Growth factor-dependent phosphorylation of membrane proteins in cultured human retinal pigment epithelial cells.
Recently, growth factors are known to phosphorylate tyrosine residues of proteins to regulate cellular functions. We investigated growth factor-dependent phosphorylation of membrane proteins in cultured human retinal pigment epithelial (RPE) cells. The phosphorylation experiments were done in membrane preparations of cultured RPE cell and the reaction was started by applying [32P]adenosine 5'-triphosphate (ATP) at 0 degrees C, and terminated after 0, 1, 5, 15, and 30 min. The samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were analyzed by autoradiography. Many proteins showed time-dependent phosphorylation. Among them, a 170 kDa protein showed platelet-derived growth factor (PDGF)-specific phosphorylation with both time- (up to 30 min) and dose-(maximal effect at 50 ng/ml) dependence. On the other hand, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) showed no specific phosphorylation. Phosphoaminoacids of the 170 kDa protein were analyzed by thin layer chromatography and autoradiography. Phosphotyrosine showed much higher radioactivity than phosphoserine or phosphothreonine. Consequently, PDGF induced phosphorylation of the 170 kDa protein which mainly consisted of phosphotyrosine. The data suggest that tyrosine phosphorylation of membrane protein is involved in signal transduction of PDGF in human RPE cells.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001345:Autoradiography; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005136:Eye Proteins / Q000378:metabolism; D005333:Fetus; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D005136:Eye Proteins; D008565:Membrane Proteins; D010982:Platelet-Derived Growth Factor; D016222:Fibroblast Growth Factor 2; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate