mTORC1-dependent regulation of the CycC/CDK8 complex

描述(由申请人提供)：肝脏新生脂肪生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密切相关。 肝脏新生脂肪生成的调控是一个复杂的过程，它依赖于营养/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，特别是对于该应用而言，固醇反应元件结合蛋白-1(SREBP-1c)转录因子对造脂基因表达的转录控制。SREBP-1c是限速酶的关键激活物，参与肝脏脂肪酸和甘油三酯的生物合成，是胰岛素诱导肝细胞新生脂肪生成的主要作用。胰岛素和喂食在三个不同的水平上强烈地调节SREBP-1c的转录活性：1)增加SREBP-1c的转录；2)从最初位于内质网膜的前体蛋白裂解成熟；3)增加核SREBP-1c蛋白的稳定性。最近，我们发现了一个新的胰岛素/营养调节的信号通路，它主要负责核SREBP-1c的稳定性 蛋白。我们发现，在基础状态(禁食状态)，细胞周期蛋白依赖的蛋白-8(CDK8)与其激活剂细胞周期蛋白C(CycC)一起磷酸化核内的SREBP-1c，从而导致核内SREBP-1c泛素化和蛋白酶体的快速降解。相反，再喂食导致CDK8/CycC蛋白复合体的Dow调节，从而稳定核SREBP-1c蛋白，从而增强成脂基因的表达，并与胰岛素的其他作用一起从头开始脂肪生成。清华，营养/激素下调CDK8/CycC复合体功能导致核SREBP-1c蛋白稳定，这是推动肝脏造脂基因表达的复杂机制的一部分。最近，我们观察到CDK8蛋白的稳定性与mTORC1的激活状态成反比。基于这些最新发现，我们提出如下工作假设：mTORC1-信号通路的激活通过直接或间接的磷酸化迅速下调CDK8/CycC蛋白复合体，进而导致CDK8/CycC复合体的解离，随后CDK8和CycC泛素化和蛋白酶体介导的降解。在这项建议中，我们建议确定最初的营养信号通路和分子机制，调节CDK8/CycC蛋白复合体和核SREBP-1c的稳定性，以控制脂肪生成基因的表达和新的脂肪生成。这将通过确定CDK8/CycC复合体的激素/营养下调发生在：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依赖的调节CDK8/CycC复合体的组装状态；以及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的磷酸化/去磷酸化；2)确定负责CDK8/CycC下调的mTORC1依赖的激酶(S)；3)定位CKD8和/或CycC中的特定磷酸化位点；以及4)分析磷酸化缺陷突变对CDK8/CycC稳定性、核SREB-1c稳定性和致脂基因表达的影响，从而确定CDK8、CycC或两者都是激素/营养刺激mTORC1信号转导的靶点。最后，我们将通过1)检测肝脏特异性Raptor和LKB1基因敲除小鼠的CDK8/CycC复合体、核SREBP-1c稳定性、成脂基因表达和新脂肪生成；2)确定CDK8/CycC降解抗性突变体在小鼠肝脏中的功能；以及3)确定mTORC1调控机制是否有助于胰岛素抵抗状态下新生脂肪生成的正常生理和病理生理调节；以及3) 分析CDK8/CycC复合体在高脂饮食诱导的胰岛素抵抗和遗传性糖尿病db/db小鼠中的调节。

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.