Context-specific Functions of CDK8

描述（由申请人提供）：高度保守的辅助因子中介复合物，包括多达30个亚基，作为转录激活因子和核心转录机制之间的分子桥梁，被认为是大多数RNA聚合酶ii依赖性转录所必需的。CDK8（细胞周期蛋白依赖性激酶8）是介导复合物中唯一已知的酶亚基，可以正调控或负调控转录。然而，CDK8是作为每个基因的激活因子还是抑制因子，还是在不同类型的基因上发挥不同的作用，是一个关键的未探索领域，这将极大地改善我们对Mediator如何促进转录的看法。此外，了解CDK8的功能和调控对于阐明CDK8及其调控伙伴CycC （Cyclin C）的失调如何导致各种人类癌症至关重要。CDK8的缺失有效地阻断了黑色素瘤和结直肠癌的生长，这强调了CDK8在基因表达中的中心地位，并证明了为什么CDK8被认为是一个有吸引力和有希望的癌症治疗靶点。目前，CDK8-CycC的失调如何促进肿瘤发生尚不清楚。为了确定这些蛋白的正常和失调功能，有必要确定CDK8-CycC的下游效应物和上游调节物。由于缺乏多细胞生物中CDK8活性的表型，对CDK8功能和调控的分析一直受到阻碍。我们通过使用果蝇解决了这一挑战，果蝇在体内操纵CDK8活性方面提供了无与伦比的复杂性。我们最近发现了CDK8的两个重要下游反激活因子，SREBP（甾醇调节元件结合蛋白）和EcR（外皮甾体受体），它们分别在调节脂肪生成和发育成熟中起重要作用。我们的分析表明，CDK8抑制srebp依赖的转录，但激活ecr激活的基因表达。因此，本提案的目的是确定CDK8如何在调节SREBP和EcR的活性中发挥根本不同的作用。具体来说，我们将通过确定CDK8与SREBP和EcR相互作用的分子机制，以及CDK8在体内磷酸化SREBP和EcR的作用，阐明CDK8如何调节SREBP和EcR介导的转录。我们还将通过胰岛素信号通路检查CDK8- cycc的生理调节，并分析CDK8在协调脂质稳态和发育时间中的作用。这项研究将极大地促进我们对CDK8在脂肪形成和发展中如何发挥独特作用的理解。由于CDK8和CycC在真核生物中很好地保守，我们在果蝇中发现的分子机制极有可能为人类研究提供工作模型。这个项目的重要性被这样一个事实所强调，即发育过程中脂质稳态失调与糖尿病、肥胖、心血管疾病和某些类型的癌症等疾病密切相关。

DESCRIPTION (provided by applicant): The highly conserved co-factor Mediator Complex, comprising up to 30 subunits, serves as a molecular bridge between transcriptional activators and the core transcription machinery, and is thought to be required for most RNA polymerase II-dependent transcription. As the only known enzymatic subunit of Mediator complex, CDK8 (Cyclin-Dependent Kinase 8) can either positively or negatively regulate transcription. However, whether CDK8 acts as an activator or repressor at every gene, or whether it acts differently at different classes of genes, is a key unexplored field that would dramatically refine our view of how Mediator facilitates transcription. Furthermore, understanding the function and regulation of CDK8 is critical to elucidate how dysregulation of both CDK8 and its regulatory partner CycC (Cyclin C) contributes to a variety of human cancers. Depletion of CDK8 effectively blocks the growth of melanoma and colorectal cancer, which underscores the centrality of CDK8 in gene expression and demonstrates why CDK8 is considered an attractive and promising target for cancer treatment. Currently, how dysregulation of CDK8-CycC contributes to tumorigenesis is poorly understood. To determine the normal and dysregulated functions of these proteins, it is essential to identify the downstream effectors and upstream regulators of CDK8-CycC. Analyses of function and regulation of CDK8 have been hampered by the lack of phenotypes for CDK8 activity in multicellular organisms. We have solved this challenge by using Drosophila, which provides unparalleled sophistication in manipulating CDK8 activity in vivo. We have recently identified two important downstream transactivators of CDK8, SREBP (Sterol Regulatory Element-Binding Protein) and the EcR (Ecdysteroid Receptor), which play essential roles in regulating lipogenesis and developmental maturation, respectively. Our analyses suggest that CDK8 inhibits SREBP-dependent transcription but activates EcR-activated gene expression. Thus, the objective of this proposal is to determine how CDK8 plays fundamentally different roles in modulating the activities of SREBP and EcR. Specifically, we will elucidate how CDK8 regulates SREBP- and EcR-mediated transcription by determining the molecular mechanism underlying the interactions between CDK8 and both SREBP and EcR, and the effects of SREBP and EcR phosphorylation by CDK8 in vivo. We will also examine the physiological regulation of CDK8-CycC by the insulin-signaling pathway and analyze the role of CDK8 in coordinating lipid homeostasis and developmental timing. This study will significantly advance our understanding of how CDK8 plays distinct roles in lipogenesis and development. Because CDK8 and CycC are well conserved in eukaryotes, the molecular mechanisms that we identify in Drosophila are highly likely to provide working models in human studies. The importance of this project is highlighted by the fact that dysregulation of lipid homeostasis durin development is closely linked to diseases such as diabetes, obesity, cardiovascular diseases, and certain types of cancers.