Role of Greatwall in mitotic entry and progression – GREATWALL

This project will be performed in human cells in culture and in Xenopus egg extracts (South Africa Frog). Oocytes of Xenopus are very useful model for biochemical studies since and for the analysis of post-translational protein modifications. Moreover, they synchronised at the same phase of the cell cycle. In our laboratory we have successfully used this model in the past and we dispose of all the required tools to performed all the experiments proposed in this project.
We will also use human cells in culture. We will knockdown and overexpress Greatwall in these cells and we will investigate the induced phenotype by different types of microscopy.

Our laboratory has recently described the different steps involved in the regulation of the cell cycle by Greatwall. Our data show that three different proteins participate to this signalisation pathway. The upstream one is Greatwall that will phosphorylate the Arpp19 protein. Once phosphorylated by Greatwall, Arpp19 will bind and inhibit the phosphatase PP2A promoting, like this, mitotic entry.
We also show that Greatwall is submitted to phosphorylation in different sites and that these phosphorylations are essential for its activation and for mitotic entry. Similarly, we report that dephosphorylation of the different residues of Greatwall at mitotic exit is also essential to promote reactivation of PP2A and dephosphoryaltion of mitotic substrates.

In a next future, our aim will be the characterization of the protein kinase/s responsible for Greatwall phosphorylation at mitotic entry and of the protein phosphatase/s essential for the dephosphorylation of this protein at mitotic exit.
We will also try to understand by which mechanism the association of Arpp19 to PP2A results in the inactivation of this phosphatase.
Finally, these two studies could allow us to identify new drugs that could specifically target this new pathway of the cell cycle and that could allow us to send to cell death the cells with an abnormal cell division.

Publication
Lorca T and Castro A. (2012). The Greatwall kinase: a new pathway in the control of the cell cycle. Oncogene. (In press) (This publication corresponds to a Review in which this new Greatwall-dependent signalisation pathway discovered in part by our laboratory has been reported).
Patent
An international patent has been submitted for the discovery of the two new substrates of Greatwall.

Greatwall is a new characterised kinase required in Xenopus egg extracts for mitotic entry and progression. The depletion of Greatwall in these extracts induces mitotic exit, whereas the same depletion in cycling egg extracts prevents mitotic entry. Greatwall also plays an important role in mitotic progression in Drosophila. Mutations in Drosophila Greatwall cause improper chromosome condensation and delay in cell cycle progression. Despite its major role in cell division, little is known about the pathway by which this kinase controls cell cycle. In this project we will try to understand how Greatwall promotes and maintains the mitotic state and how it is regulated. Our results developed in Xenopus metaphase II-arrested oocytes and in human cells clearly demonstrate that Greatwall promotes and maintains mitosis by inhibiting PP2A, the phosphatase responsible of the dephosphorylation of cyclin B-Cdk1. However, our results indicate that this inhibition is probably indirectly performed by a third protein. In this project we will try to characterize the substrates of Greatwall, that are completely unknown so far and that are probably responsible of PP2A inhibition. Moereover, we will also investigate the different impaired pathways in Greatwall-knockdown cells that are responsible of the two major phenotypes observed in these cells, sister chromatid segregation defect and centriole number defect.
Finally, since Greatwall promotes mitotic entry and exit, the correct timing of activation and inactivation of this kinase is essential to promote a correct cell division. Greatwall is phosphorylated at mitotic entry and dephoshorylated at mitotic exit concomitantly with kinase activation and inactivation respectively. Therefore, it is likely that Greatwall activity is regulated by phosphorylation. Another objective of this project will be the characterization of the mechanisms inducing Greatwall phosphorylation and dephosphorylation. To do that, we will determine by phosphomapping-mass spectrometry and by directed mutagenesis the role of each of the different phosphorylated sites in the activation of this kinase and we will try to identify the phosphatase responsible of this inactivation during mitotic exit.