Context-specific Functions of CDK8

描述(申请人提供)：高度保守的辅因子介体复合体，由多达30个亚基组成，是转录激活物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依赖的转录所必需的。CDK8(Cyclin-Dependent Kinase 8)作为已知的唯一一种介体复合体的酶亚基，可以正向或负向地调控转录。然而，CDK8是否在每个基因上都扮演着激活或抑制的角色，或者它在不同类别的基因中是否发挥不同的作用，这是一个关键的未探索的领域，将极大地完善我们对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)的失调如何导致多种人类癌症至关重要。CDK8的缺失有效地阻止了黑色素瘤和结直肠癌的生长，这突显了CDK8在基因表达中的中心地位，并说明了为什么CDK8被认为是一个有吸引力和有前途的癌症治疗靶点。目前，CDK8-CycC的异常调控如何在肿瘤发生中起作用还知之甚少。要确定这些蛋白的正常和失调功能，必须确定CDK8-CycC的下游效应因子和上游调控因子。由于缺乏多细胞生物体中CDK8活性的表型，对CDK8的功能和调控的分析一直受到阻碍。我们已经通过使用果蝇解决了这一挑战，它在体内操纵CDK8活性方面提供了无与伦比的复杂性。我们最近发现了CDK8的两个重要的下游反式激活因子，SREBP(甾醇调节元件结合蛋白)和ECR(蜕皮激素受体)，它们分别在调节脂肪生成和发育成熟方面发挥重要作用。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，这项提议的目的是确定CDK8如何在调节SREBP和ECR的活动中发挥根本不同的作用。具体地说，我们将通过确定CDK8与SREBP和ECR相互作用的分子机制，以及CDK8在体内对SREBP和ECR磷酸化的影响来阐明CDK8如何调控SREBP和ECR介导的转录。我们还将通过胰岛素信号通路研究CDK8-CycC的生理调节，并分析CDK8在协调脂质平衡和发育时机方面的作用。这项研究将极大地促进我们对CDK8在脂肪形成和发育中的不同作用的理解。由于CDK8和CycC在真核生物中非常保守，我们在果蝇中识别的分子机制很有可能为人类研究提供工作模型。该项目的重要性在于，体内脂质平衡失调与糖尿病、肥胖症、心血管疾病和某些类型的癌症密切相关。

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.