T cell adhesion to P-selectin induces tyrosine phosphorylation of pp125 focal adhesion kinase and other substrates.
Lymphocyte binding to endothelial surface adhesion molecules is an important early step in inflammation, which is mediated initially by P-selectin and E-selectin. We tested the hypothesis that lymphocyte binding to the selectin adhesion molecules induces intracellular signaling by tyrosine phosphorylation. We used an adhesion assay, which relied on cell binding to chimeric proteins consisting of the extracellular domains for P-selectin and E-selectin. Tyrosine phosphorylation was determined using anti-phosphotyrosine Abs by confocal microscopy and Western blot. Binding to P-selectin induced a significant increase in anti-phosphotyrosine immunoreactivity. The P-selectin effect was time dependent with an early response after 10 min and a maximum effect at 30 min. Western blot showed a time-dependent phosphorylation of two distinct 68- and 125-kDa proteins. These proteins were pp125 focal adhesion kinase (FAK) and paxillin, as shown by immunoprecipitation and colocalization. Phosphorylation of pp125 FAK was time dependent reaching a maximum after 30 min. Incubation with the tyrosine kinase inhibitor genistein, and, to a lesser extent, with the protein kinase C inhibitor staurosporine, resulted in decreased pp125 FAK phosphorylation. Our results are the first to demonstrate that lymphocyte binding to P-selectin induces tyrosine phosphorylation of distinct proteins. Thus, lymphocyte activation may occur already at the initial contact with surface adhesion molecules.
Haller
H
H
Franz Volhard Clinic at the Max Delbrück Center for Molecular Medicine, Humboldt University of Berlin, Germany.
Kunzendorf
U
U
Sacherer
K
K
Lindschau
C
C
Walz
G
G
Distler
A
A
Luft
F C
FC
D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D019040:E-Selectin / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D051416:Focal Adhesion Protein-Tyrosine Kinases; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism*; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins; D000080506:Sialyl Lewis X Antigen; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D019040:E-Selectin; D009844:Oligosaccharides; D019007:P-Selectin; D051419:Paxillin; D010750:Phosphoproteins; D011994:Recombinant Proteins; D000080506:Sialyl Lewis X Antigen; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases