Function of Cdk8 in Eukaryotic Cell Growth and Differentiation

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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.