Understanding the Role of Cdk8 in Glycolysis Gene Expression Regulation via Gcr2

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Raymond, Mary-Elizabeth
Cdk8 , Glycolysis , Gcr2 , Transcription regulation , Cancer , Mediator Complex
Transcription regulation is an essential molecular process that organisms use to respond to internal and external cues, the dysregulation of which can result in disease. The cyclin dependent kinase 8 (Cdk8) regulates transcription in a variety of molecular mechanisms including association with the Mediator complex and direct phosphorylation of a number of substrates including many transcription factors. In humans, Cdk8 functions as a colorectal cancer oncogene by regulating glycolysis gene expression although the molecular mechanisms by which Cdk8 regulates glycolysis remains unknown. Previous work showed that Cdk8 could phosphorylate Serine 365 of Gcr2, a transcriptional co-activator of glycolysis genes in budding yeast. To understand how Cdk8 regulates glycolysis gene expression regulation in vivo, I will: (1) determine if Gcr2-S365 is phosphorylated in vivo and contributes to normal Gcr2 function; (2) identify the conditions under which Gcr2 is phosphorylated by Cdk8 (3); and understand Gcr2 regulation by examining the Gcr2 posttranslational modification (PTM) landscape. Utilizing a Gcr2-S365 phosphorylation-specific antibody, this work shows CDK8-dependent Gcr2-S365 phosphorylation in vivo. Using phospho-mutant alleles, I found that Gcr2-S365 phosphorylation regulates the mRNA levels of representative glycolysis genes in a condition-specific manner. I also found that a switch from fermentable to non-fermentable carbon sources led to increases in Gcr2 phosphorylation, which was partially dependent on CDK8, but it did not involve changes to S365 phosphorylation. To identify phosphorylation events on Gcr2 that mediate the response to growth in non-fermentable carbon, I leveraged recent mass spectrometry studies. This analysis identified 22 residues on Gcr2 that could be phosphorylated in vivo, underscoring the complexity of Gcr2 regulation. Consistently, examination of C-terminal truncations that progressively remove clusters of phosphorylated residues revealed a condition-specific dominant negative effect on growth that highlights the importance of the C-terminal tail of GCR2 for normal function. Together, this work showed CDK8 dependent Gcr2-S365 phosphorylation in vivo and demonstrates a role for this residue in transcription regulation in response to non-fermentable carbon. This work also indicates that Gcr2 regulation is complex, involving condition-specific phosphorylation at multiple residues.
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