Comprehensive Identification of CDK8 Kinase Targets Using SILAC Phosphoproteomics

描述(申请人提供)：普遍表达的人CDK8激酶是一种有效的癌基因，是哺乳动物发育所必需的。CDK8的活性是其生物学功能所必需的；然而，很少有底物被鉴定出来。理解任何一种酶的生物学作用的一个基本的第一步是确定它修饰的底物(并因此调节)。这里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，这两个实验室的不同专业领域将协同解决这一重要但具有挑战性的问题。为了确定CDK8激酶的靶点，我们将在结肠癌细胞系中进行基于SILAC的比较蛋白组学分析，这些细胞系经有效和高选择性的CDK8激酶抑制剂处理(或未处理)。我们还将在缺乏CDK8或表达野生型CDK8、死于激酶的CDK8或结构性活性CDK8的细胞中完成比较的磷蛋白质组分析。目前只鉴定了几种CDK8激酶底物(如组蛋白H3、E2F1)，但已知每一种底物都在一般和基因特异性的转录控制中发挥重要作用。因为CDK8可逆地与介体复合体(RNA聚合酶II的全基因组调节因子)结合，所以它被招募到整个基因组的调节点。对CDK8激酶靶标的全球、公正的评估将为CDK8激酶如何调控转录提供前所未有的洞察力。此外，识别的CDK8底物可能揭示新的靶点和策略来阻断癌细胞中依赖CDK8的通路。

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.