Date of Award

Spring 1-1-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Dylan J. Taatjes

Second Advisor

Deborah S. Wuttke

Third Advisor

James Goodrich

Fourth Advisor

William Old

Fifth Advisor

Joaquin Espinosa

Abstract

(Pol II) transcription that can interact with gene-specific transcription factors and the general transcription machinery. Little is known about the molecular mechanisms by which Mediator functions. Within this multi-subunit, 1.2MDa assembly there is only one known enzymatic activity, which is carried out by CDK8 and its paralog CDK19. These kinases function in the cell as “modules” consisting of CDK8 or CDK19, Cyclin C, MED12 and MED13. This module can reversibly associate with the Mediator complex to form CDK8/19-Mediator, and can modulate the structure and activity of the assembly upon binding.

Few cellular CDK8/19 substrates are known, owing in part to a lack of specific small-molecule inhibitors for these kinases. Here, we assist in the characterization of a new CDK8 substrate, the activation domain of the transcription factor STAT1. We identify the site of phosphorylation using kinase assays and mutagenesis, and show that it is phosphorylated at a single site. We then identify a potent and specific small-molecule inhibitor of CDK8/19 activity. This inhibitor does not inhibit other transcriptional CDKs, and it inhibits CDK8/19 activity with an IC50 in the low nanomolar range. Based upon a co-crystal structure of the small molecule bound to CDK8-CCNC, we identify and test a CDK8/19 mutant that conveys resistance to the inhibitor.

To more comprehensively identify substrates of CDK8/19, we used inhibitor treatment coupled with phosphoproteomics to elucidate novel kinase targets from human cells. The biological function of most of these substrates involves Pol II transcription and includes transcription factors, Mediator subunits and chromatin-modifying enzymes. Further use of quantitative mass spectrometry experiments shows that phosphorylation by Mediator kinases is able to regulate protein abundance in select cases for identified substrates. Additionally, targeted inhibition of CDK8/19 alters the expression of a small number of genes, some of which can be linked to phosphoproteomic changes.

Collectively, these data represent the first large-scale identification of CDK8/19 kinase substrates to date and point to regulatory functions for CDK8/19 activity. This analysis has helped expand our understanding of the diverse mechanisms by which CDK8/19 control transcription.

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