Regulation of cell differentiation by RNA Polymerase II-associated Cdk8

细胞的生长和分化受到环境信号的高度调控。发芽酵母为研究细胞分化如何通过环境信号调控转录因子提供了一个重要的模型。在营养限制的条件下，酵母从正常的球形分化为细长的丝状假菌丝细胞形态，能够钻入生长底物以便于锻造营养。我们的研究表明，CDK8，一种与RNA聚合酶II介体相关的细胞周期蛋白依赖的蛋白激酶，在控制细胞分化的决定中发挥着关键作用。CDK8磷酸化并抑制至少两个序列特异性DNA结合蛋白，包括STE12和PhD1，这两个蛋白负责激活细胞分化所需的基因。当细胞遇到不利的营养条件，以氮源或碳源不足为代表时，CDK8的活性受到抑制，这反过来又缓解了CDK8对这些因素的负面影响，从而允许驱动分化的基因过度激活。这些结果确定CDK8是控制细胞分化的信号的主要靶点，我们的目标是确定营养信号控制CDK8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-Tpk2依赖的信号对CDK8的调节作用。我们的结果表明，CDK8被直接磷酸化，并受到蛋白激酶A(PKA)亚型Tpk2的调节，以响应氮的限制。使用遗传和生化策略，我们将定义氮限制如何具体调控膜相关GTP酶RAS和Gpa2下游的PKA途径的这一分支。 目的2：Tpk2依赖的磷酸化破坏CDK8的机制。CDK8在T37处的磷酸化导致其降解，这种降解是由序列特异的蛋白酶启动的。这一机制将通过对CDK8上的顺式序列和反式作用基因产物的遗传鉴定来检验，这是这一过程所必需的。这些因素与Ras-PKA/Tpk2信号通路之间的关系将被确定。 目标3：碳限制调节CDK8的平行途径。遗传观察表明，CDK8活性和假菌丝分化也受平行途径的控制，这些途径对碳质量的限制做出反应。我们将使用CDK8反应的报告系统来识别这些信号通路，并结合后续的生化分析来识别控制CDK8活性的分子机制。 长期目标：CDK8底物的全球鉴定。我们将使用ATP类似物敏感的CDK8等位基因结合MS蛋白质组学策略来全球识别CDK8靶蛋白。将确定其丰度受到CDK8直接抑制的影响的磷酸肽。此外，全球基因表达分析将用于确定营养限制诱导分化过程中依赖CDK8的靶基因的完整谱。 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个蛋白激酶来迫使细胞分化提供了一个新的视角。CDK8在真核生物进化过程中高度保守，考虑到人们对其在后生动物中的功能知之甚少，本研究将为更复杂物种的发育控制机制提供有价值的见解。

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.