Identification of Mediator kinase substrates in human cells using cortistatin A and quantitative phosphoproteomics

Zachary C. Poss; Christopher C. Ebmeier; Aaron T. Odell; Anupong Tangpeerachaikul; Thomas Lee; Henry E. Pelish; Matthew D. Shair; Robin D. Dowell; William M. Old; Dylan J. Taatjes

doi:10.1016/j.celrep.2016.03.030

Figure 3

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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.