Phosphopeptide Metabolism in Adipocytes

描述（由申请人提供）：胰岛素/IGF系统在后生动物中进化，以协调营养利用与细胞生长和增殖，在发育和成年生活中。胰岛素/IGF系统的控制叠加在系统发育上较老的、营养驱动的信号通路上并相互调节。这种双重控制的一个范例是被称为TOR的巨型蛋白激酶。首先在酵母中发现，TOR调节转录、核糖体生物发生、mRNA翻译和蛋白质周转，以响应营养可利用性。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（尤其是亮氨酸）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包含多肽raptor、底物结合亚基和lst8，主要与胰岛素/IGF和生长的营养调节有关，是本研究的重点。mTORC1的近似调节因子是小的GTPase Rheb；胰岛素/IGF通过Akt激酶抑制结节性硬化症异源二聚体GTPase激活剂功能，促进Rheb转化为活性状态。在AIM1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催化结构域和内源性mTOR抑制剂FKBP38相互作用，将mTOR催化结构域转化为活性形式。我们将定义这种转化的机制，并使用纯化的成分在体外重建这种调节。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的访问。我们将定义这些磷酸化的调控，并确定它们对mTORC1信号的整体激活的贡献。对于细胞内亮氨酸调节mTORC1的机制，除了亮氨酸戒断干扰Rheb-GTP激活mTORC1的能力外，知之甚少。在AIM2中，我们将使用全基因组RNA干扰来生成mTORC1促进40S核糖体蛋白S6磷酸化能力所需的细胞组分目录。这将揭示许多以前未被重视的mTORC1输入；然后，我们将使用更精细的二次分析来关注最可能介导亮氨酸调节的元素。最近，一些在普通人群中导致2型糖尿病易感性的基因已经被确定。其中有一种RNA结合蛋白IMP2，首次被发现是由于它能够结合主要在胎儿期表达的IGF2 mRNA，其翻译受mTORC1调节。我们发现大多数imp2相关mrna的翻译受mTORC1调控。在AIM3中，我们建议鉴定IMP2相关rna， mTORC1调控IGF2 mRNA转录的机制以及IMP2在该调控中的作用。更深入地了解IMP2的功能和调控将有助于理解IMP2是如何通过调控IGF2或其他待鉴定的rna导致2型糖尿病的易感性。公共卫生相关性：TOR复合体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复合体1的活性。我们还将确定TOR复合体1如何控制与IMP2蛋白结合的rna的翻译，IMP2基因的变异似乎赋予了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.