Regulation of nitrogen catabolic gene expression in S cerevisiae

描述(申请人提供)：在模式生物酿酒酵母中对GATA家族转录因子Gln3和Gat1的研究对于加深我们对两个高度重视的临床领域的分子事件的了解非常重要：(I)人GATA家族转录因子的调控，越来越多地发现与人类疾病有关，如骨髓增殖性疾病和急性巨核细胞白血病；以及(Ii)全球调节蛋白激酶mTOR的细胞和分子生物学，mTOR，雷帕霉素及其类似物，西罗莫司，everolimus和CCI-779。这些药物目前正处于第二阶段和第三阶段的临床试验，评估它们在支架治疗冠状动脉疾病、作为治疗雌激素引起的乳腺癌和肾癌的抗肿瘤药物以及作为器官移植手术后的免疫抑制剂的用途。在酿酒酵母中，Gln3和Gat1是负责选择性利用氮源的转录因子。当细胞在过量的氮中培养时，它们是细胞质和无功能的，在氮饥饿或在有限的氮中生长时，它们在细胞核内积聚并激活转录。用Tor抑制剂雷帕霉素处理细胞，即使有多余的氮，也会诱导Gln3去磷酸化和核积聚。这些特点使得Gln3的定位和磷酸化成为Tor功能的主要检测手段，因此研究Gln3对Tor的调控具有重要意义。一种啮合模型认为，Tor信号转导通路通过2A型相关磷酸酶Sit4的作用来调节Gln3的定位。当Tor激活时，Sit4不活跃，Gln3被磷酸化，与Ure2络合并定位于细胞质。当Tor被雷帕霉素或氮饥饿抑制时，Sit4变得活跃，Gln3去磷酸化，与Ure2解离，并在细胞核内积聚。在过去的授权期内产生的数据清楚地表明，上述控制Gln3的Tor1、2的当前模型需要进行重大修改。例如，Tor的调节被认为是通过Mks1发生的，它被认为是Ure2的负调节因子和Gln3的正调节因子。我们证明了Mks1只是通过对同化氮源(氨)所需的a-酮戊二酸的产生进行负调控来间接影响Gln3的定位，而Sit4主动去磷酸化Gln3而不考虑氮源的可用性。这个应用中的实验确定了哪些地方需要额外的改变，解释了当前模型产生的预测没有得到满足的情况，并演示了Tor途径的这一部分是如何受到调控的。这一新信息将使人们更准确地了解Gln3受Tor调控以及它对外部环境做出反应的机制。更重要的是，它将作为一个有效的模型系统来产生信息，其中大部分将直接适用于哺乳动物细胞，因为Tor途径在这些生物之间非常保守。

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.