Use of an antiserum against phosphotyrosine for the identification of phosphorylated components in human fibroblasts stimulated by platelet-derived growth factor.
In search for possible intracellular mediators of the mitogenic signal induced by platelet-derived growth factor (PDGF), we have investigated tyrosine-specific phosphorylation stimulated by PDGF in intact human fibroblasts. Cells were metabolically labeled, either with [32P] orthophosphoric acid or with [35S]methionine, and thereafter treated with PDGF for various times. Lysates from the cell cultures were then immunoprecipitated with an antiserum specifically recognizing phosphotyrosine. Analysis of the precipitated radioactivity by sodium dodecyl sulfate-gel electrophoresis and autoradiography or fluorography showed the appearance of a 185-kDa protein in cells stimulated with PDGF; maximum yield was at about 5 min after the addition of PDGF. This component was found to have several characteristics in common with the PDGF receptor, including similar Mr, binding to immobilized wheat germ agglutinin, and incorporation of phosphate on tyrosine residues after exposure to PDGF. We conclude that the 185-kDa component probably represents the PDGF receptor proper. Phosphoamino acid analysis of the 185-kDa protein/PDGF receptor, precipitated with the antiphosphotyrosine immune serum, revealed that it, in addition to phosphotyrosine, also contained phosphoserine. PDGF also consistently stimulated the phosphorylation of components of Mr values of 300,000 to 200,000, 115,000, 72,000, 54,000, 45,000, and 35,000. Some of these components may be involved in the intracellular transmission of the PDGF-induced mitogenic signal.
D000936:Antigen-Antibody Complex; D002460:Cell Line; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D007231:Infant, Newborn; D008297:Male; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D019000:Phosphotyrosine; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D012867:Skin / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D000936:Antigen-Antibody Complex; D007106:Immune Sera; D010446:Peptide Fragments; D010982:Platelet-Derived Growth Factor; D019000:Phosphotyrosine; D014443:Tyrosine