Deciphering the role of disordered regulatory motifs in activator and inhibitor of the G2 to M transition cell-cycle checkpoint

• Applicant: Ricarda Toerner, Ph.D.
• Subject Area: Structural Biology ; Biochemistry
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 528834085

Cell proliferation is a tightly monitored process. During the cell cycle, two cell cycle checkpoints, G1 - S and G2 - M, serve as roadblocks to ensure that only genomically intact cells in a healthy environment replicate. These cell-cycle checkpoints are regulated by a complex, intertwined network of kinases and phosphatases, which transmit signals via phosphorylation or dephosphorylation. Cell-cycle associated kinases and phosphatases are highly deregulated in cancer cells, which makes them important drug targets. One way that cancer cells evade chemotherapy involves the G2 – M cell cycle checkpoint. Components of the so-called DNA damage response are typically upregulated as a result of genotoxic cancer therapies, leading to a temporary arrest in the G2-M cell cycle transition. This arrest allows cancer cells to evade apoptosis by allowing for sufficient DNA repair to proceed to cell division, despite still being heavily damaged. There are two main strategies of targeting this checkpoint for cancer therapy: i) Overactivation of this transition despite accrued DNA damage can push cells into mitotic catastrophe, thereby eliminating cancer cells or ii) Inhibition of the transition, which leads to the induction of apoptosis due to stagnation. In order to investigate important cancer drug targets, I focus my studies on the two decisive proteins at this cell cycle checkpoint, the activator Cdc25c and the inhibitor Wee1, which act on the effector kinase Cdk1. The kinase Wee1 inhibits Cdk1 by phosphorylating it in a region which reduces its substrate affinity, whilst Cdc25c, a phosphatase, removes the phosphorylation. My approach is novel in that I focus on the extensive disordered regions surrounding the folded enzymatic domain. These regions have been found to be important for the regulation of the activity of these enzymes, but remain very understudied because of their disordered nature, which makes them challenging for mechanistic investigations. These regulatory regions are subject to extensive phosphorylation by upstream kinases. They are also hubs for protein interactions and might play an important role in substrate recruitment. In order to study the structure of these highly dynamic regions I will employ NMR spectroscopy, which is the only structural technique which does not necessitate a stable three-dimensional structure. I will connect the cell cycle state with phosphorylation patterns and enzyme activity and characterize changes in intermolecular interactions. With this approach I will uncover the molecular mechanisms that drive the function and regulation of Cdc25c and Wee1 and gain a comprehensive understanding of their self-regulation. This information will inform new strategies for effectively modulating the activity of these enzymes to improve cancer treatment.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Dr. Haribabu Arthanari