FAK may mediate phosphotyrosine formation during these processes.
Romer
L H
LH
Department of Pediatrics, University of North Carolina at Chapel Hill 27599.
McLean
N
N
Turner
C E
CE
Burridge
K
K
D002396:Catechols / Q000494:pharmacology; D002448:Cell Adhesion / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure*; D005109:Extracellular Matrix / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020032:Tyrphostins
D002396:Catechols; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D009570:Nitriles; D020032:Tyrphostins; C060152:tyrphostin 47; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human