Regulation of cell differentiation by RNA Polymerase II-associated Cdk8

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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.