Signal transduction for cell proliferation, apoptosis and differentiation via receptor-type tyrosine kinase and Shc molecule.

在我们之前的研究中，我们已经证明缺乏所有自磷酸化酪氨酸残基的EGF受体突变体仍然通过Shc接头分子激活Ras-MAP激酶途径。在本项目中，我们进一步扩展了egf受体和vegf受体-2 （KDR/Flk-1）信号转导的研究，并检查了受体的阳性或阴性信号。EGFR负信号转导。人上皮癌细胞系A431过表达EGFR，高浓度的EGF已被证明可诱导生长抑制和凋亡细胞死亡。在这个系统中，我们发现泛蛋白激酶- c抑制剂和pkc - δ抑制剂抑制了EGFR的负信号，而经典的pkc抑制剂则没有。这些结果表明PKC-delta参与了这一信号传导。vegfr的信号转导。使用过表达VEGFR （Flt-1或KDR）的NIH3T3细胞和原代内皮细胞，我们清楚地发现这两种受体在生化上有很大的不同。Flt-1对VEGF具有高亲和力（Kd=约10 nM），而KDR对配体的亲和力低一个数量级。在激酶活性方面，KDR具有较强的酪氨酸激酶活性，而Flt-1具有较弱的酪氨酸激酶活性。此外，KDR通过plc - γ- pkc途径而非Ras途径产生有丝分裂信号。

In our previous studies, we have shown that EGF receptor mutant lacking all autophosphorylation tyrosine residues still activates Ras-MAP kinase pathway via Shc adaptor molecule. In this project, we further extended our research on the signal transduction from EGF-receptor and VEGF-receptor-2 (KDR/Flk-1), and examined positive or negative signal from the receptors.1). Negative signal transduction from EGFR.Human epithelial carcinoma cell line A431 overexpresses EGFR, and a high concentration of EGF has been shown to induce grown suppression and apoptotic cell death. In this system we found that pan-Protein kinase-C inhibitor and PKC-delta-inhibitor, but not classical PKC-inhibitor suppressed this negative signal from EGFR. These results suggest that PKC-delta is involved in this signaling.2). Signal transduction from VEGFRs.Using NIH3T3 cells overexpressing VEGFR (Flt-1 or KDR) as well as primary endothelial cells, we clearly showed that these two receptors are biochemically quite different. Flt-1 has a high affinity (Kd= about 10 nM) to VEGF, whereas KDR showed one order lower affinity to the ligand. In terms of the kinase activity, however, KDR had a strong tyrosine kinase activity but Flt-1 had a very weak one. In addition, KDR was found to generate a mitotic signal via PLCγ-PKC pathway but not Ras pathway.