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）：肝脏脂肪形成的失调在肥胖症中普遍存在，并与胰岛素抵抗，2型糖尿病和心血管疾病紧密相关。 肝脏中从头脂肪生成的调节是一个复杂的过程，取决于养分/激素状态，底物前体的可用性，关键酶活性活性步骤的变构调节以及通过转录量CHREBP的转录基因表达的转录控制，尤其是该应用程序的型号响应元素 - 固醇元素蛋白质1（sy）1C（SROD）1C（SREP）。 SREBP-1C是限制速率酶的关键活化剂，负责脂肪酸和甘油三酸酯的肝生物合成，它是胰岛素诱导的DE从肝细胞中胰岛素诱导的DE脂肪生成的主要作用。胰岛素和喂养急性调节SREBP-1C转录活性在三个不同的水平上1）增加SREBP-1C转录； 2）最初位于内质网膜中的前体的原性成熟，3）增加了核SREBP-1C蛋白稳定性。最近，我们发现了一种新型的胰岛素/营养调节信号通路，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）依赖性激酶8（CDK8）中，其激活剂Cyclin c（cycc）磷酸化的核SREBP-1C磷酸化，从而诱导核SREBP-1C泛素化和快速的蛋白酶体介导的介导的降解。相比之下，再进程会导致CDK8/CYCC蛋白复合物的DOW调节，从而稳定核SREBP-1C蛋白，从而增强脂肪生成基因表达和从头脂肪生成，并结合其他胰岛素作用。 THU，CDK8/CYCC复合物的营养/激素减少调节在功能上会导致核SREBP-1C蛋白的稳定，这是驱动肝脂肪基因表达的复杂机制的一部分。最近，我们观察到CDK8蛋白稳定性成反比MTORC1激活状态。 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.在此提案中，我们建议确定调节CDK8/CYCC蛋白复合物和核SREBP-1C稳定性的初始养分信号通路和分子机制，以控制脂肪生成基因表达和从头脂肪生成。这将通过确定CDK8/CYCC复合物的激素/养分减少来实现，这是通过1）激活MTORC1的激活以及随后的蛋白酶体介导的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/dephosphoration来确定CDK8，CYCC或两者是MTORC1信号传导的激素/营养刺激的靶标； 2）识别负责CDK8/CYCC调节的MTORC1依赖性激酶； 3）映射CKD8和/或CYCC中的特定磷酸化位点； 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。 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）确定小鼠肝脏中CDK8/CYCC降解突变体的功能； 3） 分析在高脂肪饮食诱导的胰岛素耐药性和遗传性糖尿病DB/DB小鼠中的CDK8/CYCC复合物的调节。