Dissection of the TORC1 Signaling Network in Yeast

描述(申请人提供)：真核细胞利用一个复杂的信号通路网络来调节细胞生长。众所周知，这种网络可以控制衰老，它的故障会导致一系列疾病。该网络的中心是雷帕霉素复合体1(TORC1)途径的靶点。在适当的激素和营养物质存在的情况下，这一途径是活跃的，通过诱导核糖体和蛋白质合成并抑制分解代谢来推动生长。然而，当营养或激素水平较低，或者细胞暴露在有害压力下时，TORC1信号被失活，生长放缓。TORC1活性是如何被调节的在很大程度上仍然是未知的，尤其是不确定(1)复杂的分支TOR通路在胁迫中有什么输出，(2)哪些胁迫信号通路参与了TOR调节，以及(3)它们如何影响TOR通路本身。在这里，我们建议在模式生物酿酒酵母中解决这些问题，利用TORC1途径的结构和功能从酵母到人类高度保守的事实。在初步研究中，我们研究了渗透胁迫中的TORC1信号，发现与营养剥夺和雷帕霉素等其他刺激不同，压力改变了TORC1途径的输出。此外，我们还发现TORC1途径的抑制显著依赖于应激激活的MAPK Hog1/p38的活性。在这些结果的基础上，我们现在将使用多管齐下的方法构建TOR通路调控和应激输出的量化模型。首先，我们将使用突变菌株的DNA微阵列分析来构建TOR/Hog1信号通路的模型，以解释压力和Hog1如何改变通过TORC1和其他生长途径传递的信号。然后，我们将使用高通量筛选来确定TORC1调节因子的额外上游，然后再次使用微阵列分析来确定这些蛋白质如何相互作用以及TOR途径。最后，我们将通过研究我们发现的调控事件背后的蛋白质-蛋白质相互作用和翻译后修饰，为我们的模型增加机械细节。这项拟议的研究将为真核生物中依赖压力的生长/TORC1调控的信号通路提供第一个详细的视角。这不仅将揭示一个基本且鲜为人知的监管系统，而且由于TORC1和应激途径的高度保守，我们的数据和模型可能会对我们对TOR和细胞生长相关疾病(如癌症、衰老、肥胖和糖尿病)的理解产生深远影响。

DESCRIPTION (provided by applicant): Eukaryotic cells utilize a complex network of signaling pathways to regulate cell growth. This network is known to control aging and its malfunction leads to a wide range of diseases. At the center of the network lies the target of rapamycin complex 1 (TORC1) pathway. In the presence of the appropriate hormones and nutrients, this pathway is active and drives growth by inducing ribosome and protein synthesis and repressing catabolic metabolism. However, when nutrient or hormone levels are low, or cells are exposed to noxious stress, TORC1 signaling is inactivated and growth is slowed. How TORC1 activity is regulated remains largely unknown, and in particular it is uncertain (1) what output the complex branched TOR pathway has in stress, (2) which stress signaling pathways are involved in TOR regulation, and (3) how they impinge on the TOR pathway itself. Here we propose to address these questions in the model organism S. cerevisiae, taking advantage of the fact that the structure and function of the TORC1 pathway is highly conserved from yeast to human. In preliminary studies, we have examined TORC1 signaling in osmotic stress and find that stress alters TORC1 pathway output differently than other stimuli, such as nutrient deprivation and rapamycin. In addition, we find that TORC1 pathway repression depends significantly on the activity of the stress activated MAPK Hog1/p38. Building on these results, we will now construct a quantitative model of TOR pathway regulation and output in stress using a multipronged approach. First, we will use DNA microarray analysis of mutant strains to construct a model of the TOR/Hog1 signaling circuit to explain how stress and Hog1 alter signals transmitted through TORC1 and other growth pathways. We will then use high-throughput screens to identify additional upstream of regulators of TORC1, and then determine how these proteins interact with each other and the TOR pathway, again using microarray analysis. Finally we will add mechanistic detail to our model by studying the protein-protein interactions and post-translational modifications underlying the regulatory events we uncover. The proposed research will provide the first detailed view of the signaling pathways underlying stress dependent growth/TORC1 regulation in eukaryotes. This will not only shed light onto a fundamental and poorly understood regulatory system, but due to the high level of conservation in the TORC1 and stress pathways, our data and model will likely have a profound influence on our understanding of TOR and cell growth related diseases such as cancer, aging, obesity and diabetes.