Function of Cdk8 in Eukaryotic Cell Growth and Differentiation

Cdk8在真核细胞生长和分化中的功能

• 批准号: RGPIN-2020-05362
• 负责人: Sadowski, Ivan
• 金额: $ 2.33万
• 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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744276
• 关键词: Function; Cdk8; Eukaryotic; Cell; Growth

Our research examining Cdk8 in yeast, a protein kinase of the RNA Pol II mediator complex, has revealed its central role in regulating specific gene expression in response to global physiological signals. Cdk8 activates or inhibits gene-specific transcription factors by direct phosphorylation. Under ideal nutritional conditions, Cdk8 phosphorylates Gal4 to promote full GAL induction, but inhibits factors involved in filamentous differentiation. Nutrient limitation inhibits Cdk8 function, which limits GAL induction, but conversely allows accumulation of factors promoting differentiation. Despite its central role in response to global signals, the mechanisms regulating Cdk8 activity have not been identified. We have used genetic strategies to identify regulators of Cdk8, which revealed that it is controlled by multiple pathways that regulate stability and activity of the kinase, and phosphorylation of its substrates. We found that Cdk8 stability may be regulated by Tpk2, a PKA isomer, and that phosphorylation of Gal4 is opposed by the PP2A-Cdc55 phosphatase regulated by Tor signalling. Central to this research is a collection of mutants (gft) which were isolated in a screen for defects in Cdk8-dependent GAL gene expression. We propose that characterization of these mutants will reveal mechanisms controlling this kinase, which are likely to be conserved in all eukaryotes. Aim 1: Role of PKA signalling for Cdk8. We will confirm that Tpk2 directly phosphorylates Cdk8, and characterize the function of this phosphorylation for protein stability. Proteins required for degradation of Cdk8 in response to nitrogen limitation will be identified. Aim 2: Role of Cdc55-PP2A for GAL induction. The effect of gft and tor signalling mutants on PP2A-Cdc55 activity will be determined, and the ability of PP2A-Cdc55 to dephosphorylate Gal4 at the regulatory S699 phosphorylation will be examined. Aim 3: Effect of Tor and AMPK/ Snf1 on Cdk8. Effects of AMPK/ Snf1 and cross-talk between the Tor and PKA pathways on Cdk8 activity will be examined using in vitro kinase reactions and analysis of substrate phosphorylation in vivo. Aim 4: Characterization of additional gft mutations. The remaining 11 gft mutants isolated in our screen will be identified and characterized. One of these was recently identified as med2; we will characterize the effect of this mutant on Cdk8 activity and association with the mediator complex. 5 of the mutants affect Cdk8 kinase activity, while the remainder inhibit Gal4 phosphorylation and GAL induction without affecting kinase activity of Cdk8. We expect that identification of these additional mutations will provide novel insight into regulation of Cdk8 activity for nutrient and stress response. Cdk8 is highly conserved in eukaryotes and represents an important regulator of gene expression in response to environmental signals. We expect this research will reveal novel signalling mechanisms regulating its activity.

我们的研究检查Cdk 8在酵母中，RNA Pol II介体复合物的蛋白激酶，揭示了其在调节特定基因表达响应全球生理信号的核心作用。cdk 8通过直接磷酸化激活或抑制基因特异性转录因子。在理想的营养条件下，Cdk 8磷酸化Gal 4，以促进完全GAL诱导，但抑制因子参与丝状分化。营养限制抑制Cdk 8功能，这限制了GAL诱导，但相反地允许促进分化的因子积累。尽管它在响应全球信号中发挥着核心作用，但调节Cdk 8活性的机制尚未确定。 我们已经使用遗传策略来确定Cdk 8的调节因子，这表明它是由调节激酶的稳定性和活性以及其底物的磷酸化的多个途径控制的。我们发现Cdk 8的稳定性可能受PKA异构体Tpk 2的调节，Gal 4的磷酸化受到Tor信号调节的PP 2A-Cdc 55磷酸酶的反对。这项研究的核心是收集突变体（gft），这些突变体是在Cdk 8依赖性GAL基因表达缺陷的筛选中分离出来的。我们建议，这些突变体的表征将揭示控制这种激酶，这可能是在所有真核生物中保守的机制。目的1：PKA信号转导对Cdk 8的作用。 我们将证实Tpk 2直接磷酸化Cdk 8，并表征这种磷酸化对蛋白质稳定性的功能。将确定响应氮限制的Cdk 8降解所需的蛋白质。目的2：Cdc 55-PP 2A对GAL诱导的作用。将测定gft和tor信号传导突变体对PP 2A-Cdc 55活性的影响，并将检查PP 2A-Cdc 55在调节S699磷酸化时使Gal 4去磷酸化的能力。 目的3：Tor和AMPK/Snf 1对Cdk 8的影响。 AMPK/Snf 1和Tor和PKA通路之间的串扰对Cdk 8活性的影响将使用体外激酶反应和体内底物磷酸化分析进行检查。目的4：表征额外的gft突变。将鉴定和表征在我们的筛选中分离的其余11个gft突变体。其中之一最近被确定为Med 2，我们将描述这种突变体对Cdk 8活性的影响，并与介体复合物相关联。其中5个突变体影响Cdk 8激酶活性，而其余突变体抑制Gal 4磷酸化和GAL诱导，而不影响Cdk 8的激酶活性。我们希望这些额外的突变的鉴定将提供新的见解调节Cdk 8活性的营养和应激反应。cdk 8在真核生物中高度保守，是响应环境信号的基因表达的重要调节因子。我们希望这项研究将揭示调节其活性的新信号机制。