Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.
Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-150,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after 15 to 30 min. Phosphoamino acid analysis of the Mr-150,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-150,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-150,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.
Friesel
R
R
Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockeville, Maryland 20855.
Burgess
W H
WH
Maciag
T
T
D000818:Animals; D002460:Cell Line; D016220:Fibroblast Growth Factor 1; D006133:Growth Substances / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D011975:Receptors, Mitogen / Q000378:metabolism; D040262:Receptors, Vascular Endothelial Growth Factor; D015398:Signal Transduction / Q000187:drug effects; D013696:Temperature; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D006133:Growth Substances; D011506:Proteins; D011975:Receptors, Mitogen; D016220:Fibroblast Growth Factor 1; D014638:Vanadates; D014443:Tyrosine; D006493:Heparin; D040262:Receptors, Vascular Endothelial Growth Factor