Heparin-Binding Growth Factor 1 Stimulates Tyrosine Phosphorylation in NIH 3T3 Cells

Abstract

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 M_r 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 M_r-150,000 M_r-130,000, and M_r-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 M_r-150,000 and M_r-130,000 proteins was detected within 30 s, whereas phosphorylation of the M_r-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 M_r-150,000 and M_r-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the M_r-150,000 and M_r-90,000 proteins occurred when cells were exposed to HBGF-1 at 37°C but not at 4°C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the M_r-150,000, M_r-130,000, and M_r-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 eluted with phenyl phosphate as two components: M_r 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the ¹²⁵I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked ¹²⁵I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the ¹²⁵I-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.