Integrin-mediated signal transduction in human endothelial cells: analysis of tyrosine phosphorylation events.
Adhesion of human umbilical endothelial cells to fibronectin resulted in increased tyrosine phosphorylation of a group of proteins with molecular mass ranging from 100 to 130 kDa and of a 70 kDa protein. This pattern of tyrosine phosphorylation was also observed when endothelial cells adhered to vitronectin, collagen IV, collagen I and laminin or to culture dishes coated with antibodies directed to either beta 1, alpha 3, alpha 5, alpha 6 or beta 3 integrin subunits. Increased phosphorylation of the 100-130 kDa proteins was detectable as early as 30 sec after adhesion, reached maximal level after 15 min, and remained high as long as the cells adhere to culture dishes. The 70 kDa protein was phosphorylated with a slower kinetics and its phosphorylation increased over a period of 3 h. Using specific monoclonal antibodies, the major component of the 100-130 kDa complex was identified as the focal adhesion tyrosine kinase p125FAK. The phosphorylation of the p125FAK was also observed by inducing beta 1 integrin clustering in non adherent HEC, indicating that this is a primary signalling event induced by integrins. Using tyrosine kinase inhibitors, we show a direct correlation between integrin-stimulated tyrosine kinases and assembly of focal adhesions and actin fibres.
Defilippi
P
P
Departimento di Genetica, Biologia e Chimica Medica, University of Torino, Italy.
Bozzo
C
C
Volpe
G
G
Romano
G
G
Venturino
M
M
Silengo
L
L
Tarone
G
G
D000906:Antibodies / Q000494:pharmacology; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D003094:Collagen; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D016326:Extracellular Matrix Proteins; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006023:Glycoproteins; D006801:Humans; D016023:Integrins / Q000276:immunology / Q000502:physiology*; D007797:Laminin; D046911:Macromolecular Substances; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*; D014471:Umbilical Veins; D019096:Vitronectin
D000906:Antibodies; D015815:Cell Adhesion Molecules; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D016023:Integrins; D007797:Laminin; D046911:Macromolecular Substances; D010750:Phosphoproteins; D019096:Vitronectin; D019000:Phosphotyrosine; D014443:Tyrosine; D003094:Collagen; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human