INTERACTION OF LIPIDS WITH EGFR SIGNALING PATHWAY

本项目的重点是研究的机制（S），其中，拟南芥素和亚油酸代谢产物增强EGF有丝分裂信号在成纤维细胞系。我们最近更详细地研究了信号蛋白的表达和EGF信号通路的酪氨酸磷酸化程度在两个SHE细胞变体。在EGFR磷酸化时，含有SH-2的信号传导蛋白与受体上的特异性酪氨酸磷酸化位点结合。含SH-2的酪氨酸磷酸酶SHP-2与supB+中的磷酸化EGFR结合，但与supB-细胞中的磷酸化EGFR结合的动力学不同。我们对来自SHE细胞变体的SHP-2和EGFR受体进行了测序，在这两种蛋白质的氨基酸序列中没有发现supB+和supB-之间的任何差异。13（S）-HpODE在supB+细胞中以浓度依赖性方式增加SHP-2与EGFR和其他磷酸化蛋白的结合，而在supB-细胞中几乎没有影响。SHP-2与EGFR相互作用的差异可能部分解释了supB+和supB-细胞之间生长速率和对EGF反应性的表型差异。EGF的加入诱导EGFR与SHP-2的快速和短暂的关联。EGFR与Gab-1在EGF不存在的情况下相关联，在EGF加入细胞后结合增强。SHP-2与Gab-1结合，并与SHP-2形成持续15分钟的复合物。免疫耗竭实验显示SHP-2和Gab-1结合相同的EGFR分子并形成三元复合物。在supB+和supB-细胞之间的结合动力学是不同的。我们通过免疫沉淀和免疫印迹检测了Gab-1在supB+和supB-中的表达。SupB+显示Gab-1（100 kD）的表达，其在EGF刺激后与EGFR以及SHP-2相关。一种新的87 kD的蛋白质，它与两个抗Gab-1对不同的表位反应，也只在supB-。随着细胞传代，87-kD表达逐渐增加，100-kD表达逐渐减少。北方分析显示在supB+中仅表达5.2-kb mRNA，在supB-中表达4.6-kb mRNA以及5.2-kb mRNA。EGF刺激后，87-kD形式也与EGFR和SHP-2相关。此外，在软琼脂中的细胞生长揭示了Gab-1表达与细胞响应EGF和胰岛素的殖民地形成效率的密切关系;在supB+中为3.9%，在supB-的早期传代中为7.4%，在supB-的后期传代中为26.4%。我们还在表征87 kD形式的Gab-1的序列。这些结果表明Gab-1参与了SHE细胞肿瘤进展过程中对生长因子和亚油酸代谢产物的反应性改变。- 表皮生长因子，脂质，信号通路，酪氨酸磷酸化，磷酸酶

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