A precisely positioned MED12 activation helix stimulates CDK8 kinase activity

Abstract

The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association of Mediator and RNA polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and regulatory subunit MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, cross-linking coupled to mass spectrometry, and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an “activation helix” close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene-expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Finally, functional assays in the presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase inhibitors. Taken together, our results not only allow us to propose a revised model of how CDK8 activity is regulated by MED12, but also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form.

Structure and mechanism of the RNA polymerase II transcription machinery

“…Biochemical studies suggested that the N terminus of MED12, which is a hot spot for oncogenic mutations (Makinen et al 2011;Lim et al 2014), interacted with CCNC as part of the MED12-dependent activation mechanism (Turunen et al 2014). However, these findings were contradicted by CXMS data obtained with partial assemblies of the human CDK8 module (Klatt et al 2020). The CXMS results supported an interaction between the MED12 N terminus (residues 30-42) and a disordered CDK8 activation loop (residues 173-203).…”

“…Similar activation loops (also known as T-loops) exist in other CDKs, but the CDK8 sequence contains a D instead of a typical T at residue 191, suggesting a phosphorylation-independent activation mechanism. The MED12 N terminus (residues 19-50) is predicted to adopt an α-helical structure, and a model was proposed in which negatively charged residues (e.g., E33) in this MED12 "activation helix" mimicked CDK8 activation loop phosphorylation as a means to activate CDK8 (Klatt et al 2020). The structural discrepancies between these models for MED12-dependent activation of CDK8 could reflect two distinct mechanisms of activation.…”

Manipulating the Mediator complex to induce naïve pluripotency

“…Completing the kinase module are: cyclin C (CCNC), and subunits MED12 and MED13. CDK8/19 activity appears restricted by its requirement for proper quaternary structure [ 23 ]. For full activity, CDK8/19 must associate simultaneously with cyclin C and with MED12, the latter embraces CDK8 allowing it to attain proper opening of its T-loop [ 23 ].…”

“…CDK8/19 activity appears restricted by its requirement for proper quaternary structure [ 23 ]. For full activity, CDK8/19 must associate simultaneously with cyclin C and with MED12, the latter embraces CDK8 allowing it to attain proper opening of its T-loop [ 23 ]. Evidence suggests that only CDK8, or CDK19, can occupy Mediator at one time, and the same is true for the paralog subunits MED12L and MED13L (reviewed elsewhere) [ 8 , 9 , 22 ].…”

“…Also, we and others have found that CDK8/19 can phosphorylate itself and several other Mediator subunits [ 10 , 30 ,[ 36 ] 37 ], raising the possibility that the kinase activity of CDK8/19 may somehow influence its structural role hindering the approach of Pol II to the Mediator complex. In reciprocal, extensive Mediator structural rearrangements re-position CDK8/19 with respect to RNA Pol II [ 18 , 19 , 23 ].…”