Bradykinin-induced tyrosine phosphorylation of proteins in cultured human keratinocytes.
The stimulating effect of bradykinin on phosphorylation of proteins at tyrosine residues was visualized on human keratinocytes in primary culture. Keratinocytes were subjected either to short-time (30 s) or to long-time stimulation (4 h) with 200 nM bradykinin. Especially keratinocytes of the G1 phase showed bright immunofluorescence with monoclonal anti-phosphotyrosine antibody. Solubilized membrane proteins were fractionated by gel filtration and tested for tyrosine phosphorylation by ELISA. Short-time stimulation induced a broad peak with a shoulder at 90 kDa, the main peak at about 60 kDa and a second shoulder at 44 kDa. After long-time stimulation an additional distinct peak at 180 kDa appeared, phosphorylation at 90, 60 and 44 kDa was less pronounced. Tyrosine phosphorylated proteins were further characterized by SDS-polyacrylamide gel electrophoresis, Western blotting and detection by monoclonal anti-phosphotyrosine antibody. After short-time stimulation with bradykinin tyrosine phosphorylation was confined to distinct bands at 82, 76, 70, 57, 54, 48, 40 and 39 kDa and a diffuse band at 62 kDa. After long-time stimulation tyrosine phosphorylation increased for the 76 kDa band and the bands at 48 and 40 kDa became more diffuse, the 39 kDa band remained and the others disappeared. Among these proteins, MAP kinase, actin, paxillin and the EGF receptor were the most likely candidates for bradykinin-induced tyrosine phosphorylation. Therefore, these effects in keratinocytes might be associated with events related to mitosis, adhesion and variation in cell shape.
Schremmer-Danninger
E
E
Kinderklinik im Dr von Haunerschen Kinderspital, Abteilung für Klinische Biochemie, München, Germany. E.Schremmer@kk-i.med.uni-muenchen.de
Toepfer-Petersen
E
E
Fritz
H
H
Roscher
A A
AA
D000911:Antibodies, Monoclonal; D001920:Bradykinin / Q000494:pharmacology*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000276:immunology; D011506:Proteins; D014443:Tyrosine / Q000187:drug effects*
D000911:Antibodies, Monoclonal; D011506:Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D001920:Bradykinin