Regulation of cell differentiation by RNA Polymerase II-associated Cdk8

RNA 聚合酶 II 相关的 Cdk8 对细胞分化的调节

• 批准号: 418261-2013
• 负责人: Sadowski, Ivan
• 金额: $ 2.94万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=573563
• 关键词: Regulation; cell; differentiation; RNA; Polymerase

Cellular growth and differentiation are highly regulated by signals from the environment. The budding yeast Saccharomyces cerevisiae has provided an important model to study molecular details of how cellular differentiation is controlled through regulation of transcription factors by signals from the environment. Under conditions of nutrient limitation, yeast differentiate from their normal spherical shape into an elongated filamentous pseudohyphal cellular morphology, which are capable of burrowing into the growth substrate to facilitate forging for nutrients. Our research has shown that Cdk8, a cyclin-dependent protein kinase associated with the RNA Polymerase II mediator, plays a critical role in controlling the decision to undergo cellular differentiation. Cdk8 phosphatorylates and inhibits at least two sequence-specific DNA binding proteins, including Ste12 and Phd1, which are responsible for activation of genes necessary for cellular differentiation. When cells encounter unfavorable nutrient conditions, typified by poor sources of nitrogen or carbon, Cdk8 activity is inhibited, which in turn relieves the negative effect of Cdk8 on these factors to allow hyperactivation of genes that drive differentiation. These results identify Cdk8 as a primary target for signals controlling cellular differentiation, and our objectives are to determine the molecular mechanisms by which nutrient signals control Cdk8 function, and to characterize the complete spectrum of its target substrate proteins for controlling differentiation. Aim 1: Regulation of Cdk8 by Ras-Tpk2-dependent signaling. Our results show that Cdk8 is directly phosphorylated and regulated by Tpk2, a protein kinase A (PKA) isoform, in response to nitrogen limitation. Using genetic and biochemical strategies, we will define how nitrogen limitation specifically regulates this branch of the PKA pathway downstream of the membrane-associated GTPases Ras and Gpa2. Aim 2: Mechanism for destruction of Cdk8 by Tpk2-dependent phosphorylation. Phosphorylation of Cdk8 at T37 causes its degradation that is initiated by a sequence-specific protease. This mechanism will be examined by genetic identification of the cis-sequences on Cdk8, and trans-acting gene products, that are necessary for the process. The relationship between these factors and signaling through the Ras-PKA/ Tpk2 pathway will be determined. Aim 3: Parallel pathways for regulation of Cdk8 by carbon limitation. Genetic observations indicate that Cdk8 activity, and pseudohyphal differentiation, are also controlled by parallel pathways responsive to limitations in carbon quality. We will use Cdk8-responsive reporter systems to identify these signaling pathways, along with subsequent biochemical analysis to identify molecular mechanisms for control of Cdk8 activity. Longer term aim: global identification of Cdk8 substrates. We will use an ATP-analog-sensitive CDK8 allele in combination with MS proteomics strategies to globally identify Cdk8 target proteins. Phosphopeptides whose abundance is affected upon direct inhibition of Cdk8 will be identified. Additionally, global gene expression analysis will be used to define the complete spectrum of Cdk8-dependent target genes during nutrient-limitation induced differentiation. Our research provides a novel perspective on how environmental signals can force cellular differentiation through regulation of a single protein kinase that is closely associated with the general transcription factor machinery. Cdk8 is highly conserved throughout eukaryotic evolution, and considering that little is known regarding its function in metazoans, this research will provide valuable insight into mechanisms controlling development of more complicated species.

细胞的生长和分化受到来自环境的信号的高度调节。 芽殖酵母Saccharomyces cerevisiae为研究细胞分化的分子细节提供了一个重要的模型，细胞分化是如何通过环境信号调节转录因子来控制的。 在营养限制的条件下，酵母从它们的正常球形分化成细长的丝状假菌丝细胞形态，其能够钻入生长基质以促进锻造营养。 我们的研究表明，Cdk 8，一种与RNA聚合酶II介体相关的细胞周期蛋白依赖性蛋白激酶，在控制细胞分化的决定中起着关键作用。 Cdk 8磷酸化并抑制至少两种序列特异性DNA结合蛋白，包括Ste 12和Phd 1，它们负责细胞分化所需基因的激活。 当细胞遇到不利的营养条件时，典型的是氮或碳来源不足，Cdk 8活性受到抑制，这反过来又减轻了Cdk 8对这些因子的负面影响，从而使驱动分化的基因超活化。 这些结果确定Cdk 8作为控制细胞分化的信号的主要目标，我们的目标是确定营养信号控制Cdk 8功能的分子机制，并表征其控制分化的靶底物蛋白的完整谱。 目的1：Ras-Tpk 2依赖性信号对Cdk 8的调控。 我们的研究结果表明，Cdk 8直接磷酸化和调节TPK 2，蛋白激酶A（PKA）亚型，在响应氮限制。 使用遗传和生化策略，我们将确定如何氮限制，特别是调节这个分支的PKA通路下游的膜相关的GTP酶Ras和Gpa 2。 目的2：通过Tpk 2依赖性磷酸化破坏Cdk 8的机制。 Cdk 8在T37处的磷酸化导致其降解，该降解由序列特异性蛋白酶启动。 这一机制将通过对Cdk 8上的顺式序列和反式作用基因产物的遗传鉴定来检查，这是该过程所必需的。 将确定这些因子与通过Ras-PKA/Tpk 2途径的信号传导之间的关系。 目的3：通过碳限制调节Cdk 8的平行途径。 遗传观察表明，Cdk 8的活性，和假菌丝分化，也控制了平行的途径，碳质量的限制。 我们将使用Cdk 8响应报告系统，以确定这些信号通路，沿着与随后的生化分析，以确定控制Cdk 8活性的分子机制。 长期目标：Cdk 8底物的全球识别。 我们将使用一个ATP类似物敏感的CDK 8等位基因结合MS蛋白质组学策略，在全球范围内确定Cdk 8靶蛋白。 将鉴定其丰度受Cdk 8直接抑制影响的磷酸肽。 此外，全局基因表达分析将用于定义在营养限制诱导分化期间Cdk 8依赖性靶基因的完整谱。 我们的研究为环境信号如何通过调节与一般转录因子机制密切相关的单一蛋白激酶来迫使细胞分化提供了一个新的视角。 cdk 8在整个真核生物进化过程中高度保守，考虑到对它在后生动物中的功能知之甚少，这项研究将为控制更复杂物种发育的机制提供有价值的见解。