mTORC1-dependent regulation of the CycC/CDK8 complex

CycC/CDK8 复合物的 mTORC1 依赖性调节

• 批准号: 8856228
• 负责人: JEFFREY E. PESSIN
• 金额: $ 31万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8856228
• 关键词: ADD-1 protein; Address; Allosteric Regulation; Anabolism; Applications Grants; Atherosclerosis; Automobile Driving; Binding Proteins; Carbohydrates; Cardiovascular Diseases; Cause of Death; Centers for Disease Control and Prevention (U.S.); Complex; Cyclin-Dependent Kinases; Data; Diabetes Mellitus; Diagnosis; Diet; Dietary Fatty Acid; Dissociation; Down-Regulation; Dyslipidemias; Elements; Endoplasmic Reticulum; Enzymes; Fasting; Fatty Acids; Fatty acid glycerol esters; Gene Expression; Genetic Transcription; Health; Heart Diseases; Hepatic; Hepatocyte; Homeostasis; Hormones; Hypertriglyceridemia; Insulin; Insulin Resistance; Knockout Mice; Lipids; Lipolysis; Liver; Liver diseases; Lysine; Maps; Mediating; Membrane; Molecular; Mus; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Nonesterified Fatty Acids; Nuclear; Nutrient; Obesity; Pathway interactions; Peripheral; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Process; Protein Dephosphorylation; Proteins; Public Health; Raptors; Rattus; Regulation; Resistance; Response Elements; Risk Factors; Role; STK11 gene; Series; Signal Pathway; Signal Transduction; Sterols; Transcriptional Regulation; Triglycerides; Ubiquitination; Work; base; citrate carrier; cyclin C; db/db mouse; diabetic; diabetic patient; fatty acid biosynthesis; feeding; human disease; improved; in vivo; lipid biosynthesis; multicatalytic endopeptidase complex; mutant; non-alcoholic fatty liver; novel; protein complex; transcription factor

DESCRIPTION (provided by applicant): Dysregulation of hepatic de novo lipogenesis is prevalent in obesity and closely associates with insulin resistance, Type 2 Diabetes and cardiovascular disease. The regulation of de novo lipogenesis in the liver is a complex process that is dependent upon the nutrient/hormone state, availability of substrate precursors, allosteric regulation of key enzymati activity steps, and the transcriptional control of lipogenic gene expression through the transcripton factors ChREBP and in particular for this application the sterol response element-binding protein-1 (SREBP-1c) transcription factor. SREBP-1c is a pivotal activator of rate-limiting enzymes that are responsible for hepatic biosynthesis of fatty acids and triglycerides and it is the primary effecto of insulin-induced de novo lipogenesis in hepatocytes. Insulin and feeding acutely regulates SREBP-1c transcriptional activity at three distinct levels by 1) increasing SREBP-1c transcription; 2) protelytic maturation from its precursor that is initially located in endoplasmic reticulum membrane, and 3) increased nuclear SREBP-1c protein stability. Recently, we have uncovered a novel insulin/nutrient regulated signaling pathway that is primarily responsible for the stability of the nuclear SREBP-1c protein. We have found that in the basal (fasted state) cyclin- dependent kinase-8 (CDK8) in comple with its activator cyclin C (CycC) phosphorylates nuclear SREBP-1c that induces nuclear SREBP-1c ubiquitination and rapid proteasome mediated degradation. In contrast, refeeding results in the dow-regulation of CDK8/CycC protein complex thereby stabilizing nuclear SREBP-1c protein to enhance lipogenic gene expression and de novo lipogenesis in conjunction with other actions of insulin. Thu, the nutrient/hormone down regulation of the CDK8/CycC complex functionally results in the stabilizaion of the nuclear SREBP-1c protein as part of the complex mechanisms driving hepatic lipogenic gene expression. More recently, we have observed that that CDK8 protein stability is inversely related t the mTORC1 activation state. Based upon these recent findings, we propose the following working hypothesis: Activation of the mTORC1-signaling pathway rapidly down regulates the CDK8/CycC protein complex through either a direct or indirect phosphorylation, that in turn leads to dissociaion of the CDK8/CycC complex, subsequent CDK8 and CycC ubiquitination and proteasome-mediated degradation. In this proposal we propose to determine the initial nutrient signaling pathways and molecular mechanisms regulating the CDK8/CycC protein complex and nuclear SREBP-1c stability in the control of lipogenic gene expression and de novo lipogenesis. This will be accomplished by determining hormone/nutrient down regulation of CDK8/CycC complex occurs through 1) activation of mTORC1 and subsequent proteasome-mediated degradation of the CDK8 and CycC proteins; mTORC1-dependent regulation CDK8/CycC complex assembly state; and 3) ubiquitination of lysine residues in CDK8 and CycC. We will then determine whether CDK8, CycC or both are the targets of hormone/nutrient stimulation of mTORC1 signaling by 1) analyzing CDK8 and/or CycC phosphorylation/dephosphorylation; 2) identifying the mTORC1-dependent kinase(s) responsible for CDK8/CycC down regulation; 3) mapping the specific phosphorylation sites in CKD8 and/or CycC; and 4) analyzing the effect of phosphorylation defective mutations on CDK8/CycC stability, nuclear SREB- 1c stability and lipogenic gene expression. Lastly, we will determine if the mTORC1 regulatory mechanism contributes to the normal physiologic and pathophysiologic regulation of de novo lipogenesis in insulin resistant states by 1) examining the CDK8/CycC complex, nuclear SREBP-1c stability, lipogenic gene expression and de novo lipogenesis in liver-specific Raptor and LKB1 knocout mice; 2) determining the function of CDK8/CycC degradation-resistant mutants in mouse liver; and 3) analyzing the regulation of the CDK8/CycC complex in high fat diet-induced insulin resistant and genetically diabetic db/db mice.

描述（由申请人提供）：肝脏从头脂肪生成失调在肥胖症中普遍存在，并且与胰岛素抵抗、2型糖尿病和心血管疾病密切相关。 肝脏从头脂肪生成的调节是一个复杂的过程，依赖于营养/激素状态、底物前体的可用性、关键酶活性步骤的变构调节以及通过转录因子 ChREBP 和特别是对于该应用，甾醇反应元件结合蛋白-1 (SREBP-1c) 转录因子。 SREBP-1c 是限速酶的关键激活剂，负责脂肪酸和甘油三酯的肝脏生物合成，并且是胰岛素诱导的肝细胞从头脂肪生成的主要效应物。胰岛素和喂养在三个不同水平上急剧调节 SREBP-1c 转录活性：1) 增加 SREBP-1c 转录； 2) 最初位于内质网膜的其前体的蛋白水解成熟，以及 3) 核 SREBP-1c 蛋白稳定性增加。最近，我们发现了一种新的胰岛素/营养调节信号通路，该通路主要负责核 SREBP-1c 的稳定性 蛋白质。我们发现，在基础（禁食状态）下，细胞周期蛋白依赖性激酶 8 (CDK8) 与其激活剂细胞周期蛋白 C (CycC) 一起磷酸化核 SREBP-1c，从而诱导核 SREBP-1c 泛素化和快速蛋白酶体介导的降解。相反，再喂食会导致 CDK8/CycC 蛋白复合物的下调，从而稳定核 SREBP-1c 蛋白，从而与胰岛素的其他作用结合增强脂肪生成基因表达和从头脂肪生成。因此，CDK8/CycC 复合物的营养/激素下调在功能上导致核 SREBP-1c 蛋白的稳定，作为驱动肝脂肪生成基因表达的复杂机制的一部分。最近，我们观察到 CDK8 蛋白稳定性与 mTORC1 激活状态呈负相关。基于这些最新发现，我们提出以下工作假设：mTORC1信号通路的激活通过直接或间接磷酸化快速下调CDK8/CycC蛋白复合物，进而导致CDK8/CycC复合物解离，随后 CDK8 和 CycC 泛素化和蛋白酶体介导的降解。在本提案中，我们建议确定调节 CDK8/CycC 蛋白复合物和核 SREBP-1c 稳定性在控制脂肪生成基因表达和从头脂肪生成中的初始营养信号传导途径和分子机制。这将通过确定 CDK8/CycC 复合物的激素/营养素下调来实现，其通过以下方式发生：1) mTORC1 的激活以及随后蛋白酶体介导的 CDK8 和 CycC 蛋白的降解； mTORC1依赖性调节CDK8/CycC复合体组装状态； 3) CDK8 和 CycC 中赖氨酸残基的泛素化。然后，我们将通过 1) 分析 CDK8 和/或 CycC 磷酸化/去磷酸化来确定 CDK8、CycC 或两者是否是 mTORC1 信号传导的激素/营养素刺激的靶标； 2) 鉴定负责 CDK8/CycC 下调的 mTORC1 依赖性激酶； 3)绘制CKD8和/或CycC中特定磷酸化位点的图谱； 4)分析磷酸化缺陷突变对CDK8/CycC稳定性、核SREB-1c稳定性和脂肪生成基因表达的影响。最后，我们将通过以下方法确定 mTORC1 调节机制是否有助于胰岛素抵抗状态下从头脂肪生成的正常生理和病理生理调节：1) 检查胰岛素抵抗状态下的 CDK8/CycC 复合体、核 SREBP-1c 稳定性、脂肪生成基因表达和从头脂肪生成。肝脏特异性 Raptor 和 LKB1 基因敲除小鼠； 2)确定CDK8/CycC降解抗性突变体在小鼠肝脏中的功能；和 3) 分析高脂饮食诱导的胰岛素抵抗和遗传性糖尿病 db/db 小鼠中 CDK8/CycC 复合物的调节。