Dissection of the TORC1 Signaling Network in Yeast

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， 真核生物过去20年来的工作揭示了激素的潜在机制， 以及向TORC 1发出的氨基酸信号，但目前尚不清楚其他关键信号（例如葡萄糖饥饿）的情况 传递到这个高度保守的复合体。在上一个授权期，我们检查了TORC 1信号， 芽殖酵母，并发现PKC，Gcn 2，Sit 4和CK 2信号通路与差距一起工作 SEAC（人GATOR 1/2）通过高度保守的GTP酶Gtr 1/2（Rag A/B和C/D）抑制TORC 1 在人类中）。这反过来又释放TORC 1，使其移动到边缘处的单个非活动主体中。 液泡/溶酶体-依赖于TORC 1结合蛋白Pib 2的事件。在这一框架的基础上， 我们现在希望：（1）确定和表征与GTR 1/2平行工作的蛋白质和途径，以调节 （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的细胞中纯化了TORC 1 条件，并确定了许多新的互动者。最值得注意的是 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， 需要它的运动进入，或出，不活跃的机构。我们现在建议研究这些功能 T0 RC 1结合蛋白的详细研究，测试以下假设：（i）Ydl 180 w是T0 RC 1的阻遏物， 与Gtr 1/2竞争以控制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的磷酸依赖性激活剂。为了解决第二个问题，我们净化了少校 Gtr 1/2调节因子SEAC，并绘制了其在葡萄糖和氮饥饿条件下的磷酸化。这导致 鉴定了超过150个磷酸化位点，其中许多在磷酸化过程中是高磷酸化或低磷酸化的。 葡萄糖和/或氮饥饿。在这些数据的基础上，我们现在希望测试假设， 保守的Gcn 2、PKC、Sit 4、CK 2和其他激酶/磷酸酶通过磷酸化（去磷酸化）抑制TORC 1， 启动SEAC我们还计划探索一种新的联系，我们确定之间的关键丝氨酸合成 酶Ser 3/33（人类中的PHGDH）和TORC 1调节因子Pib 2-检验Ser 3/33 在丝氨酸存在但不缺乏的情况下，通过Pib 2激活TORC 1。我们的建议是创新的， 使用最先进的系统，蛋白质组学，和 生物化学方法。拟议的研究是重要的，因为它有望阐明 在一个重要的模式生物中，细胞生长控制和复杂信号整合的潜在机制- 这对（a）理解TORC 1相关疾病如癌症、癫痫、糖尿病和 肥胖，因为许多研究中的蛋白质和途径是保守的，以及（B）开发药物 选择性地阻止病原真菌的生长。

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.