Regulation and roles of Extracellular Regulated Kinases 1&2 (ERK1&2) and Polo-like kinase 1 (Plk1) during G2/M progression – G2Progress

A human body performs about 10,000 trillion cell divisions in a lifetime. Deciphering how is precisely controlled the “decision” to enter into mitosis during each cell cycle is a major challenge in cell biology that will create new therapeutic perspectives. Irreversible entry into mitosis is under the control of checkpoint mechanisms; a G2 DNA damage checkpoint that will arrest cells in G2 phase in the presence of DNA lesions and an antephase checkpoint sensing stress conditions up to early prophase. These control mechanisms delay Cyclin B1-Cdk1 activation, the master kinase orchestrating entry into mitosis, and prevent genetic instability as a consequence of chromosome separation with unreplicated or damaged DNA. Experiments performed in yeast to human showed that deregulation of Cyclin B-Cdk1 activity or overexpression of its activator(s) can trigger entry into mitosis of S phase cells still containing unreplicated DNA, enlightening the importance of the tight control of entry into mitosis for genomic stability.
When the checkpoint mechanisms are satisfied, Cyclin B1-Cdk1 activation takes place and trigger entry into mitosis. We previously developed a FRET (Förster Resonance Energy Transfer)-based specific CyclinB1-Cdk1 activity biosensor to demonstrate that its initial activation is taking place at a very reproducible set time in each individual living cell. What are the immediate upstream mechanisms that reproductively trigger its initial activation during each G2 phase is still a fundamental unanswered question. More generally, the core molecular machinery taking place after the completion of DNA replication during a normal G2 phase progression and ultimately leading to Cyclin B1-Cdk1 activation is poorly understood.
In the present project, we aim to investigate the spatio-temporal regulation and roles of ERK (Extracellular Regulated Kinases) 1&2 and Plk1 (Polo-like kinase 1) in the cell cycle progression from early G2 to mitosis. Recent reports suggest that ERK1&2 activities regulate a gene expression program in early G2 specifically in epithelial versus fibroblast cells. Because temporal and intensity-modulated ERK1&2 activities strongly affect their ability to activate downstream events, we will analyze in real time their activation signature during G2 progression using our recently developed FRET-based specific activity reporter. We will next combine the use of this biosensor with ERK inhibitor(s) to visualize in each individual living cell the extent of ERK1&2 inhibition and the consequences for G2 phase progression and/or mitotic entry in cells from different tissue origins. We thus expect to clarify their involvement in the regulation of G2/M progression.
Plk1 is known to participate in the regulation of entry into mitosis in mammals but the underlying mechanisms are not fully understood. We identified a main target, among the Cyclin B1-Cdk1 regulators, and observed that its phosphorylation is taking place from late G2 cells, mostly at centrosomes. We will determine if a burst of Plk1 kinase activity is taking place just before entry into mitosis during each cell cycle and if Plk1 dependent phosphorylation of the Cyclin B1-Cdk1 regulator triggers entry into mitosis. We will evaluate if the centrosome is a "platform" facilitating the initial activation of CyclinB1-Cdk1 and entry into mitosis using optogenetic inducible recruitment of this regulator.
Finally, an ambitious aim of this project will be to analyze the conservation of our findings concerning the regulation and roles of ERK1&2 and Plk1 during G2 to mitosis progression in Xenopus embryonic epithelial tissues as a model of cell proliferation in vertebrate tissues using our corresponding FRET-based activity reporters and implemented frequency domain FLIM FRET microscopy approaches.
We thus expect to significantly improve our knowledge of the successive molecular steps taking place during G2 to mitosis progression and which are potential therapeutic targets.