The focus of this project is to examine the mechanism(s) by which prostaglandins and linoleic acid metabolites potentiate the EGF mitogenic signal in fibroblast cell lines. We have recently examined in more detail the expression of signaling proteins and the extent of their tyrosine phosphorylation of the EGF signaling pathway in the two SHE cell variants. On phosphorylation of EGFR, SH-2 containing signaling protein binds to a specific tyrosine phosphorylation site on the receptor. The SH-2 containing tyrosine phosphatase SHP-2 binds to the phosphorylated EGFR in the supB+ but the kinetics of binding to the phosphorylated EGFR in the supB- cells are different. We sequenced the SHP-2 and the EGFR receptor from the SHE cell variants and did not find any differences between supB+ and supB- in the amino acid sequence of these two proteins. 13(S)-HpODE increased the association of the SHP-2 with the EGFR and others phosphorylated proteins in a concentration dependent manner in the supB+ cells with little effect in the supB- cells. Differences in SHP-2 interaction with EGFR may account, in part, for phenotypic differences in the growth rates and responsiveness to EGF between the supB+ and supB- cells.We have investigated the linkage of SHP-2 to the EGFR. The addition of EGF induced a rapid and transient association of EGFR with SHP-2. EGFR was associated with Gab-1 in absence of EGF with enhanced binding after EGF addition to the cells. SHP-2 associated with Gab-1 and formed a sustained complex for 15 min. with SHP-2. Immunodepletion experiments revealed SHP-2 and Gab-1 bind to the same EGFR molecule and form a ternary complex. The kinetic of association was different between the supB+ and supB- cells. We examined the Gab-1 expression in supB+ and supB- by immunoprecipitation and immunoblotting. SupB+ showed the expression of Gab-1 (100 kD) which became associated with EGFR as well as SHP-2 upon EGF stimulation. A novel 87-kD protein, which reacted with two anti-Gab-1 against different epitopes, was also detected exclusively in supB-. The expression of 87-kD form gradually increased during cell passages, while the expression of 100-kD form decreased. Northern analysis revealed the expression of 5.2-kb mRNA alone in supB+, and 4.6-kb mRNA as well as 5.2-kb in supB-. 87-kD form also became associated with EGFR and SHP-2 upon EGF stimulation. Moreover, cell growth in soft agar revealed the close relationship of the Gab-1 expression to the colony forming efficiency of the cells in response to EGF and insulin; 3.9% in supB+, 7.4% in the early passage of supB-, and 26.4% in the later passage of supB-. We are also in the process of characterizing the sequence of the 87-kD form of Gab-1. These results indicate the involvement of Gab-1 in the altered responsiveness to growth factors and linoleic acid metabolites during neoplastic progression of SHE cells. - EGF, lipids, signalling pathway, tyrosine phosphorylation, phosphotases
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