Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, SC 29208, USA.
Senex Biotechnology, Inc. Columbia, SC 29208, USA.
Nucleic Acids Res. 2023 Aug 11;51(14):7288-7313. doi: 10.1093/nar/gkad538.
We have conducted a detailed transcriptomic, proteomic and phosphoproteomic analysis of CDK8 and its paralog CDK19, alternative enzymatic components of the kinase module associated with transcriptional Mediator complex and implicated in development and diseases. This analysis was performed using genetic modifications of CDK8 and CDK19, selective CDK8/19 small molecule kinase inhibitors and a potent CDK8/19 PROTAC degrader. CDK8/19 inhibition in cells exposed to serum or to agonists of NFκB or protein kinase C (PKC) reduced the induction of signal-responsive genes, indicating a pleiotropic role of Mediator kinases in signal-induced transcriptional reprogramming. CDK8/19 inhibition under basal conditions initially downregulated a small group of genes, most of which were inducible by serum or PKC stimulation. Prolonged CDK8/19 inhibition or mutagenesis upregulated a larger gene set, along with a post-transcriptional increase in the proteins comprising the core Mediator complex and its kinase module. Regulation of both RNA and protein expression required CDK8/19 kinase activities but both enzymes protected their binding partner cyclin C from proteolytic degradation in a kinase-independent manner. Analysis of isogenic cell populations expressing CDK8, CDK19 or their kinase-inactive mutants revealed that CDK8 and CDK19 have the same qualitative effects on protein phosphorylation and gene expression at the RNA and protein levels, whereas differential effects of CDK8 versus CDK19 knockouts were attributable to quantitative differences in their expression and activity rather than different functions.
我们对 CDK8 及其同工酶 CDK19（与转录中介体复合物相关的激酶模块的替代酶成分，与发育和疾病有关）进行了详细的转录组学、蛋白质组学和磷酸化蛋白质组学分析。这项分析是使用 CDK8 和 CDK19 的遗传修饰、选择性 CDK8/19 小分子激酶抑制剂和一种有效的 CDK8/19 PROTAC 降解剂进行的。在暴露于血清或 NFκB 或蛋白激酶 C（PKC）激动剂的细胞中抑制 CDK8/19 会减少信号响应基因的诱导，表明中介体激酶在信号诱导的转录重编程中具有多效性作用。在基础条件下抑制 CDK8/19 最初会下调一小部分基因，其中大多数基因可被血清或 PKC 刺激诱导。长时间抑制 CDK8/19 或突变会上调更大的基因集，同时核心中介体复合物及其激酶模块的蛋白质组成也会发生转录后增加。RNA 和蛋白质表达的调节都需要 CDK8/19 激酶活性，但这两种酶以非激酶依赖的方式保护其结合伴侣 cyclin C 免受蛋白水解降解。对表达 CDK8、CDK19 或其激酶失活突变体的同基因细胞群进行分析表明，CDK8 和 CDK19 对蛋白质磷酸化和 RNA 和蛋白质水平的基因表达具有相同的定性影响，而 CDK8 与 CDK19 敲除的差异影响归因于它们表达和活性的定量差异，而不是不同的功能。
