mTORC1-dependent regulation of the CycC/CDK8 complex

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.