Phosphopeptide Metabolism in Adipocytes

描述（由申请人提供）：在后生动物中进化的胰岛素/IGF系统在发育和成年期协调营养利用与细胞生长和增殖。胰岛素/IGF系统的控制叠加在遗传学上较老的营养驱动的信号通路上并受其交叉调节。这种双重控制的范例是被称为TOR的巨大蛋白激酶。首先在酵母中发现，TOR调节转录，核糖体生物合成，mRNA翻译和蛋白质周转以响应营养可用性。在哺乳动物细胞中，TOR保留了这些功能，但胰岛素/IGF系统的调节叠加在氨基酸，特别是亮氨酸的调节上。在酵母和人类中，TOR在两种物理上不同且独立调节的复合物中起作用; TOR复合物1，其含有多肽raptor、底物结合亚基和lst 8，主要与胰岛素/IGF和生长的营养调节有关，并且是该提议的焦点。mTORC 1的最接近调节因子是小GTdR heb;胰岛素/IGF通过激酶Akt抑制肥大性硬化症异源二聚体的GTdR活化剂功能，促进Rheb转化为活性状态。在AIM 1中，我们建议详细阐明Rheb-GTP激活mTOR复合物1的机制。我们假设一个两步过程;首先，Rheb-GTP与mTOR催化结构域和FKBP 38（mTOR的内源性抑制剂）相互作用，将mTOR催化结构域转化为活性形式。我们将定义这种转换的机制，并使用纯化的成分在体外重新创建这种调节。第二步涉及mTOR催化的自身和raptor的磷酸化，这增强了底物对raptor的接近。我们将定义这些磷酸化的调节，并确定它们对mTORC 1信号传导的整体激活的贡献。除了亮氨酸撤回干扰Rheb-GTP激活mTORC 1的能力外，细胞内亮氨酸调节mTORC 1的机制知之甚少。在AIM 2中，我们将使用全基因组RNA干扰来产生mTORC 1促进40 S核糖体蛋白S6磷酸化的能力所需的细胞组分目录。这将揭示许多以前未被认识到的mTORC 1输入;然后，我们将使用更精细的二级检测来关注最有可能介导亮氨酸调节的元素。最近，一些赋予普通人群2型糖尿病易感性的基因已被确定。其中有一种RNA结合蛋白IMP 2，首次发现它能够与主要在胎儿期表达的IGF 2 mRNA结合，其翻译受mTORC 1调节。我们发现大多数IMP 2相关mRNA的翻译受mTORC 1调控。在AIM 3中，我们提出鉴定IMP 2相关RNA，mTORC 1调节IGF 2 mRNA转录的机制以及IMP 2在此调节中的作用。对IMP 2功能和调节的更深入理解将有助于理解IMP 2如何通过调节IGF 2或其他待鉴定的RNA赋予2型糖尿病的易感性。公共卫生相关性：TOR复合物1是一种蛋白激酶，是骨骼肌和β细胞发育和生长的主要决定因素。我们将定义胰岛素和氨基酸亮氨酸如何共同控制TOR复合物1的活性。我们还将确定TOR复合物1如何控制与蛋白质IMP 2结合的RNA的翻译，IMP 2的基因变体似乎赋予2型糖尿病的易感性。

DESCRIPTION (provided by applicant): The insulin/IGF system evolved in metazoans to coordinate nutrient utilization with cell growth and proliferation, both in development and adult life. Control by the insulin/IGF system is superimposed upon and cross-regulated by phylogenetically older, nutrient-driven signaling pathways. A paradigm for such dual control is the giant protein kinase known as TOR. First identified in yeast, there TOR regulates transcription, ribosomal biogenesis, mRNA translation and protein turnover in response to nutrient availability. In mammalian cells TOR retains these functions, but regulation by the insulin/IGF system is superimposed on regulation by amino acids, especially leucine. In both yeast and man TOR functions in two physically distinct and independently regulated complexes; TOR complex 1, which contains the polypeptides raptor, the substrate binding subunit and lst8, is primarily concerned with insulin/IGF and nutrient regulation of growth, and is the focus of this proposal. The proximate regulator of mTORC1 is the small GTPase Rheb; insulin/IGF, through the kinase Akt, suppresses the GTPase activator function of the Tuberous Sclerosis heterodimer promoting the conversion of Rheb to the active state. In AIM1, we propose to elucidate in detail the mechanism by which Rheb-GTP activates mTOR complex 1. We hypothesize a two step process; first, Rheb-GTP interacts with the mTOR catalytic domain and with FKBP38, an endogenous inhibitor of mTOR, to convert the mTOR catalytic domain into an active form. We will define the mechanism of this conversion and recreate this regulation in vitro using purified components. The second step involves the mTOR-catalyzed phosphorylation of itself and of raptor, which enhances the access of substrates to raptor. We will define the regulation of these phosphorylations and establish their contribution to the overall activation of mTORC1 signaling. Little is known about the mechanism by which intracellular leucine regulates mTORC1, apart from the ability of leucine withdrawal to interfere with Rheb-GTP activation of mTORC1. In AIM2 we will use genome-wide RNA interference to generate a catalog of cellular components required for the ability of mTORC1 to promote phosphorylation of the 40S ribosomal protein S6. This will uncover many previously unappreciated inputs to mTORC1; we will then use more refined secondary assays to focus on elements most likely to mediate leucine regulation. Recently some of the genes that confer susceptibility to type 2 diabetes in the general population have been identified. Among them is an RNA binding protein IMP2, first discovered by its ability to bind to an IGF2 mRNA expressed primarily in fetal life, whose translation is regulated by mTORC1. We find that translation of most IMP2-associated mRNAs is regulated by mTORC1. In AIM3 we propose to identify IMP2-associated RNAs, the mechanisms by which mTORC1 regulates the IGF2 mRNA transcript and the role of IMP2 in this regulation. A deeper understanding of IMP2 function and regulation will contribute to the understanding of how IMP2, perhaps through the regulation of IGF2 or other to-be- identified RNAs, confers susceptibility to type 2 diabetes. PUBLIC HEALTH RELEVANCE: The TOR complex 1 is a protein kinase that is major determinant of the development and growth of both skeletal muscle and beta cells. We will define how insulin and the amino acid leucine jointly control the activity of TOR complex 1. We will also determine how TOR complex 1 controls the translation of RNAs that bind to the protein IMP2, variants of whose gene appear to confer susceptibility to type 2 diabetes.