ARPP19 Phosphorylations by PKA and Greatwall: The Yin and the Yang of the Cell Decision to Divide

Entry into mitosis and meiosis is orchestrated by the phosphorylation of thousands of mitotic substrates under the control of active Cdk1-cyclin B complexes. To avoid futile cycles of phosphorylation/dephosphorylation, the specific Cdk1-antagonizing phosphatase, PP2A-B55δ, must be simultaneously inactivated. This process is achieved by the activation of the kinase Greatwall (Gwl), which phosphorylates ARPP19. Gwl-phosphorylated ARPP19 then inactivates PP2A-B55δ to allow Cdk1 activation as well as to secure the phosphorylation state of mitotic substrates. This chapter discusses a series of recent works showing that ARPP19 is also phosphorylated by another kinase, PKA. Phosphorylated by PKA, ARPP19 arrests Xenopus oocytes in G2 before the first meiotic division. Therefore, depending on its phosphorylation state by either PKA or Gwl, ARPP19 either restrains or activates Cdk1 in Xenopus oocytes. Beyond the understanding of the mechanisms of meiotic and mitotic cell division, the control of ARPP19 by its dual phosphorylation enlightens the cAMP-regulated signalization pathways that control vital functions in numerous eukaryotic cell types

Mos in the Oocyte: How to Use MAPK Independently of Growth Factors and Transcription to Control Meiotic Divisions

In many cell types, the mitogen-activated protein kinase (MAPK) also named extracellular signal-regulated kinase (ERK) is activated in response to a variety of extracellular growth factor-receptor interactions and leads to the transcriptional activation of immediate early genes, hereby influencing a number of tissue-specific biological activities, as cell proliferation, survival and differentiation. In one specific cell type however, the female germ cell, MAPK does not follow this canonical scheme. In oocytes, MAPK is activated independently of growth factors and tyrosine kinase receptors, acts independently of transcriptional regulation, plays a crucial role in controlling meiotic divisions, and is under the control of a peculiar upstream regulator, the kinase Mos. Mos was originally identified as the transforming gene of Moloney murine sarcoma virus and its cellular homologue was the first proto-oncogene to be molecularly cloned. What could be the specific roles of Mos that render it necessary for meiosis? Which unique functions could explain the evolutionary cost to have selected one gene to only serve for few hours in one very specific cell type? This review discusses the original features of MAPK activation by Mos and the roles of this module in oocytes

Unfertilized Xenopus Eggs Die by Bad-Dependent Apoptosis under the Control of Cdk1 and JNK

Ovulated eggs possess maternal apoptotic execution machinery that is inhibited for a limited time. The fertilized eggs switch off this time bomb whereas aged unfertilized eggs and parthenogenetically activated eggs fail to stop the timer and die. To investigate the nature of the molecular clock that triggers the egg decision of committing suicide, we introduce here Xenopus eggs as an in vivo system for studying the death of unfertilized eggs. We report that after ovulation, a number of eggs remains in the female body where they die by apoptosis. Similarly, ovulated unfertilized eggs recovered in the external medium die within 72 h. We showed that the death process depends on both cytochrome c release and caspase activation. The apoptotic machinery is turned on during meiotic maturation, before fertilization. The death pathway is independent of ERK but relies on activating Bad phosphorylation through the control of both kinases Cdk1 and JNK. In conclusion, the default fate of an unfertilized Xenopus egg is to die by a mitochondrial dependent apoptosis activated during meiotic maturation

The G2-to-M transition from a phosphatase perspective: a new vision of the meiotic division

International audienceCell division is orchestrated by the phosphorylation and dephosphorylation of thousands of proteins. These post-translational modifications underlie the molecular cascades converging to the activation of the universal mitotic kinase, Cdk1, and entry into cell division. They also govern the structural events that sustain the mechanics of cell division. While the role of protein kinases in mitosis has been well documented by decades of investigations, little was known regarding the control of protein phosphatases until the recent years. However, the regulation of phos-phatase activities is as essential as kinases in controlling the activation of Cdk1 to enter M-phase. The regulation and the function of phosphatases result from post-translational modifications but also from the combinatorial association between conserved catalytic subunits and regulatory subunits that drive their substrate specificity, their cellular localization and their activity. It now appears that sequential dephosphorylations orchestrated by a network of phosphatase activities trigger Cdk1 activation and then order the structural events necessary for the timely execution of cell division. This review discusses a series of recent works describing the important roles played by protein phos-phatases for the proper regulation of meiotic division. Many breakthroughs in the field of cell cycle research came from studies on oocyte meiotic divisions. Indeed, the meiotic division shares most of the molecular regulators with mitosis. The natural arrests of oocytes in G2 and in M-phase, the giant size of these cells, the variety of model species allowing either biochemical or imaging as well as genetics approaches explain why the process of meiosis has served as an historical model to decipher signalling pathways involved in the G2-to-M transition. The review especially highlights how the phosphatase PP2A-B55δ critically orchestrates the timing of meiosis resumption in amphibian oocytes. By opposing the kinase PKA, PP2A-B55δ controls the release of the G2 arrest through the dephosphorylation of their substrate, Arpp19. Few hours later, the inhibition of PP2A-B55δ by Arpp19 releases its opposing kinase, Cdk1, and triggers M-phase. In coordination with a variety of phosphatases and kinases, the PP2A-B55δ/Arpp19 duo therefore emerges as the key effector of the G2-to-M transition

The greatwall kinase is dominant over PKA in controlling the antagonistic function of ARPP19 in Xenopus oocytes

International audienceThe small protein ARPP19 plays a dual role during oocyte meiosis resumption. In Xenopus, ARPP19 phosphorylation at S109 by PKA is necessary for maintaining oocytes arrested in prophase of the first meiotic division. Progesterone downregulates PKA, leading to the dephosphorylation of ARPP19 at S109. This initiates a transduction pathway ending with the activation of the universal inducer of M-phase, the kinase Cdk1. This last step depends on ARPP19 phosphorylation at S67 by the kinase Greatwall. Hence, phosphorylated by PKA at S109, ARPP19 restrains Cdk1 activation while when phosphorylated by Greatwall at S67, ARPP19 becomes an inducer of Cdk1 activation. Here, we investigate the functional interplay between S109 and S67-phosphorylations of ARPP19. We show that both PKA and Gwl phosphorylate ARPP19 independently of each other and that Cdk1 is not directly involved in regulating the biological activity of ARPP19. We also show that the phosphorylation of ARPP19 at S67 that activates Cdk1, is dominant over the inhibitory S109 phosphorylation. Therefore our results highlight the importance of timely synchronizing ARPP19 phosphorylations at S109 and S67 to fully activate Cdk1

Translational Control of Xenopus Oocyte Meiosis: Toward the Genomic Era

International audienceThe study of oocytes has made enormous contributions to the understanding of the G 2 /M transition. The complementarity of investigations carried out on various model organisms has led to the identification of the M-phase promoting factor (MPF) and to unravel the basis of cell cycle regulation. Thanks to the power of biochemical approaches offered by frog oocytes, this model has allowed to identify the core signaling components involved in the regulation of M-phase. A central emerging layer of regulation of cell division regards protein translation. Oocytes are a unique model to tackle this question as they accumulate large quantities of dormant mRNAs to be used during meiosis resumption and progression, as well as the cell divisions during early embryogenesis. Since these events occur in the absence of transcription, they require cascades of successive unmasking, translation, and discarding of these mRNAs, implying a fine regulation of the timing of specific translation. In the last years, the Xenopus genome has been sequenced and annotated, enabling the development of omics techniques in this model and starting its transition into the genomic era. This review has critically described how the different phases of meiosis are orchestrated by changes in gene expression. The physiological states of the oocyte have been described together with the molecular mechanisms that control the critical transitions during meiosis progression, highlighting the connection between translation control and meiosis dynamics

Time-course of morphological changes characterizing the spontaneous death of progesterone matured oocytes.

<p>Fully-grown prophase oocytes were isolated from the ovaries and incubated in the presence (+Pg) or in the absence (−Pg) of 1 µM progesterone. (A) Oocytes originating from one female were photographed at the indicated times. (B) Time-course of death of either prophase-blocked oocytes (−Pg, white circles) or progesterone-matured oocytes (+Pg, black circles) from eight different females. Oocytes were considered as dead when their external morphology was similar to the one occurring at 48 h in panel A.</p

Bad and JNK control egg apoptosis in a cooperative manner.

<p>(A) and (B) Prophase oocytes were microinjected with mRNAs encoding either GFP (black circles), Bax (triangles) or Bad (squares) and were stimulated (B) or not (A) with progesterone one hour later. Egg death was monitored by external egg morphology at the indicated times. (C) and (D) Oocytes were microinjected with mRNAs encoding either GFP (white circles), the JNK-JJ (black diamonds), Bad (black triangles), a dominant negative form of Bad (BAD151A, black squares) or a mix of mRNA encoding for both JNK-JJ and Bad (crosses). One hour later, oocytes were incubated with progesterone (time 0). (C) Death time-course was monitored by egg morphological changes at the indicated times. (D) Western blot analysis of egg lysates using antibodies directed against ERK1, the active phosphorylated form of JNK (p-JNK) and active caspase 3 (casp-3). Note that the JNK-JJ form migrates slower than endogenous JNK. These data are representative experiments repeated on oocytes and eggs from three different females.</p