Cdk8 directly regulates glycolysis via phosphorylation of Gcr2

CDK8 encodes an evolutionarily conserved Mediator complex kinase subunit that functions in general and context-specific transcription regulation by phosphorylating core components of the transcription machinery and gene-specific transcription factors. To better understand the role Cdk8 in transcription regulation, we performed high-resolution gene expression time course analysis following nuclear depletion of Cdk8. Focusing on the earliest gene expression alterations revealed dysregulation of genes encoding glycolysis enzymes, suggesting a functional link to Gcr1 and Gcr2, key transcriptional activators of these genes. Consistently, we found that nuclear depletion of Cdk8 altered the mRNA levels of glycolysis genes as well as the promoter occupancy of Gcr2, but not Gcr1. Examination of the Gcr2 protein sequence revealed a putative Cdk8 phosphorylation site at serine 365, which we confirmed using in vitro and in vivo assays. Importantly, phospho-mutant GCR2 recapitulated the growth and gene expression defects of the GCR2 deletion mutant, effects not observed with a phospho mimetic mutant. As such, our work highlights Gcr2 as a new Cdk8 substrate, revealing that its phosphorylation is critical for the activation of genes encoding glycolysis enzymes.