Comprehensive Identification of CDK8 Kinase Targets Using SILAC Phosphoproteomics

描述（由申请人提供）：普遍表达的人CDK 8激酶是一种强效致癌基因，是哺乳动物发育所必需的。CDK 8激酶活性对其生物学功能至关重要;然而，已鉴定的激酶底物很少。理解任何激酶的生物学作用的基本第一步是确定它修饰（并因此调节）的底物。本文描述的实验将使用强大的方法组合来解决这些重要问题;该项目涉及两个实验室之间的合作，其不同的专业领域将协同解决这个重要而具有挑战性的问题。为了鉴定CDK 8激酶靶点，我们将在用强效和高选择性CDK 8激酶抑制剂处理（或未处理）的结肠癌细胞系中进行基于SILAC的磷酸化蛋白质组学比较分析。我们还将在缺乏CDK 8或表达野生型CDK 8、激酶死亡CDK 8或组成型活性CDK 8的细胞中完成比较磷酸化蛋白质组学分析。只有少数CDK 8激酶底物已被确定（如组蛋白H3，E2 F1），但每一个已知的一般和基因特异性的方式在控制转录中发挥重要作用。因为CDK 8可逆地与介体复合物（RNA聚合酶II的全基因组调节物）结合，所以它被募集到整个基因组的调节位点。对CDK 8激酶靶点的全球性、无偏见的评估将为CDK 8激酶如何调节转录提供前所未有的见解。此外，鉴定的CDK 8底物可能揭示阻断癌细胞中CDK 8依赖性通路的新靶点和策略。

DESCRIPTION (provided by applicant): The ubiquitously expressed human CDK8 kinase is a potent oncogene, and is required for mammalian development. CDK8 kinase activity is essential for its biological function; however, few kinase substrates have been identified. A basic first ste in understanding the biological role for any kinase is to identify the substrates that it modifies (and therefore regulates). The experiments described herein will address these important issues using a powerful combination of approaches; this project involves collaboration between two labs whose diverse areas of expertise will synergize to tackle this important yet challenging problem. To identify CDK8 kinase targets, we will perform comparative SILAC-based phosphoproteomic analyses in colon cancer cell lines treated (or not treated) with a potent and highly selective CDK8 kinase inhibitor. We will also complete comparative phosphoproteomic analyses in cells that lack CDK8, or express wild-type CDK8, kinase-dead CDK8, or constitutively active CDK8. Only a handful of CDK8 kinase substrates have been identified (e.g. histone H3, E2F1), yet each is known to play major roles in controlling transcription in both general and gene-specific ways. Because CDK8 reversibly associates with the Mediator complex (a genome-wide regulator of RNA polymerase II), it is recruited to regulatory loci throughout the genome. A global, unbiased assessment of CDK8 kinase targets will provide unprecedented insight into how the CDK8 kinase regulates transcription. Moreover, the identified CDK8 substrates may reveal new targets and strategies to block CDK8-dependent pathways in cancer cells.