In a major advance for molecular biology and cancer research, scientists have uncovered the molecular mechanisms that control a key step during the activation of cyclin-dependent kinases (CDKs) – the master regulators of cell division.
Specifically, the study provides high-resolution structural data that reveal how an enzyme called CDK-activating kinase (CAK) recognises CDKs through a newly identified interface, enabling their full activation. The discovery opens new avenues for therapeutic intervention in cancer, where CDK regulation is often disrupted.
The findings of the study, which was led by scientists at The Institute of Cancer Research, London, were published in the journal Science. The research was primarily funded by The Institute of Cancer Research (ICR), which is both a research institute and a charity. Additional funding came from the Medical Research Council and Cancer Research UK (CRUK).
Resolving long-standing puzzles
CDKs are enzymes that control the progression of cells through the cell cycle. Their activation typically requires two steps: binding to a cyclin partner and phosphorylation of a conserved threonine residue within the activation segment known as the T-loop. This phosphorylation is carried out by CAK, a complex composed of the proteins CDK7, cyclin H and MAT1.
As CDKs are known to be frequently dysregulated in cancer, they are prime targets for therapeutic intervention. Several next-generation therapeutics targeting the CAK, which aim to inhibit CDK activity by blocking T-loop phosphorylation, are currently undergoing clinical evaluation.
However, until now, it was not known how the CAK binds and recognises its CDK-type clients to activate them. A puzzling observation was that the sequence of the T-loop – the part of the CDK that actually receives the phosphorylation – does not appear to play a role in substrate recognition. How, then, does the CAK recognise its targets?
Cryo-EM reveals a critical interface
Using a powerful imaging technique called cryogenic electron microscopy (cryo-EM), the team determined the structures of CAK bound to CDK2, both when CDK2 is bound to a cyclin and when it is not.
The researchers were most interested in the interface between CDK2 and the CDK7 subunit of CAK, where they were able to identify two key clusters of molecular interactions in specific areas of the kinases. They also noted that the proteins interacted ‘head to head’ rather than ‘head to tail’ as previously thought.
Importantly, the T-loop of CDK2 did not contribute meaningfully to this interface, explaining how CAK can recognise CDKs independently of their T-loop sequence.
Together, these discoveries highlight the power of modern structural biology to provide answers to long-standing mechanistic questions.
Delving deeper
To validate their structural findings, the researchers introduced targeted mutations into the interaction clusters. They found that mutations in one particular region of the interface – called the C-lobe – prevented CDK7 from phosphorylating CDK2, thereby confirming the critical role of this area. Similarly, equivalent mutations in CDK2 that disrupted the interface prevented activation by CAK
These experiments demonstrate that the kinase-kinase interface itself is sufficient for substrate recognition, independent of the T-loop sequence, which then allows activation of the target CDK by phosphorylation.
The team then extended its analysis to other CDKs, including CDK1 and CDK11, both of which CAK is also known to act on during biochemical reactions. Cryo-EM structures of CAK bound to CDK1-cyclin B and CDK11 revealed nearly identical interfaces, suggesting that this mechanism is conserved across multiple CDKs.
Computational predictions supported this conclusion, showing similar interaction patterns for other CDKs that are being activated by CAK, but not for those that do not rely on CAK for activation. This suggests that the newly described interface represents a general architecture for CAK-mediated CDK activation.
Interestingly, the study also shed light on a second way in which CAK can interact with CDK-cyclin complexes: a particular sequence of amino acids at the very end of CDK7 can interact with cyclins. This sequence follows a known pattern of amino acids – known as a motif – and appears to be conserved across species, including fungi and plants. This sequence may play a role in the phosphorylation of CDK7 targets, or it may serve as a recognition signal for enzymes involved in regulating CAK itself.
“Even well-studied pathways can hold surprising secrets”
First author Victoria Cushing, formerly a PhD student in the Division of Structural Biology at the ICR, said:
“It will be interesting to explore the therapeutic potential of targeting the newly identified interface. We are hopeful that, in the longer term, key structural insights such as those achieved in this study will guide the design of new drugs that offer a new approach to modulating CDK activity.”
Senior author Dr Basil Greber, Group Leader of the Structural Biology of DNA Repair Complexes Group at the ICR, said:
“We were excited to see how neatly our structural results explain previous experimental observations. As we continue to explore the intricacies of kinase signalling, this study serves as a powerful reminder that even well-studied pathways that are central to cellular function hold secrets waiting to be uncovered.”
Image credit: Gerd Altmann from Pixabay