Mediator is a large complex composed of 25–30 proteins arranged in structural modules that is thought to act as a molecular bridge between DNA-binding transcription factors and RNAPII (reviewed in refs. 43
). Mediator can restore activator-dependent transcription in cell free assays and is considered a quasi-universal regulator of RNAPII transcription. Although numerous interactions of Mediator with DNAbinding proteins and GTFs have been identified, the precise mechanism by which Mediator transduces regulatory signals to RNAPII remains poorly characterized. CDK8 associates in a dynamic fashion with the rest of Mediator as part of a foursubunit subcomplex dubbed the CDK-module (CDK8, cyclin C, MED12 and MED13).1,45–49
Mediator complexes lacking or containing the CDK-module will be referred to here as core and CDK8-Mediator, respectively (). The observation that up to 30% of the CDK-module can be purified free of core Mediator supports the notion of reversible association but also raises the possibility that CDK8 may function independently of core Mediator.1,2
Figure 1 Hierarchical map of CDK8 interactions. CDK8 interacts directly with subunits of the CDK-module, which connects to core Mediator via MED13. Analysis of endogenous CDK-module purified away from core Mediator revealed interactions with GCN1L, TriC, MED14 (more ...)
Importantly, vertebrates have a CDK8 paralog with high amino acid sequence conservation, recently renamed CDK19 (previously known as CDK8-like, CDK8L or CDC2L6).26,27,50
Although CDK19 was sporadically referred to as CDK11,27,50,51
it should not be confused with the ‘splicing kinase’ CDK11.52
Although both CDK8 and CDK19 associate with seemingly identical Mediator complexes,50,51
it is likely they are not functionally redundant, as indicated by the fact that CDK8 knockout leads to early embryonic lethality in mice,53
and that CDK8 knockdown alone produces clear phenotypes in human cell cultures.18,20–22
In fact, it has been proposed that CDK8 and CDK19 play opposite roles in VP16-dependent transcription.51,54
While the kinase and cyclin binding domains are highly conserved between CDK8 and CDK19, the proteins differ in their C-terminal regions,50,51
which raises the possibility that differential interactions mediated by the C-terminal peptide might alter access to substrates or incorporation into complexes. Intriguingly, MED12 and MED13 have also undergone independent gene duplications to generate MED12L and MED13L,50,55
and it is unknown which combinations of subunits are assembled in vivo or whether they have differential effects on the function of CDK8/19 or Mediator.
Biochemical analysis of endogenous and reconstituted human CDK-module by the Taatjes lab has generated several insights into CDK8 regulation.1–3
First, it was found that MED12—but not MED13—was required for kinase activity of the reconstituted CDK8-module toward the RNAPII CTD and other targets.2
Second, it was revealed that MED13 mediates the interaction between the CDK-module and the rest of Mediator, likely via MED14,1,2
and that MED13 itself can be phosphorylated by CDK8.2
Third, association of the CDK-module with core Mediator enabled CDK8 to phosphorylate histone H3 on chromatin.2
These observations are significant as they suggest that not only is CDK8 regulated at the level of kinase activity by association with other subunits of the module, but also by substrate accessibility via association with core Mediator. Mass spectrometry analysis of purified endogenous CDK-module revealed its association with additional factors including GCN1L and the TriC chaperonin (), the latter of which may be able to sequester the free CDK-module.2
Most intriguingly, unlike the recombinant CDK-module, the endogenous CDK-module could not phosphorylate RNAPII CTD, suggesting that these novel interactors might serve a regulatory role, perhaps by inhibiting the activity of the free CDK-module until its association with core Mediator.
These studies reveal several possible layers of CDK8 regulation and prompt key questions. What are the roles of GCN1L and TriC in overall CDK8 biological activity? Is there a differential impact of MED12 versus MED12L or MED13 versus MED13L on the regulation of CDK8? What is the role of MED13 phosphorylation and does it affect association of the CDK-module with core Mediator? Is CDK19 regulated in a similar fashion to CDK8? Future studies combining biochemical reconstitution and genetic dissection in cells will be required to address these issues.