Regulation of nitrogen catabolic gene expression in S cerevisiae

描述（由申请人提供）：gata家族转录因子Gln3和Gat1在模式生物酿酒酵母中的研究对于我们进一步了解两个高度优先的临床领域的分子事件非常重要：(i)人类GATA-家族转录因子调控，越来越多地被发现与人类疾病相关，如骨髓增生性疾病和急性巨核母细胞白血病，以及（ii）全球调控蛋白激酶mTor的细胞和分子生物学，mTor是药物、雷帕霉素及其类似物、西罗莫司、依维莫司和CCI-779的体内靶点。这些药物目前正处于II期和III期临床试验，评估其在冠状动脉疾病支架治疗中的应用，作为雌激素诱导的乳腺癌和肾细胞癌治疗中的抗肿瘤药物，以及作为器官移植手术后的免疫抑制剂。在酿酒酵母中，Gln3和Gat1是负责选择性氮源利用的转录因子。当细胞在过量氮条件下培养时，它们是细胞质的，无功能的，在氮饥饿或限氮条件下生长时，它们在细胞核中积累并激活转录。用Tor抑制剂雷帕霉素处理细胞，诱导Gln3去磷酸化和核积累，即使在有过量氮可用的情况下。这些特征导致Gln3的定位和磷酸化被用作Tor功能的主要检测手段，因此研究Tor对Gln3的调控具有重要意义。一个引人入胜的模型假设Tor信号转导通路通过与2a型相关的磷酸酶Sit4的作用调节Gln3的定位。当Tor活跃时，Sit4不活跃，Gln3磷酸化，与Ure2络合并定位到细胞质中。当Tor被雷帕霉素或氮饥饿抑制时，Sit4变得活跃，Gln3被去磷酸化，与Ure2分离，并在细胞核中积累。过去资助期的数据清楚地表明，上述tor1,2控制Gln3的当前模型需要进行重大修订。例如，Tor调控被认为是通过Mks1发生的，Mks1被认为是Ure2的负调节因子和Gln3的正调节因子。我们证明Mks1仅间接影响Gln3定位，通过其负调控吸收氮源（氨）所需的a-酮戊二酸的产生，而Sit4不考虑氮源的可用性而积极地使Gln3去磷酸化。本应用程序中的实验确定了需要额外改变的地方，解释了当前模型产生的预测不满足的实例，并演示了Tor途径的这一部分是如何调节的。这一新信息将使我们对Gln3受Tor调控及其对外部环境作出反应的机制有更准确的了解。更重要的是，它将作为一个有效的模型系统来产生信息，其中大部分信息将直接适用于哺乳动物细胞，因为Tor途径在这些生物之间是如此的保守。

DESCRIPTION (provided by applicant): Studies of GATA-family transcription factors Gln3 and Gat1 in the model organism S. cerevisiae are important to further our understanding of the molecular events in two high priority clinical areas: (i) human GATA- family transcription factor regulation, increasingly found associated with human diseases, such as myeloproliferative disorder and acute megakaryoblastic leukemia, and (ii) the cellular and molecular biology of the global regulatory protein kinase, mTor, the in vivo target of the drugs, rapamycin and its analogues, sirolimus, everolimus and CCI-779. These drugs are currently in phase II and III clinical trials evaluating their use in stents to treat coronary artery disease, as antineoplastic agents in the treatment of estrogen-induced breast cancer and renal cell carcinoma, and as immunosuppressants following organ transplant surgery. In S. cerevisiae, Gln3 and Gat1 are the transcription factors responsible for selective nitrogen source utilization. They are cytoplasmic and non-functional when cells are cultured in excess nitrogen, and accumulate in the nucleus and activate transcription during nitrogen starvation or growth in limiting nitrogen. Treating cells with the Tor inhibitor, rapamycin, induces Gln3 dephosphorylation and nuclear accumulation even when excess nitrogen is available. These characteristics have resulted in the use of Gln3 localization and phosphorylation as a principal assay of Tor function, hence the importance of studying Tor regulation of Gln3. An engaging model posits that the Tor signal transduction pathway regulates Gln3 localization through the action of type- 2A-related phosphatase, Sit4. When Tor is active, Sit4 is inactive, Gln3 is phosphorylated, complexed with Ure2 and localized to the cytoplasm. When Tor is inhibited by rapamycin or nitrogen starvation, Sit4 becomes active, Gln3 is dephosphorylated, dissociates from Ure2, and accumulates in the nucleus. Data generated in the past grant period clearly demonstrate the above current model for Tor1, 2 control of Gln3 requires significant revision. For example, Tor regulation was thought to occur via Mks1, which was posited to be a negative regulator of Ure2 and positive regulator of Gln3. We demonstrated Mks1 affects Gln3 localization only indirectly through its negative regulation of a-ketoglutarate production required to assimilate the nitrogen source (ammonia), and that Sit4 actively dephosphorylates Gln3 irrespective of nitrogen source availability. Experiments in this application identify where additional alterations are required, explain instances in which predictions generated by the current model are not fulfilled, and demonstrate how that segment of the Tor pathway is regulated. This new information will generate a more accurate understanding of the mechanisms through which Gln3 is regulated by Tor and by which it responds to its external environment. More importantly it will serve as an efficient model system that generates information, much of which will be directly applicable to mammalian cells because the Tor pathway is so well conserved between these organisms.