Acidic and basic fibroblast growth factors stimulate tyrosine kinase activity in vivo.
We assessed the ability of acidic and basic fibroblast growth factor (FGF) to stimulate tyrosine kinase activity in intact cells. Immunoblot with polyclonal antiphosphotyrosine antibodies detected a 90-kDa phosphotyrosine-bearing protein in lysates of Swiss 3T3 cells exposed to pituitary-derived FGF, recombinant acidic FGF, or recombinant basic FGF, but not from unstimulated cells or cells exposed to epidermal growth factor or platelet-derived growth factor. Phosphotyrosine and its analogue phenyl phosphate, but not phosphoserine, phosphothreonine, or tyrosine itself, blocked recognition of the 90-kDa protein by antiphosphotyrosine antiserum. A monoclonal antiphosphotyrosine antibody also recognized the 90-kDa protein and was used to partially purify the protein by immunoaffinity chromatography. Phosphoamino acid analysis of the 90 kDa band revealed that it contained 20% phosphotyrosine, 35% phosphothreonine, and 45% phosphoserine. Tyrosine phosphorylation of the 90-kDa protein was detectable within 30 s and reached a plateau within 10 min of FGF addition. The addition of suramin, which blocks the interaction of FGF with its receptor, caused rapid disappearance of the 90 kDa band. Cell fractionation experiments were consistent with the 90-kDa protein being membrane-associated, but cross-linking studies revealed that the FGF receptor had an Mr between 145 and 210 kDa in Swiss 3T3 cells, distinct from the 90-kDa major substrate for tyrosine phosphorylation. These data demonstrate that both acidic and basic FGF activate a tyrosine kinase in vivo leading to phosphorylation of a unique 90-kDa substrate, and they suggest that protein modification by phosphorylation at tyrosine is involved in eliciting the mitogenic effect of FGF.
D000818:Animals; D002460:Cell Line; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D007106:Immune Sera; D007163:Immunosorbent Techniques; D051379:Mice; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor; D013498:Suramin / Q000378:metabolism; D013696:Temperature; D014443:Tyrosine / Q000031:analogs & derivatives / Q000276:immunology
D007106:Immune Sera; D011956:Receptors, Cell Surface; D017468:Receptors, Fibroblast Growth Factor; D019000:Phosphotyrosine; D014443:Tyrosine; D013498:Suramin; D005346:Fibroblast Growth Factors; D011505:Protein-Tyrosine Kinases