SIGNAL TRANSDUCTION PATHWAYS IN NORMAL AND AGED CELLS

工作总结：本项目以信号为重点 介导分子应激反应的信号转导途径 正常细胞和衰老细胞。过去一年的研究集中在 在三个主题上。(1)确定上游调解人在 亚砷酸盐引发的ERK级联反应。在此之前，我们演示了 亚砷酸盐可激活ERK、JNK和p38 MAP激酶。近期 研究主要集中在生长因子受体在体内的作用。 介导ERK的激活。我们已经证明了亚砷酸盐治疗 表皮生长因子受体快速激活的结果 (EGFR)，Shc接头的酪氨酸磷酸化，以及 EGFR-Shc-Grb2复合体的形成。这些事件，以及 ERK的激活均因下调而显著降低 对EGFR活性的影响。这些结果提供了第一个证据表明 EGFR和Shc是细胞外信号转导通路激活的关键介质 Ras/ERK信号通路被亚砷酸盐激活，提示该肿瘤 启动子在很大程度上通过篡改这种生长因子信号来发挥作用。 路径。(2)MAP激酶的结构基础 磷酸酶-1(MKP-1)。MAP激酶磷酸酶是一组 细胞外刺激诱导的双特异性磷酸酶 包括生长因子和压力。他们可以表现出选择性 MAP激酶家族的不同成员之间的关系。的目标是 本研究旨在了解MKP-1底物的结构基础 专一性。MKP-1的不同结构域正在被交换 PAC-1或MKP-3的相应区域产生嵌合体。 分析这些嵌合体的结合和酶的特异性 对于不同的贴图，可能会提供有关 底物选择性的结构基础。(3)年龄相关 大鼠肝细胞内信号通路的改变。以前我们 证明了衰老与ERK的减少有关 MAP激酶和p70 S6激酶活性跟随生长因子 治疗。这两种激酶活性的下降表明 老化小区可能在早期上游事件中显示变化 在这些小路上是常见的。我们已经比较了最早的 对EGF刺激的反应发生的信号事件 年轻和衰老的细胞。在年轻的肝细胞中，EGF迅速触发 EGFR和Shc的酪氨酸磷酸化及其复合体的形成 在他们之间。这种复合体的形成对EGF的反应是 在老化细胞中显著减少，尽管在 观察到EGFR或Shc的酪氨酸磷酸化。这个 生长因子受体复合体的改变可能起到一定作用 衰老细胞增殖能力下降的原因。

Summary of work: This project focuses on signal transduction pathways mediating the molecular reponse to stress in normal and aged cells. Studies over the past year have concentrated on three topics. (1) Identification of upstream mediators in arsenite-triggered ERK cascade. Previously we demonstrated that arsenite can activate ERK, JNK and p38 MAP kinases. Recent studies have focused on the role of growth factor receptors in mediating ERK activation. We have shown that arsenite treatment results in the rapid activation of epidermal growth factor receptor (EGFR), tyrosine phosphorylation of the Shc adaptor, and the formation of EGFR-Shc-Grb2 complexes. These events, as well as activation of ERK, were all drastically reduced by down-regulation of EGFR activity. These results provide the first evidence that the EGFR and Shc are critical mediators in the activation of the Ras/ERK signaling cascade by arsenite and suggest that this tumor promoter acts largely by usurping this growth factor signaling pathway. (2) Structural basis of MAP kinase phosphatase-1(MKP-1). MAP kinase phosphatases are a group of dual specificity phosphatases induced by extracellular stimuli including growth factors and stress. They can exhibit selectivity towards different members of the MAP kinase family. The goal of this study is to understand the structural basis for MKP-1 substrate specificity. Various domains of MKP-1 are being swapped with corresponding regions of PAC-1 or MKP-3 to generate chimeras. Analyzing the binding and enzymatic specificities of these chimeras for different MAP kinases may provide important information about the structural basis for the substrate selectivity. (3) Age-associated alteration in signaling pathways in rat hepatocytes. Previously we demonstrated that aging is correlated with decreases in both ERK MAP kinase and p70 S6 kinase activities following growth factor treatment. A decline in the activities of both kinases suggests that aged cells may display an alteration in an early upstream event common to these pathways. We have compared the earliest signaling events which occur in response to EGF stimulation in young and aged cells. In young hepatocytes, EGF triggers rapid tyrosine phosphorylation of EGFR and Shc, and complex formation between them. Formation of this complex in response to EGF is significantly reduced in aged cells, although no difference in tyrosine-phosphorylation of either EGFR or Shc is observed. The alteration in the growth factor receptor complexes may contribute to the decline in proliferation capacity in aged cells.