Dissection of the TORC1 Signaling Network in Yeast

酵母中 TORC1 信号网络的剖析

• 批准号: 10798367
• 负责人: Andrew Paul Capaldi
• 金额: $ 5.04万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10798367
• 关键词: Address; Amino Acids; Binding; Binding Proteins; Biochemical; Cells; Cellular Metabolic Process; Complex; Data; Development; Diabetes Mellitus; Disease; Dissection; Enzymes; Epilepsy; Eukaryota; Event; Exposure to; Glucose; Grant; Growth; Guanosine Triphosphate Phosphohydrolases; Hormones; Human; Investigation; Lysosomes; Malignant Neoplasms; Maps; Membrane Proteins; Mental Depression; Movement; Nitrogen; Obesity; Pathway interactions; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Proteins; Proteomics; Public Health; Regulation; Research; Role; Saccharomycetales; Serine; Signal Pathway; Signal Transduction; Sirolimus; Starvation; Stress; Sulfur; System; Testing; Vacuole; Work; Yeasts; cell growth; innovation; inorganic phosphate; model organism; pathogenic fungus; transmission process

SUMMARY/ABSTRACT The Target of Rapamycin kinase Complex I (TORC1) is a master regulator of cell growth and metabolism in eukaryotes. Work carried out over the last 20 years has shed light on the mechanisms underlying hormone and amino acid signaling to TORC1, but it is still unclear how other key signals, such as glucose starvation, are transmitted to this highly conserved complex. In the last grant period, we examined TORC1 signaling in budding yeast, and found that the PKC, Gcn2, Sit4, and CK2 signaling pathways work together with the GAP SEAC (GATOR1/2 in humans) to inhibit TORC1 via the highly-conserved GTPases, Gtr1/2 (Rag A/B and C/D in humans). This in turn releases TORC1 to move into a single inactive body at the edge of the vacuole/lysosome— an event that depends on the TORC1 binding protein, Pib2. Building on this framework, we now wish to: (1) Identify and characterize the proteins and pathways work in parallel with Gtr1/2 to regulate TORC1, and (2) determine how the conserved Gcn2, PKC, Sit4, CK2 pathways, regulate TORC1 via Gtr1/2. To address the first question, we purified TORC1 from cells exposed to a variety of stress and starvation conditions, and identified numerous new interactors. The most notable are the uncharacterized vacuolar/lysosomal membrane proteins Ydl180w, Ygr125w and Syg1, since they bind tightly to TORC1 and are required for its movement into, or out of, the inactive bodies. We now propose to study the function of these TORC1 binding proteins in detail, testing the hypotheses that: (i) Ydl180w is repressor of TORC1 and competes with Gtr1/2 to control TORC1 activity, (ii) Ygr125w is a sulfur dependent activator of TORC1, and (iii) Syg1 is a phosphate dependent activator of TORC1. To address the second question, we purified the major Gtr1/2 regulator SEAC, and mapped its phosphorylation in glucose and nitrogen starvation conditions. This led to the identification of over 150 phosphorylation sites, many of which are hyper- or hypo-phosphorylated during glucose and/or nitrogen starvation. Building on these data, we now wish to test the hypothesis that the conserved Gcn2, PKC, Sit4, CK2, and other kinases/phosphatases inhibit TORC1 by (de)phosphorylating and activating SEAC. We also plan to explore a new connection we identified between the key serine synthesis enzymes Ser3/33 (PHGDH in humans) and the TORC1 regulator Pib2—testing the hypothesis that Ser3/33 activate TORC1 via Pib2 in the presence, but not absence, of serine. Our proposal is innovative in that we study new and unexplored aspects of TORC1 signaling using state-of-the-art systems, proteomic, and biochemical approaches. The proposed research is significant in that it promises to shed light on the mechanisms underlying cell growth control, and complex signal integration, in an important model organism—with implications for (a) understanding TORC1 related diseases such as cancer, epilepsy, diabetes and obesity, since many of the proteins and pathways under investigation are conserved and (b) developing drugs that selectively block the growth of pathogenic fungi.

总结/摘要 雷帕霉素激酶复合物I（TORC 1）的靶标是细胞生长和代谢的主要调节剂， 真核生物在过去20年中进行的工作已经阐明了激素和 氨基酸信号传递到TORC 1，但目前还不清楚其他关键信号，如葡萄糖饥饿， 传递到这个高度保守的复合体。在上一个资助期，我们研究了TORC 1信号在出芽过程中的作用。 酵母，并发现PKC，Gcn 2，Sit 4和CK 2信号通路与差距SEAC一起工作 （人类中的GATOR 1/2）通过高度保守的GTP酶Gtr 1/2（人类中的Rag A/B和C/D）抑制TORC 1。 人类）。这反过来又释放TORC 1移动到液泡/溶酶体边缘的单个无活性体中。 依赖于TORC 1结合蛋白Pib 2的事件。在这个框架的基础上，我们现在希望：（1） 鉴定和表征与Gtr 1/2平行工作以调节TORC 1的蛋白质和途径，以及（2） 确定保守的Gcn 2，PKC，Sit 4，CK 2通路如何通过Gtr 1/2调节TORC 1。为了解决第一个 问题，我们从暴露于各种应激和饥饿条件的细胞中纯化了TORC 1，并鉴定了 许多新的互动。最值得注意的是未表征的空泡/溶酶体膜蛋白 Ydl 180 w、Ygr 125 w和Syg 1，因为它们与TORC 1紧密结合，并且是TORC 1移入或移出所需的， 不活跃的身体。我们现在建议详细研究这些TORC 1结合蛋白的功能，测试TORC 1结合蛋白的功能。 假设：（i）Ydl 180 w是TORC 1的阻遏物，并与Gtr 1/2竞争控制TORC 1活性，（ii） Ygr 125 w是TORC 1的硫依赖性激活剂，和（iii）Syg 1是TORC 1的磷酸盐依赖性激活剂。 为了解决第二个问题，我们纯化了主要的Gtr 1/2调节因子SEAC，并绘制了其磷酸化图谱 在葡萄糖和氮饥饿条件下。这导致了超过150个磷酸化位点的鉴定， 其中许多在葡萄糖和/或氮饥饿期间被高磷酸化或低磷酸化。根据这些 数据，我们现在希望测试的假设，保守的Gcn 2，PKC，Sit 4，CK 2，和其他 激酶/磷酸酶通过（去）磷酸化和激活SEAC来抑制TORC 1。我们还计划探索 我们发现了关键丝氨酸合成酶Ser 3/33（人类PHGDH）与 TORC 1调节因子Pib 2-检验Ser 3/33在存在Pib 2的情况下通过Pib 2激活TORC 1，但不激活的假设。 没有丝氨酸。我们的提议是创新的，因为我们研究了TORC 1信号传导的新的和未探索的方面 使用最先进的系统、蛋白质组学和生物化学方法。该研究具有重要意义， 它有望阐明细胞生长控制和复杂信号整合的潜在机制， 在一个重要的模式生物-与影响（a）了解TORC 1相关的疾病，如 癌症，癫痫，糖尿病和肥胖，因为许多正在研究的蛋白质和途径是 保守和（B）开发选择性阻断病原真菌生长的药物。