Understanding the role and the regulation of Arpp19, a central player of cell division – KiARP

The KiARP project used the oocyte of an amphibian, the Xenopus, an invertebrate, the jellyfish Clytia, cell-free extracts and mouse cells. This has enabled us to study the role of Arpp19 in a variety of animal species and in different types of cell division, in particular those called meiotic, which are specific to reproductive cells. We have used a wide range of techniques to elucidate Arpp19 function as a brake of cell division. Cell biology approaches enabled us to manipulate the cells by microinjections and to analyze the consequences of Arpp19 perturbations on cell division by imaging technics. We have developed molecular tools by producing proteins, Arpp19 or its partners, with or without mutations, to understand their interactions and functions. Finally, we have identified proteins that interact with Arpp19 or specific regions of this protein that are important for its role in cell division, using biochemical (chromatography purification) or proteomic (mass spectrometry) techniques.

We have elucidated the enzymes that control Arpp19 and give it its function as a brake on cell division. We also understood how Arpp19 exerted this role: by interactions of different regions within the same Arpp19 molecule. Our results thus enrich fundamental knowledge of cell division and open up new avenues for understanding pathological processes such as tumorigenesis or female infertility, as well as being a potential and unexpected source of new biotechnological tools.

To further our understanding of the roles of Arpp19 in cell division, we need to perform: (1) comparative structural analyses of Arpp19 between different species, such as Clytia and Xenopus, in order to understand the acquisition of new properties by Arpp19 during evolution; (2) the study of the intramolecular interaction between different motifs of the protein; (3) the study of the initiation of PP2A repression and Cdk1 activation in the context of the resumption of meiosis in the oocyte. The aim will be to build on recent work by one of the two project teams to understand the balance used between two PP2A-B55 inhibitors, Arpp19 and FAM122A, and which meiotic Cdk1 primer replaces the mitotic role played by Cyclin A-Cdk1.
In the long term, our results should have an impact on issues relating to human health: (1) Human infertility: our studies on the oocyte reveal the molecular pathways presiding over the oocyte's decision to resume meiotic divisions, naturally arrested in G2, and then to carry out these divisions. This meiotic maturation step of oogenesis plays a key role in the origins of many female human infertilities, such as POCS (polycystic ovary syndrome) and POI (primary ovarian insufficiency). Our studies reveal potential candidates whose dysfunction may be linked to the origin of such infertility. (2) Cancer: Our studies of the key players regulating cell division also reveal players potentially involved in tumorigenesis. It is well established that the phosphatase PP2A is deregulated in many types of cancer and considered to be a tumour suppressor. Understanding its regulation by proteins such as Arpp19, which is also deregulated in certain types of cancer, and the structural mechanics governing its catalytic properties is particularly important for understanding how its alterations are involved in tumorigenesis. (3) Biotechnological tools: our studies on the structure of the Arpp19 protein have revealed that short sequences of the protein have either anti-mitotic or division-inducing properties. These peptide sequences have biotechnological potential that needs to be exploited.

Our results have been published in high-impact journals (Nature Communications, Cell Reports, an article under review in Development), and deposited in open archives, ensuring maximum dissemination. We have also placed our results in the context of cell division, thereby enriching its overall vision, through the publication of 6 review articles, including one for the general public. Finally, we have presented our findings in oral and poster presentations at international conferences.

Cells proliferate by means of the mitotic cell cycle that supports growth, development and maintenance of all living organisms. Understanding the control of cell cycle has fascinated biologists for many years. Cell division is orchestrated by the phosphorylation of mitotic proteins, under the control of two master enzymes: the kinase Cdk1 and its antagonizing phosphatase, PP2A-B55delta. While the regulation of these enzymes has been widely studied since their discovery, there is still a surprising lack of knowledge on cell division. In particular, the switch-like behavior of M-phase transitions that depends on an antagonistic interplay between kinases and phosphatases remains largely unknown.
The KiPARP project aims at elucidating the regulatory circuits that underlie the cellular decision to divide or not, the conservation of these molecular networks among species and the specific adaptations of the mitotic molecular controls ensuring a meiotic division. To get new insights into these questions, our project is based on a newly identified player of cell division, the small protein Arpp19, which plays a dual function in controlling the switch between Cdk1 and PP2A-B55delta-depending on its phosphorylation. When phosphorylated at S109 by the cAMP-dependent protein kinase, PKA, Arpp19 strongly impairs Cdk1 activation during female meiosis. Conversely, when phosphorylated by the kinase Greatwall at S67, Arpp19 becomes a potent activator of both meiosis and mitosis by inactivating PP2A-B55delta. Hence, depending on its phosphorylation by PKA or Greatwall, Arpp19 authorizes or not entry into cell division.
To decipher the molecular decision to divide, the KiPARP project will investigate how Arpp19 is converted from a negative to a positive regulator of M-phase by using distinct and powerful experimental models, oocytes of various species, cell-free extracts and an engineered mouse cell line. We will elucidate the mechanisms controlled by PKA-phosphorylated Arpp19 that block M-phase entry and further investigate how Arpp19 dephosphorylation at S109 fires the Cdk1-activatory mechanisms inducing M-phase in Xenopus oocytes and cell-free extracts. We will also study the function of a double-phosphorylated form of ARPP19 at both S67 and S109, which is generated during M-phase. We will determine whether S109 phosphorylation of ARPP19 is a conserved mechanism regulating both meiotic and mitotic division, by using physiological models as oocytes from vertebrates (mouse and human) and invertebrates (jellyfish).
The two KiPARP partners have confirmed experiences in studying meiotic and mitotic divisions and show excellent complementarity in their favored experimental models and technical approaches (biochemical/molecular and cell biology/imaging), which will drive the accomplishment of the project. By focusing on the biochemical networks that trigger the flip-flop switch in M-phase, the KiPARP project will help understanding key biological processes involved in reproduction, cell cycle regulation, signal transduction and oncogenesis.