Function of Cdk8 in Eukaryotic Cell Growth and Differentiation

我们对酵母中CDK8的研究揭示了它在调节特定基因表达以响应全球生理信号方面的核心作用。CDK8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，CDK8使Gal4磷酸化以促进完整的GAL诱导，但抑制参与丝状分化的因子。营养限制抑制CDK8功能，CDK8功能限制GAL诱导，但反过来允许促进分化的因素积累。尽管CDK8在响应全球信号方面发挥了核心作用，但调控CDK8活性的机制尚未确定。我们已经使用遗传策略来确定CDK8的调节因子，这揭示了它受多个途径控制，这些途径调节激酶的稳定性和活性，以及其底物的磷酸化。我们发现，CDK8的稳定性可能受到PKA异构体Tpk2的调节，而Gal4的磷酸化则与Tor信号调节的PP2A-CDC55磷酸酶相反。这项研究的中心是一组突变体(GFT)，它们是在CDK8依赖的GAL基因表达缺陷的筛查中分离出来的。我们认为，这些突变体的特征将揭示控制该激酶的机制，它可能在所有真核生物中都是保守的。目的1：PKA信号在CDK8中的作用。我们将证实Tpk2直接磷酸化CDK8，并表征这种磷酸化对蛋白质稳定性的作用。将确定对氮素限制反应的CDK8降解所需的蛋白质。目的2：CDC55-PP2A在GAL诱导中的作用。将确定GFT和TOR信号突变体对PP2A-CDC55活性的影响，并检测PP2A-CDC55在调节S699磷酸化时脱磷酸化Gal4的能力。目的3：Tor和AMPK/Snf1对CDK8的影响。AMPK/Snf1以及Tor和PKA通路之间的串扰对CDK8活性的影响将通过体外激酶反应和体内底物磷酸化分析来检测。目的4：其他GFT突变的特征。在我们的筛选中分离到的其余11个GFT突变体将被鉴定和鉴定。其中之一最近被鉴定为Med2；我们将表征这个突变体对CDK8活性的影响以及与介体复合体的联系。其中5个突变影响CDK8的活性，其余的抑制Gal4的磷酸化和GAL的诱导，而不影响CDK8的活性。我们期望这些额外突变的鉴定将为调节CDK8活性以调节营养和应激反应提供新的见解。CDK8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。我们预计这项研究将揭示调节其活动的新的信号机制。

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.