Regulation of nitrogen catabolic gene expression in S cerevisiae

描述（由申请人提供）：在模式生物S.酿酒酵母是重要的，以进一步我们的分子事件的理解在两个高度优先的临床领域：（i）人加塔家族转录因子调节，越来越多地发现与人类疾病如骨髓增生性疾病和急性巨核细胞白血病相关，和（ii）全局调节蛋白激酶mTor的细胞和分子生物学，mTor是药物雷帕霉素及其类似物的体内靶点，西罗莫司、依维莫司和CCI-779。这些药物目前正在进行II期和III期临床试验，评估它们在治疗冠状动脉疾病的支架中的用途，作为治疗雌激素诱导的乳腺癌和肾细胞癌的药物，以及作为器官移植手术后的免疫抑制剂。In S. Gln 3和Gat 1是负责选择性利用氮源的转录因子。当细胞在过量氮中培养时，它们是细胞质的且无功能的，并且在氮饥饿或在有限氮中生长期间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Gln 3去磷酸化和核积累，即使当过量的氮可用时。这些特征导致使用Gln 3定位和磷酸化作为Tor功能的主要测定，因此研究Gln 3的Tor调节的重要性。一个参与模型假定Tor信号转导途径通过2A型相关磷酸酶Sit 4的作用调节Gln 3定位。当Tor有活性时，Sit 4无活性，Gln 3被磷酸化，与Ure 2复合并定位于细胞质。当Tor被雷帕霉素或氮饥饿抑制时，Sit 4变得活跃，Gln 3被去磷酸化，与Ure 2解离，并在细胞核中积累。在过去的授权期间产生的数据清楚地表明，上述Tor 1，2控制Gln 3的当前模型需要进行重大修改。例如，Tor调节被认为是通过Mks 1发生的，Mks 1被认为是Ure 2的负调节器和Gln 3的正调节器。我们证明Mks 1影响Gln 3本地化仅间接通过其负调节α-酮戊二酸生产所需的同化氮源（氨），和Sit 4积极脱磷酸化Gln 3的氮源的可用性无关。本申请中的实验确定了需要额外改变的地方，解释了当前模型生成的预测未实现的情况，并演示了Tor通路的该部分是如何调节的。这一新的信息将产生一个更准确的了解的机制，通过该机制Gln 3是由Tor调节，并通过它响应其外部环境。更重要的是，它将作为一个产生信息的有效模型系统，其中大部分将直接适用于哺乳动物细胞，因为Tor途径在这些生物体之间非常保守。

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.