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.

Keywords: CDK8-Mediator; MED13; MED13L; Mediator; SILAC; SIRT1; cholesterol.

Figure 1. Quantitative phosphoproteomics in HCT116 cells ± CA

(A) Cortistatin A (CA) structure. (B) Overview of phosphoproteomics workflow used to identify Mediator kinase substrates. (C) Unique phosphopeptides identified with LC-MS/MS after ERLIC fractionation. Average of biological triplicates is represented. (D) CA treatment with quantitative phosphoproteomics reproducibly identifies Mediator kinase substrates. H/L ratios quantified in two of three biological replicates are plotted on the x- and y- axes. Plot shows proteins whose H/L ratios decrease (green) and increase (peach) upon CA treatment. (E) and (F) Representative mass spectra. Spectra shown are from replicates in which either the light (E) or heavy (F) cells were CA-treated. Differences in SILAC pairs are shown based on the labeled amino acid; Arg(10) in (E) and Lys(8) in (F). The charge is +2 for both peptides.

Figure 2. Identification of Mediator kinase (CDK8/19) substrates

(A) Functional categorization of high-confidence Mediator kinase substrates identified. (B) Volcano plot of statistically significant phosphosite changes with CA treatment using an empirical Bayes analysis.

Figure 3. In vitro validation of select CDK8/19 substrates

(A) Validation of STAT1 S727 as a Mediator kinase target in HCT116 cells. (B) Western blot validation of SIRT1 T530 as a Mediator kinase target. Levels of total SIRT1 and other proteins known to regulate CDK8 activity (MED12 or CCNC), remained unchanged. TBP is a loading control. (C) Quantitation of data in (B). Error bars are SEM; n=2 for technical replicates. (D) In vitro kinase assay with recombinant CDK8 module and SIRT1. With increasing time, SIRT1 pT530 detection increases, indicating CDK8 is phosphorylating this site. Increase is not seen in no kinase or no substrate (ns) controls. (E) In vitro kinase assay with GST-tagged TP53BP1 or RIF1 fragments. Alanine mutations at identified phosphorylation sites show reduced phosphorylation by CDK8. (F) Overview of method for identifying MED12 and MED13 phosphorylation sites using recombinant CDK8 modules. (G) Verification of MED12 S688 and MED13 S749 phosphorylation sites. (H) In vitro kinase assay using CA and GST-pol II CTD as a substrate. Whereas each kinase tested phosphorylates this substrate, CA only inhibits the CDK8 module.

Figure 4. Mediator kinase inhibition is functionally distinct from CDK8 or CDK19 knockdown

(A) Heat map of differentially expressed genes (RNA-Seq) after 3 hr CA treatment. Green font represents transcription or chromatin regulator. (B and C) Comparison with microarray data (Galbraith et al., 2013) using stable CDK8/19 knockdown (shRNA) vs. 3h CA treatment (B) or 24h treatment (C) in HCT116 cells under normal growth conditions. A 1.5-fold cutoff was used for microarray data and Cufflinks was used for CA-treated cells (no specific fold-change cutoff). (D) TFBS analysis of promoters for genes whose expression changed with 3h CA treatment (listed in A). Promoters (±2kb from the TSS of the canonical isoform) were analyzed using F-Match, part of the Transfac database. Overrepresented sites with at least 1.5 fold increase vs. control promoters are shown for Transfac vertebrate matrices. Matrix name at left.

Figure 5. Quantitative proteomics reveals pathways and proteins affected by Mediator kinase inhibition

(A) Overview of quantitative proteomics method. (B) Venn diagram of biological replicates showing number of proteins identified in the time series. Replicates show a high degree of overlap for protein IDs. (C) Volcano plot comparing protein abundance at 18 hr and 24 hr time points vs. control (0 hr). Adjusted p-values are colored according to an empirical Bayes analysis. (D) Individual analysis of t = 3hr, 6hr, 18hr, and 24hr CA treatment time points using GSEA and the hallmark gene sets from the Molecular Signatures Database. Comparison of the t = 0hr and 1hr time points showed no differences in the hallmark gene sets (not shown). The color of the heat map corresponds to the direction and magnitude of the normalized enrichment score for that gene set at each time point, compared to t = 0hr controls. ‘NA’ and the corresponding color indicate a hallmark gene set not being identified from the proteome data at the designated time. (E) Protein abundance increases for MED13 and MED13L in CA-treated cells.