Biomedical subjects

C Jessus

Publications and source records attributed to C Jessus.

At least 19 recordsLinked to original sources

PubMed2006

Deciphering the H-Ras pathway in Xenopus oocyte.

Xenopus oocytes are arrested in prophase of the first meiotic division. In response to progesterone, they re-enter meiosis and arrest again in metaphase of the second meiotic division. This process, called meiotic maturation, is under the control of the Cyclin B-Cdc2 complex, M phase promoting factor (MPF). Injection of a constitutively active Xenopus H-Ras protein activates MPF, suggesting that Ras proteins could be implicated in the progesterone transduction pathway. The aim of this study was (1) to elucidate the pathway triggered by H-Ras leading to MPF activation in Xenopus oocytes and (2) to investigate whether endogenous H-Ras is involved in the physiological process of meiotic maturation. We generated three constitutively active double mutants, each of them recruiting a single effector in mammalian cells, mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) or RalGDS. Our results show that the activation of a PI3K-related enzyme is crucial for H-Ras-induced MPF activation, whereas the recruitment of either MAPK or RalGDS is not. However, although the H-Ras/PI3K pathway is functional in Xenopus oocytes, it is not the physiological transducer of progesterone responsible for meiotic resumption.

Animals↗

PubMed2001

Membrane-anchored cyclin A2 triggers Cdc2 activation in Xenopus oocyte.

In Xenopus oocyte, the formation of complexes between neosynthesized cyclins and Cdc2 contributes to Cdc2 kinase activation that triggers meiotic divisions. It has been proposed that cytoplasmic membranes could be involved in this process. To investigate this possibility, we have injected in the oocyte two undegradable human cyclin A2 mutants anchored to the endoplasmic reticulum (ER) membrane. They encode fusion proteins between the truncated cyclin A2-Delta152 and a viral or cellular ER-targeting domain. We show that both mutants are fully functional as mitotic cyclins when expressed in Xenopus oocytes, bind Cdc2 and activate M-phase promoting factor.

Animals↗

PubMed2001

Differential regulation of Cdc2 and Cdk2 by RINGO and cyclins.

Cyclin-dependent kinases (Cdks) are key regulators of the eukaryotic cell division cycle. Cdk1 (Cdc2) and Cdk2 should be bound to regulatory subunits named cyclins as well as phosphorylated on a conserved Thr located in the T-loop for full enzymatic activity. Cdc2- and Cdk2-cyclin complexes can be inactivated by phosphorylation on the catalytic cleft-located Thr-14 and Tyr-15 residues or by association with inhibitory subunits such as p21(Cip1). We have recently identified a novel Cdc2 regulator named RINGO that plays an important role in the meiotic cell cycle of Xenopus oocytes. RINGO can bind and activate Cdc2 but has no sequence homology to cyclins. Here we report that, in contrast with Cdc2- cyclin complexes, the phosphorylation of Thr-161 is not required for full activation of Cdc2 by RINGO. We also show that RINGO can directly stimulate the kinase activity of Cdk2 independently of Thr-160 phosphorylation. Moreover, RINGO-bound Cdc2 and Cdk2 are both less susceptible to inhibition by p21(Cip1), whereas the Thr-14/Tyr-15 kinase Myt1 can negatively regulate the activity of Cdc2-RINGO with reduced efficiency. Our results indicate that Cdk-RINGO complexes may be active under conditions in which cyclin-bound Cdks are inhibited and can therefore play different regulatory roles.

Animals↗

PubMed2001

Interplay between Cdc2 kinase and the c-Mos/MAPK pathway between metaphase I and metaphase II in Xenopus oocytes.

Xenopus oocytes arrested in prophase I resume meiotic division in response to progesterone and arrest at metaphase II. Entry into meiosis I depends on the activation of Cdc2 kinase [M-phase promoting factor (MPF)]. To better understand the role of Cdc2, MPF activity was specifically inhibited by injection of the CDK inhibitor, Cip1. When Cip1 is injected at germinal vesicle breakdown (GVBD) time, Cdc25 and Plx1 are both dephosphorylated and Cdc2 is rephosphorylated on tyrosine. The autoamplification loop characterizing MPF is therefore not only required for MPF generation before GVBD, but also for its stability during the GVBD period. The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), responsible for cyclin degradation, is also under the control of Cdc2; therefore, Cdc2 activity itself induces its own inactivation through cyclin degradation, allowing the exit from the first meiotic division. In contrast, cyclin accumulation, responsible for Cdc2 activity increase allowing entry into metaphase II, is independent of Cdc2. The c-Mos/mitogen-activated protein kinase (MAPK) pathway remains active when Cdc2 activity is inhibited at GVBD time. This pathway could be responsible for the sustained cyclin neosynthesis. In contrast, during the metaphase II block, the c-Mos/MAPK pathway depends on Cdc2. Therefore, the metaphase II block depends on a dynamic interplay between MPF and CSF, the c-Mos/MAPK pathway stabilizing cyclin B, whereas in turn, MPF prevents c-Mos degradation.

Animals↗

PubMed2001

From progesterone to active Cdc2 in Xenopus oocytes: a puzzling signalling pathway.

Since almost two decades, it is known that progesterone is responsible of the release of the prophase I arrest of amphibian oocytes and leads to the activation of the universal MPF, through a puzzling transduction pathway. It involves negative regulation of the cAMP-dependent protein kinase (PKA) and synthesis of new proteins, among them the c-Mos protooncogene product. The implication of the Mos/mitogenic activated protein kinase (MAP kinase) pathway in Cdc2 activation has been extensively studied and is now at the centre of a controversial debate. In this paper, we discuss the current progress and our recent results on the molecular mechanisms allowing progesterone to activate MPF and propose a model to partly resolve the long-standing inconsistencies concerning the role of Mos/MAP kinase during this process.

Animals↗

PubMed2000

Progesterone regulates the accumulation and the activation of Eg2 kinase in Xenopus oocytes.

Xenopus prophase oocytes reenter meiotic division in response to progesterone. The signaling pathway leading to Cdc2 activation depends on neosynthesized proteins and a decrease in PKA activity. We demonstrate that Eg2 protein, a Xenopus member of the Aurora/Ipl1 family of protein kinases, accumulates in response to progesterone and is degraded after parthenogenetic activation. The polyadenylation and cap ribose methylation of Eg2 mRNA are not needed for the protein accumulation. Eg2 protein accumulation is induced by progesterone through a decrease in PKA activity, upstream of Cdc2 activation. Eg2 kinase activity is undetectable in prophase and is raised in parallel with Cdc2 activation. In contrast to Eg2 protein accumulation, Eg2 kinase activation is under Cdc2 control. Furthermore, by using an anti-sense strategy, we show that Eg2 accumulation is not required in the transduction pathway leading to Cdc2 activation. Altogether, our results strongly suggest that Eg2 is not necessary for Cdc2 activation, though it could participate in the organization of the meiotic spindles, in agreement with the well-conserved roles of the members of the Aurora family, from yeast to man.

Animals↗

PubMed1999

In vitro binding of free cdc2 and raf kinase to membrane vesicles: a possible new regulatory mechanism for cdc2 kinase activation in Xenopus oocyte.

The G2-M transition of the cell cycle is under the control of the M-phase promoting factor (MPF) formed of cdc2 kinase and cyclin B. The Xenopus prophase-blocked oocyte contains a stockpile of cyclin B2-cdc2 complexes that are maintained inactive by a double inhibitory phosphorylation on Thr-14 and Tyr-15 of cdc2. Free cdc2 molecules that are not associated with cyclin, are present in excess as compared to cyclin B2-associated cdc2. This pool of free cdc2 is permanently recruited to associate with neosynthetized cyclin B2 in the resting prophase oocyte, to feed up the pre-MPF stockpile. During re-entry into meiosis, free cdc2 could generate with newly synthesized cyclin B a small level of active MPF, that could serve as starter to initiate the conversion of pre-MPF into MPF. It was, therefore, of high interest to investigate whether free cdc2 interacts with other proteins and what could be its intracellular localization. To address these questions, we developed an in vitro system of membrane vesicles. We demonstrate here that free cdc2 is recovered in association with the external layer of membrane vesicles, whereas cyclin B2-associated cdc2 is not. Cyclin is able to associate in vitro with cdc2-containing membrane vesicles. This association does not induce the inhibitory cdc2 phosphorylations. However, it does not lead to active complexes, suggesting that membrane vesicles prevent cdc2 activation. C-Raf1, another kinase activated during reentry into meiosis, is also totally recovered in association with the membrane vesicles.

Animals↗

PubMed1999

Two distinct mechanisms control the accumulation of cyclin B1 and Mos in Xenopus oocytes in response to progesterone.

Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34(cdc2) could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34(cdc2), and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor-induced feedback. We report here that the cdk inhibitor p21(cip1), when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21(cip1), progesterone fails to induce the activation of MAPK or p34(cdc2), and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA.

Animals↗

PubMed1999

Phosphatase 2A and polo kinase, two antagonistic regulators of cdc25 activation and MPF auto-amplification.

The auto-catalytic activation of the cyclin-dependent kinase Cdc2 or MPF (M-phase promoting factor) is an irreversible process responsible for the entry into M phase. In Xenopus oocyte, a positive feed-back loop between Cdc2 kinase and its activating phosphatase Cdc25 allows the abrupt activation of MPF and the entry into the first meiotic division. We have studied the Cdc2/Cdc25 feed-back loop using cell-free systems derived from Xenopus prophase-arrested oocyte. Our findings support the following two-step model for MPF amplification: during the first step, Cdc25 acquires a basal catalytic activity resulting in a linear activation of Cdc2 kinase. In turn Cdc2 partially phosphorylates Cdc25 but no amplification takes place; under this condition Plx1 kinase and its activating kinase, Plkk1 are activated. However, their activity is not required for the partial phosphorylation of Cdc25. This first step occurs independently of PP2A or Suc1/Cks-dependent Cdc25/Cdc2 association. On the contrary, the second step involves the full phosphorylation and activation of Cdc25 and the initiation of the amplification loop. It depends both on PP2A inhibition and Plx1 kinase activity. Suc1-dependent Cdc25/Cdc2 interaction is required for this process.

14-3-3 Proteins↗

PubMed1998

Inhibition of small G proteins by clostridium sordellii lethal toxin activates cdc2 and MAP kinase in Xenopus oocytes.

The lethal toxin (LT) from Clostridium sordellii is a glucosyltransferase that modifies and inhibits small G proteins of the Ras family, Ras and Rap, as well as Rac proteins. LT induces cdc2 kinase activation and germinal vesicle breakdown (GVBD) when microinjected into full-grown Xenopus oocytes. Toxin B from Clostridium difficile, that glucosylates and inactivates Rac proteins, does not induce cdc2 activation, indicating that proteins of the Ras family, Ras and/or Rap, negatively regulate cdc2 kinase activation in Xenopus oocyte. In oocyte extracts, LT catalyzes the incorporation of [14C]glucose into a group of proteins of 23 kDa and into one protein of 27 kDa. The 23-kDa proteins are recognized by anti-Rap1 and anti-Rap2 antibodies, whereas the 27-kDa protein is recognized by several anti-Ras antibodies and probably corresponds to K-Ras. Microinjection of LT into oocytes together with UDP-[14C]glucose results in a glucosylation pattern similar to the in vitro glucosylation, indicating that the 23- and 27-kDa proteins are in vivo substrates of LT. In vivo time-course analysis reveals that the 27-kDa protein glucosylation is completed within 2 h, well before cdc2 kinase activation, whereas the 23-kDa proteins are partially glucosylated at GVBD. This observation suggests that the 27-kDa Ras protein could be the in vivo target of LT allowing cdc2 kinase activation. Interestingly, inactivation of Ras proteins does not prevent the phosphorylation of c-Raf1 and the activation of MAP kinase that occurs normally around GVBD.

Animals↗

PubMed1998

MPF amplification in Xenopus oocyte extracts depends on a two-step activation of cdc25 phosphatase.

The activation of Cdc2 kinase induces the entry into M-phase of all eukaryotic cells. We have developed a cell-free system prepared from prophase-arrested Xenopus oocytes to analyze the mechanism initiating the all-or-none activation of Cdc2 kinase. Inhibition of phosphatase 2A, the major okadaic acid-sensitive Ser/Thr phosphatase, in these extracts, provokes Cdc2 kinase amplification and concomitant hyperphosphorylation of Cdc25 phosphatase, with a lag of about 1 h. Polo-like kinase (Plx1 kinase) is activated slightly after Cdc2. All these events are totally inhibited by the cdk inhibitor p21(Cip1), demonstrating that Plx1 kinase activation depends on Cdc2 kinase activity. Addition of a threshold level of recombinant Cdc25 induces a linear activation of Cdc2 and Plx1 kinases and a partial phosphorylation of Cdc25. We propose that the Cdc2 positive feedback loop involves two successive phosphorylation steps of Cdc25 phosphatase: the first one is catalyzed by Cdc2 kinase and/or Plx1 kinase but it does not modify Cdc25 enzymatic activity, the second one requires a new kinase counteracted by phosphatase 2A. Furthermore we demonstrate that, under our conditions, Cdc2 amplification and MAP kinase activation are two independent events.

Animals↗

PubMed1998

Human retinoblastoma protein (Rb) is phosphorylated by cdc2 kinase and MAP kinase in Xenopus maturing oocytes.

Xenopus oocyte meiotic maturation combines features of G0/G1 and G2/M transitions of the cell cycle. To study the in ovo Rb kinase activity, we have microinjected human Rb into oocytes. Microinjected human Rb localizes into the nucleus, is hypophosphorylated in prophase oocytes, becomes hyperphosphorylated during meiotic maturation and is dephosphorylated as the cell reenters interphase. Inactivation or overexpression of the cyclin D-cdk4/6 complex in an oocyte extract does not affect the Rb kinase activity. This kinase activity could be attributed to both cdc2-cyclin B and MAP kinase, opening new perspectives of investigation in somatic cells.

Animals↗

PubMed1998

Ras family proteins: new players involved in the diplotene arrest of Xenopus oocytes.

Oogonia undergo numerous mitotic cell cycles before completing the last DNA replication and entering the meiotic prophase I. After chromosome pairing and chromatid exchanges between paired chromosomes, the oocyte I remains arrested at the diplotene stage of the first meiotic prophase. Oocyte growth then occurs independently of cell division; indeed, during this growth period, oocytes (4n DNA) are prevented from completing the meiotic divisions. How is the prophase arrest regulated? One of the players of the prophase block is the high level of intracellular cAMP, maintained by an active adenylate cyclase. By using lethal toxin from Clostridium sordellii (LT), a glucosyltransferase that glucosylates and inactivates small G proteins of the Ras subfamily, we have shown that inhibition of either Ras or Rap or both proteins is sufficient to release the prophase block of Xenopus oocytes in a cAMP-dependent manner. The implications of Ras family proteins as new players involved in the prophase arrest of Xenopus oocytes will be discussed here.

Animals↗

PubMed1997

Cyclin D2 arrests Xenopus early embryonic cell cycles.

Xenopus cyclin D2 mRNA is a member of the class of maternal RNAs. It is rare and stable during early embryonic development. To investigate the potential role of cyclin D2 during early embryonic cell cycles, cyclin D2 was injected into one blastomere of a two-cell embryo. This injection induced a cell cycle arrest in the injected blastomere. To analyze more precisely the mechanism of this arrest, we took advantage of cycling egg extracts that recapitulate major events of the cell cycle when supplemented with demembranated sperm heads. When Xenopus cyclin D2 is added to egg extracts, the first round of DNA replication occurs as in control extracts. However, Xenopus cyclin D2 blocks subsequent rounds of DNA replication and the oscillations of histone H1 kinase activity associated with cdc2 kinase, indicating that the cell cycle is arrested after the first S-phase. The block induced by Xenopus cyclin D2 is not due to a lack of the mitotic cyclin B2 that accumulates normally. Radiolabeled Xenopus cyclin D2 enters nuclei after completion of the first S-phase and remains stable over the entire period of the arrest. These features suggest that Xenopus cyclin D2 could play an original role during early development, controlling the G2-phase and/or the G2/M transition.

Animals↗

PubMed1997

In vivo regulation of cytostatic activity in Xenopus metaphase II-arrested oocytes.

Metaphase II arrest of Xenopus oocyte is characterized by the presence of M-phase-promoting factor (MPF) and of a microtubular spindle, both of which are stable in the presence of protein synthesis inhibitors. We studied in vivo this equilibrium state that is settled during meiotic maturation. At time of germinal vesicle breakdown (GVBD), cdc2 kinase and MAP kinase activities are stimulated. A component of the cyclin ubiquitin ligase, CDC27, is phosphorylated at the same time and remains phosphorylated until fertilization, indicating that an important component of the ligase complex is modified as early as GVBD. During a first period extending from GVBD until the cortical anchorage of the metaphase II spindle, homogeneous pools of cdc2 kinase and mitogen-activated protein (MAP) kinase activities are present in oocyte and are strictly dependent on protein turnover, since protein synthesis inhibition induces their total inactivation and drives oocytes into interphase. The metaphase II spindle, once anchored into the cortex, is no more sensitive to protein synthesis inhibition, likewise MAP kinase activity. During this cellular arrest, cdc2 kinase is divided into two distinctly regulated pools. The first one contains cyclin B that actively turns over and is subjected to a microtubular checkpoint. The second one is stable. Alteration of intracellular compartmentation of metaphase II oocytes either by gentle centrifugation or by cold shock inactivates MAP kinase and targets all cyclin B molecules for full destruction. We therefore suggest that MAP kinase participates to the cytostatic activity by preventing part of cyclin B molecules from entering the ubiquitination/degradation machinery which is still turned on in metaphase II oocytes.

Animals↗

PubMed1997

On cyclins, oocytes, and eggs.

Oocyte and egg are suitable model systems for studying cell division since meiotic maturation resembles a G2/M transition and early embryonic divisions are precisely timed and occur without zygotic transcription. The analysis of oocytes and eggs from different species provides the opportunity to understand the roles of proteins that the critical to the progression and maintenance of the cell cycle. Among them, cyclins are certainly worthy of investigation. Mitotic cyclins (cyclins A and B) are clearly implicated in meiosis and early embryonic cell cycles. More recent studies have revealed that G1-type cyclins (cyclins E and D) could also play a role in both processes and cyclin H has been suggesed to participate to CAK activity (cdc2-activating kinase) in oocytes. The study of cyclins in oocytes and eggs clearly offer insights into their roles during the cell cycle.

Animals↗

PubMed1996

Xenopus cyclin D2: cloning and expression in oocytes and during early development.

We have isolated and characterized a cDNA which contains the entire coding sequence of Xenopus laevis cyclin D2 protein. Cyclin D2 mRNA is identified as a member of the class of maternal RNAs. It is rare and stable during embryonic development at least until tadepole. In addition, a second cDNA coding for a truncated version of cyclin D2 was also isolated. Microinjection of cyclin D2 into oocytes undergoing meiotic maturation and parthenogenetic activation reveals that the protein is stable for several hours, independently of the ubiquitin-mediated degradation of cyclin B2 that takes place periodically during this process. Microinjected cyclin D2 localizes both in the cytoplasm and in the nucleus of oocyte. In somatic cells, it is well established that cyclin D2 is almost exclusively nuclear and very labile. The unusual behaviour of cyclin D2 upon injection into oocytes may provide indications about a possible role for this protein during meiosis and early development.

Amino Acid Sequence↗

PubMed1995

Inhibition of glycosphingolipid synthesis induces p34cdc2 activation in Xenopus oocyte.

In Xenopus prophase-blocked oocytes, it is assumed that progesterone interacts with the plasma membrane to initiate a signalling cascade that ultimately leads to MPF activation. Progesterone regulates negatively the cAMP pathway through an inhibition of adenylate cyclase. However, the mechanisms linking the initial action of the hormone with adenylate cyclase activity remain to be elucidated. Here, we demonstrate that PDMP, an inhibitor of glucosphingolipid synthesis, triggers oocyte meiotic maturation in a cAMP- and cycloheximide-dependent manner, whereas exogenous ceramide is unefficient. We propose that sphingolipid metabolism and targeting represent an important regulatory process of oocyte meiosis.

1-Methyl-3-isobutylxanthine↗