Activation Mechanism of Bora-Plk1 Regulating Entry into mitosis – AMBRE

we used a multiscale (from the molecule to the organism) and multidisciplinary approach combining biochemistry (protein production and purification, in vitro assay, biacore, fluorescence polarization), structural biology (NMR), proteomics (bottom-up tandem mass spectrometry), cell biology (live cell imaging) along with functional assays in three biological systems (Xenopus extracts, C. elegans, human cells).

we discovered a novel kinase activation mechanism in which the intrinsically disordered protein Bora, phosphorylated at a unique site by Cyclin A-Cdk1 (Vigneron et al., 2018) allosterically activates AURKA for mitotic commitment. We found that the phosphate adduct on Bora can substitute in trans for AURKA autophosphorylation on the A-segment (T288) to activate AURKA and trigger mitotic entry. This mechanism represents a new paradigm in mitotic kinase regulation (Tavernier et al., 2021b) and for reviews (Tavernier et al., 2021a; Pillan et al., 2022).

The mechanism we have discovered coordinates S/G2 phases and entry into mitosis. Bora thus acts as a signal molecule that couples the activity of Cyclin A-Cdk1 complexes to the activation of mitotic kinases. The discovery of the existence of different forms of AURKA, regulated by different mechanisms in time and space, could open new avenues for the development of targeted anti-cancer strategies to inactivate certain functions of AURKA. Indeed, overexpression of AURKA is observed in many cancers, associated with a poor prognosis for the patients.

1- Velez-Aguilera, G., Ossareh-Nazari, B., Van Hove, L., Joly, N., and Pintard, L. (2022). Cortical microtubule pulling forces contribute to the union of the parental genomes in the Caenorhabditis elegans zygote. Elife 11, e75382.

2- Lebrec, V., Poteau, M., Morretton, J. P., and Gavet, O. (2022). Chk1 dynamics in G2 phase upon replication stress predict daughter cell outcome. Dev Cell 57(5), 638-653.e5.

3- Pillan, A., Tavernier, N., and Pintard, L. (2022). [The kiss of life: Aurora A embraces the phosphate of its cofactor Bora to trigger mitotic entry]. Med Sci (Paris) 38(4), 345-347.

4- Tavernier, N., Sicheri, F.*, and Pintard, L*. (2021a). Aurora A kinase activation: Different means to different ends. J Cell Biol 220(9),

5- Tavernier, N., Thomas, Y., Vigneron, S., Maisonneuve, P., Orlicky, S., Mader, P., Regmi, S. G., Van Hove, L., Levinson, N. M., Gasmi-Seabrook, G., Joly, N., Poteau, M., Velez-Aguilera, G., Gavet, O., Castro, A., Dasso, M., Lorca, T., Sicheri, F.*, and Pintard, L*. (2021b). Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry. Nat Commun 12(1), 1899.

6- Velez-Aguilera, G., Nkombo Nkoula, S., Ossareh-Nazari, B., Link, J., Paouneskou, D., Van Hove, L., Joly, N., Tavernier, N., Verbavatz, J. M., Jantsch, V., and Pintard, L. (2020). PLK-1 promotes the merger of the parental genome into a single nucleus by triggering lamina disassembly. Elife 9, e59510.

7- Pintard, L., and Bowerman, B. (2019). Mitotic Cell Division in Caenorhabditis elegans. Genetics 211(1), 35-73.

8- Vigneron, S., Sundermann, L., Labbé, J. C., Pintard, L., Radulescu, O., Castro, A., and Lorca, T. (2018). Cyclin A-cdk1-Dependent Phosphorylation of Bora Is the Triggering Factor Promoting Mitotic Entry. Dev Cell 45(5), 637-650.e7.

9- Pintard, L., and Archambault, V. (2018). A unified view of spatio-temporal control of mitotic entry: Polo kinase as the key. Open Biol 8

10- Castro, A., and Lorca, T. (2018). Greatwall kinase at a glance. J Cell Sci 131(20)

11- Martino, L., Morchoisne-Bolhy, S., Cheerambathur, D. K., Van Hove, L., Dumont, J., Joly, N., Desai, A., Doye, V., and Pintard, L. (2017). Channel Nucleoporins Recruit PLK-1 to Nuclear Pore Complexes to Direct Nuclear Envelope Breakdown in C. elegans. Dev Cell 43(2), 157-171.e7.

12- Gheghiani, L., Loew, D., Lombard, B., Mansfeld, J., and Gavet, O. (2017). PLK1 Activation in Late G2 Sets Up Commitment to Mitosis. Cell Rep 19(10), 2060-2073.

Cell division (Mitosis) is a fundamental process required for the generation of multicellular organisms, for tissue renewal and homeostasis. Mitosis drives the distribution of an identical genetic material into two daughter cells. Commitment to mitosis must be tightly coordinated with DNA replication to preserve genome integrity. Consistently, unscheduled mitosis may contribute to genetic instability in numerous pathologies. Entry into mitosis is controlled by evolutionarily conserved serine/threonine kinases as well as counteracting phosphatases. How these kinase activities are regulated in space and time, and how they work in concert to trigger mitosis at the right time remain ill defined.

Polo-like kinase (Plk1) is a highly conserved mitotic kinase instrumental for the accurate segregation of chromosomes during mitosis. Plk1 controls centrosome maturation, DNA condensation, chromatids separation, spindle assembly and cytokinesis. We recently determined that Plk1 is rapidly activated shortly before mitosis and is critically required for commitment to mitosis (Gheghiani et al. Cell Reports, in revision). We found that CyclinA2-Cdk, a S-promoting factor, is acting upstream of Plk1 activation. We also found that Plk1 is actively recruited to the nuclear envelope in prophase and contribute to Nuclear Envelope Break Down (NEBD) (Martino et al. Developmental Cell, in revision). Importantly, the mechanisms activating Plk1 in space and time to trigger a timely entry into mitosis are poorly defined. Likewise, the mechanism of Plk1 recruitment to the NE and its role in NEBD remain to be determined. Plk1 activation relies on the phosphorylation of a conserved residue (Thr210) in its activation segment (T-loop) by the Aurora-A kinase (AurkA). This process requires the conserved protein Bora, which stimulates Plk1 phosphorylation by AurkA. We determined that Bora phosphorylation by Cdk1/2 is critical for Plk1 activation in vitro and in vivo, both in C. elegans embryos and human cells. The identity of the Cyclin-Cdk complex involved, spatio-temporal regulation of Bora phosphorylation and the mechanistic by which Bora promotes AurkA-dependent Plk1 activation remain crucially unknown. Here, we aim to combine a multidisciplinary approach (cell biology, genetics, biochemistry and structural biology) in three model organisms (Xenopus egg extracts, human culture cells, C. elegans) to decipher the mechanistic of Plk1 activation and functions during mitotic entry. We believe that this project will provide a significant contribution in our understanding of the mechanisms that control a timely entry into mitosis to preserve the genetic integrity.
We have the necessary complementary expertise in biochemistry, structural biology and cell biology as well as tools and methodologies to successfully achieve our objectives.

• We will elucidate Plk1 activation mechanism by Bora and AurkA. Plk1 is a central component of cell division. Understanding its activation mechanism is of prime importance for the future development of strategies to interfere with its functions. Hence, this project will have significant societal and economic implications. Developing Bora inhibitors that will be used in combination with existing Plk1 inhibitors represents an attractive strategy to robustly inhibit Plk1 catalytic activity, which is deregulated in numerous diseases including cancers.

• We will decipher the longstanding missing molecular link between CyclinA-Cdk2 and/or -Cdk1 and commitment to mitosis. Elucidating the role of CyclinA is essential to unravel how replication and mitosis are tightly coordinated during cell cycle.

• We will decipher the unexplored role of Plk1 in nuclear envelope breakdown, a critical step for chromosome segregation and successful cell division.