INSULIN REGULATION OF RAS-DEPENDENT SIGNALING PATHWAYS

描述（改编自申请人摘要）：研究者 观察到激活各种MAP激酶途径的几种药物 （包括胰岛素、血小板衍生生长因子（PDGF）、T细胞受体 活化、血清、佛波醇酯、V-ras和V-raf）导致快速的 ras GTP/GDP交换因子SOS的丝氨酸/苏氨酸磷酸化。 在这种磷酸化之后，SOS从Grb 2上解离 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 表皮生长因子（EGF）和神经生长因子受体（NGF） SOS的磷酸化，但不能诱导Grb 2-SOS的解离 复杂. 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激活方面的生理后果， 下游生物反应。 这将通过 确定SOS的丝氨酸/苏氨酸磷酸化的特定位点 从胰岛素和EGF刺激的细胞，并通过表达各种缺失和 SOS的点突变。 主要研究者将使用MAP激酶 磷酸酶（MKP 1）和各种负显性和组成活性 ERK/JNK/p38激酶途径中的激酶，以确定体内途径 负责SOS的基础和激素刺激变化 磷酸化、与Grb 2的结合以及对ras/GTP负载的影响。 的 主要研究者将调查其他可能存在的 参与Grb 2-SOS解偶联的相关效应蛋白 通过表达表位标记的突变型SOS和Grb 2蛋白来制备复合物， 在特定的相互作用基序中有缺陷。 此外，在体内 代谢标记实验将用于鉴定潜在的相关 效应蛋白 最后，首席研究员将评估 Shc、Grb 2和SOS的膜靶向对位点特异性 SOS和Shc的磷酸化，以及它们与各自的特定相互作用 其他以及与EGF和胰岛素受体酪氨酸激酶。

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.