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

在我们以前的研究中，我们已经表明，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衔接分子激活Ras-MAP激酶途径。本课题进一步研究了EGF受体和VEGF受体2（KDR/Flk-1）的信号转导，并检测了受体的正、负信号。EGFR的负信号转导：人上皮癌细胞系A431过表达EGFR，高浓度的EGF可诱导生长抑制和细胞凋亡。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，而不是经典的PKC-抑制剂抑制EGFR的这种负信号。这些结果表明PKC-δ参与了该信号传导。来自VEGFRs的信号转导：使用过表达VEGFR（Flt-1或KDR）的NIH 3 T3细胞以及原代内皮细胞，我们清楚地表明这两种受体在生物化学上是完全不同的。Flt-1对VEGF具有高亲和力（Kd=约10 nM），而KDR对配体显示出低一个数量级的亲和力。然而，就激酶活性而言，KDR具有强的酪氨酸激酶活性，而Flt-1具有非常弱的活性。KDR通过PLCγ-PKC途径而不是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.