IDENTIFICATION OF P90, A PROMINENT TYROSINE-PHOSPHORYLATED PROTEIN INFIBROBLAST GROWTH FACTOR-STIMULATED CELLS, AS 80K-H

Tyrosine phosphorylation of cellular proteins occurs rapidly upon trea tment of fibroblasts with acidic or basic fibroblast growth factors (a FGF, bFGF), suggesting a role for protein phosphorylation in the FGF s ignaling pathway. Stimulation of Swiss 3T3 cells and MRC-5 fibroblasts with bFGF results in the tyrosine phosphorylation of several proteins , of which the most prominent has been designated as p90. The phosphor ylation of p90 is observed within 30 s of treating the cells with FGF but not with other growth factors. Microsequencing of p90 resolved on two-dimensional polyacrylamide gel electrophoresis indicated an N-term inal amino acid sequence which corresponded to a protein previously na med as 80K-H. Polyclonal antibodies raised against the predicted C ter minus of 80K-H recognized p90 on all Western blots. p90 was found to b ind specifically to GRB-2-glutathione S-transferase fusion protein and to be immunoreactive with 80K-H antibody. In addition, anti-phosphoty rosine antibodies immunoprecipitated 80K-H from cell lysates of FGF-st imulated but not from control fibroblasts. The biological function of 80K-H is yet unknown, However, from this study and a previous observat ion of the obligatory dependence of p90 phosphorylation on FGF recepto r occupation, it appears that 80K-H is involved in FGF signaling.