INSULIN REGULATION OF RAS-DEPENDENT SIGNALING PATHWAYS

胰岛素对 RAS 依赖性信号通路的调节

• 批准号: 2713408
• 负责人: JEFFREY E. PESSIN
• 金额: $ 19.88万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2713408
• 关键词: 3T3 cells; CHO cells; biological signal transduction; epidermal growth factor; growth factor receptors; guanine nucleotide binding protein; guanine nucleotide exchange factors; hormone regulation /control mechanism; immunoprecipitation; insulin; insulin receptor; mitogen activated protein kinase; mixed tissue /cell culture; mutant; phosphorylation; protein tyrosine kinase; protein tyrosine phosphatase; site directed mutagenesis; transfection

DESCRIPTION (Adapted from the applicant's abstract): The investigator has observed that several agents which activate the various MAP kinase pathways (including insulin, platelet-derived growth factor (PDGF), T-cell receptor activation, serum, phorbol esters, v-ras and v-raf) result in a rapid serine/threonine phosphorylation of the ras GTP/GDP exchange factor SOS. Subsequent to this phosphorylation, there is a dissociation of SOS from Grb2 that appears to correlate with the inactivation phase of ras/GTP loading. This phenomenon, however, is not universal, as stimulation of the epidermal growth factor (EGF) and nerve growth factor receptors cause a phosphorylation of SOS, yet fails to induce the dissociation of the Grb2-SOS complex. The principal investigator proposes to determine the mechanism(s) for these apparent divergent signaling events and to directly assess the physiological consequences of these processes in terms of ras activation and downstream biological responsiveness. This will be accomplished by determining the specific sites of serine/threonine phosphorylation of SOS from insulin and EGF-stimulated cells and by expressing various deletion and point mutations of SOS. The principal investigator will use the MAP kinase phosphatase (MKP1) and various negative-dominant and constituitively active kinases in the ERK/JNK/p38 kinase pathways to determine the in vivo pathways responsible for basal and hormonal stimulated changes in SOS phosphorylation, association with Grb2 and effect on ras/GTP loading. The principal investigator will investigate the potential presence of other associated effector proteins involved in the uncoupling of the Grb2-SOS complex by expression of epitope tagged mutant SOS and Grb2 proteins that are defective in specific interactions motifs. In addition, in vivo metabolic labeling experiments will be used to identify potential associated effector proteins. Finally, the principal investigator will assess the effect of membrane targeting of Shc, Grb2 and SOS on the site-specific phosphorylation of SOS and Shc, and their specific interactions with each other as well as with the EGF and insulin receptor tyrosine kinases.

描述（改编自申请人的摘要）：研究者已 观察到几种激活各种 MAP 激酶途径的药物 （包括胰岛素、血小板衍生生长因子（PDGF）、T细胞受体 激活、血清、佛波酯、v-ras 和 v-raf）导致快速 ras GTP/GDP 交换因子 SOS 的丝氨酸/苏氨酸磷酸化。 磷酸化后，SOS 从 Grb2 解离 这似乎与 ras/GTP 负载的失活阶段相关。 然而，这种现象并不普遍，因为表皮的刺激 生长因子（EGF）和神经生长因子受体引起 SOS 磷酸化，但未能诱导 Grb2-SOS 解离 复杂的。 主要研究者建议确定机制 对于这些明显不同的信号事件并直接评估 这些过程在 ras 激活和 下游生物反应性。 这将通过以下方式完成 确定 SOS 丝氨酸/苏氨酸磷酸化的特定位点 来自胰岛素和 EGF 刺激的细胞并通过表达各种缺失和 SOS点突变。 主要研究者将使用 MAP 激酶 磷酸酶 (MKP1) 和各种负显性和组成型活性 ERK/JNK/p38 激酶通路中的激酶以确定体内通路 负责 SOS 的基础和激素刺激变化 磷酸化、与 Grb2 的关联以及对 ras/GTP 负载的影响。 这 首席研究员将调查其他潜在的存在 参与 Grb2-SOS 解偶联的相关效应蛋白 通过表位标记的突变型 SOS 和 Grb2 蛋白的表达形成复合物 特定相互作用基序有缺陷。 此外，体内 代谢标记实验将用于识别潜在的相关 效应蛋白。 最后，首席研究员将评估 Shc、Grb2 和 SOS 的膜靶向对位点特异性的影响 SOS 和 Shc 的磷酸化，以及它们与各自的特定相互作用 其他以及 EGF 和胰岛素受体酪氨酸激酶。