title: Identification of Mediator Kinase Substrates in Human Cells using Cortistatin A and Quantitative Phosphoproteomics.
abstract: Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116). We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0-24 hr), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80303, USA.
authors.forename: Zachary C
authors.initials: ZC
authors.lastname: Poss
authors.affiliation: Department of Molecular, Cell, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Ebmeier
authors.affiliation: Department of Molecular, Cell, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA; BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA.
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Odell
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Anupong
authors.initials: A
authors.lastname: Tangpeerachaikul
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80303, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lee
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Henry E
authors.initials: HE
authors.lastname: Pelish
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Shair
authors.affiliation: Department of Molecular, Cell, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA; BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA.
authors.forename: Robin D
authors.initials: RD
authors.lastname: Dowell
authors.affiliation: Department of Molecular, Cell, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
authors.forename: William M
authors.initials: WM
authors.lastname: Old
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80303, USA. Electronic address: taatjes@colorado.edu.
authors.forename: Dylan J
authors.initials: DJ
authors.lastname: Taatjes
mesh_terms: D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D045325:HCT116 Cells; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism*; D011083:Polycyclic Compounds / Q000737:chemistry / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D013379:Substrate Specificity / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D010750:Phosphoproteins; D011083:Polycyclic Compounds; D047428:Protein Kinase Inhibitors; D020543:Proteome; C531521:cortistatin A; D011494:Protein Kinases; D018844:Cyclin-Dependent Kinases
keywords: CDK8-Mediator; MED13; MED13L; Mediator; SILAC; SIRT1; cholesterol