Context-specific control of the Cdk8/19-cyclin C kinase by MTBP and Med12

• Applicant: Professor Dr. Dominik Boos
• Subject Area: Cell Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 571993187

How kinases find their substrates in the sea of cellular proteins is a central biological question. The Cdk8/19-cyclin C kinase forms the CDK-kinase module (CKM) of the Mediator of Transcription upon binding to Med12 and Med13. Whereas Mediator governs transcription broadly, the Cdk8/19-cyclin C selectively modulates gene expression during specific cell state changes such as cell differentiation. Cdk8/19-cyclin C dysregulation has been implicated in colorectal cancer, and its activation by the cofactor Med12 is compromised in the majority of uterine leiomyoma cases. Our lab has contributed significantly to the characterization of MTBP (Mdm2 binding protein), an interactor of Cdk8/19-cyclin C. We established MTBP as the vertebrate orthologue of yeast Sld7, a key regulator of DNA replication origin firing. Moreover, MTBP integrates multiple kinase signaling pathways such as the cell cycle and replication stress response, to regulate replication which is crucial for complete and accurate genome duplication and therefore faithful genetic inheritance. We recently identified that MTBP interacts with Cdk8/19-cyclin C, and described that the interaction is required to prevent replication stress in cultured human cells, though the underlying mechanisms remain unclear. Unpublished biochemical evidence suggests that MTBP is an allosteric activator of the Cdk8/19-cyclin C kinase. MTBP’s interaction with the Cdk8/19-cyclin C excludes simultaneous binding of the kinase activator Med12. This raises a new model for cellular control of Cdk8/19-cyclin C using distinct activators, MTBP and Med12, to target the kinase toward distinct cellular functions. We will address interrogate this model in aims 1 and 2. Aim 1 will dissect the context-dependent activation and function of Cdk8/19-cyclin C by generating mutant cell lines deficient in forming either the MTBP or Med12 kinase complexes, or deficient for both. We will analyze these cell lines by RNA sequencing to profile differential gene expression, phosphoproteomics to identify differential Cdk8/19-cyclin C kinase substrates, and proximity biotinylation to distinguish the molecular environments of three kinase complexes. This will globally determine activator-specific Cdk8/19-cyclin C roles in cells. Aim 2 will investigate in detail novel MTBP-dependent roles of Cdk8/19-cyclin C in replication origin firing, transcriptional control, and potentially other cellular processes. It will build partly on the MTBP-dependent pathways and kinase substrates identified in aim 1. Functional and biochemical assays will be used in genetically modified cell models for this aim. This study will establish a model of context-dependent activation of Cdk8/19-cyclin C by distinct cofactors, and reveal how MTBP and Med12 govern Cdk8/19-cyclin C kinase function in transcription and replication. These insights have implications for cancer biology, where targeting factor-specific functions may represent novel therapeutic opportunities.

• DFG Programme: Research Grants