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Claude Prigent

Publications and source records attributed to Claude Prigent.

13 recordsLinked to original sources
PubMed2003

Phosphorylation of serine 10 in histone H3, what for?

Eukaryotic cells must possess mechanisms for condensing and decondensing chromatin. Chromatin condensation is particularly evident during mitosis and cell death induced by apoptosis, whereas chromatin decondensation is necessary for replication, repair, recombination and transcription. Histones are among the numerous DNA-binding proteins that control the level of DNA condensation, and post-translational modification of histone tails plays a critical role in the dynamic condensation/decondensation that occurs during the cell cycle. Phosphorylation of Ser10 in the tails of histone H3 has been extensively studied in many organisms. Interestingly, this modification is involved in both transcription and cell division, two events requiring opposite alterations in the degree of chromatin compaction. How does one and the same modification of histone H3 fulfil such roles? For instance, in interphase, phosphorylation of H3 correlates with chromatin relaxation and gene expression, whereas in mitosis it correlates with chromosome condensation. What is the kinase and under what circumstances does Ser10 becomes phosphorylated? Most importantly, what are the consequences of phosphorylation of this residue?

Animals↗
PubMed2003

Aurora A and mitotic commitment.

A remarkable study published in this issue of Cell reveals a key role of Aurora A protein kinase in G2/M progression. To achieve this role, Aurora A acts in conjunction with the LIM protein Ajuba, which functions as an activating factor.

Animals↗
PubMed2003

Cdc2-cyclin B triggers H3 kinase activation of Aurora-A in Xenopus oocytes.

Xenopus oocytes are arrested in meiotic prophase I and resume meiotic divisions in response to progesterone. Progesterone triggers activation of M-phase promoting factor (MPF) or Cdc2-cyclin B complex and neosynthesis of Mos kinase, responsible for MAPK activation. Both Cdc2 and MAPK activities are required for the success of meiotic maturation. However, the signaling pathway induced by progesterone and leading to MPF activation is poorly understood, and most of the targets of both Cdc2 and MAPK in the oocyte remain to be determined. Aurora-A is a Ser/Thr kinase involved in separation of centrosomes and in spindle assembly during mitosis. It has been proposed that in Xenopus oocytes Aurora-A could be an early component of the progesterone-transduction pathway, acting through the regulation of Mos synthesis upstream Cdc2 activation. We addressed here the question of Aurora-A regulation during meiotic maturation by using new in vitro and in vivo experimental approaches. We demonstrate that Cdc2 kinase activity is necessary and sufficient to trigger both Aurora-A phosphorylation and kinase activation in Xenopus oocyte. In contrast, these events are independent of the Mos/MAPK pathway. Aurora-A is phosphorylated in vivo at least on three residues that regulate differentially its kinase activity. Therefore, Aurora-A is under the control of Cdc2 in the Xenopus oocyte and could be involved in meiotic spindle establishment.

Animals↗
PubMed2003

Preparation and characterization of a human aurora-A kinase monoclonal antibody.

We have developed monoclonal antibodies against the human aurora-A serine/threonine kinase. After immunization of a mouse, a fusion was performed to obtain hybridomas that were selected because they produced immunoglobulin positively reacting against the protein used for immunization. We isolated one particular monoclonal that we named 35C1 using a series of selective assays. The first criteria of the screen for monoclonals was an ELISA (Enzyme Linked Immunosorbant Assay) assay performed in 96-well plates against the purified recombinant histidine-tagged aurora-A. The second was a positive Western blot against the same recombinant protein. The third criteria was a positive western blot against an HeLa cell extract, the selected monoclonal should detect only one protein migrating at 46 kDa (kiloDalton) on SDS (Sodium Dodecyl Sulfate)-polyacrylamide gel electrophoresis. Finally, the monoclonal had to bind to duplicated centrosomes and spindle poles in human MCF7 cultured cells by indirect immunofluorescence. At this stage several monoclonals were still positive. We then increased the selectivity by searching for antibodies that were able to cross-react with the mouse aurora-A kinase both by western blot and indirect immunofluorescence. We selected and cloned the 35C1 hybridoma to produce the antibody. Further characterization of the 35C1 antibody revealed that it was able to immunoprecipitate the kinase, that it did not inhibit the aurora-A kinase activity and consequently could be used to measure the aurora-A kinase activity in vivo after immunoprecipitation.

Animals↗
PubMed2003

A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly.

The activated form of Ran (Ran-GTP) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-alpha and -beta. We report here that Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. We show that TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-alpha and -beta, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. As the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, we hypothesize that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics.

Animals↗
PubMed2002

The D-Box-activating domain (DAD) is a new proteolysis signal that stimulates the silent D-Box sequence of Aurora-A.

We have demonstrated previously that Xenopus Aurora-A is degraded at late mitosis by the APC/Fizzy-Related in a D-Box-dependent manner. Here we demonstrate that, although Aurora-B possesses the same D-Box as Aurora-A, Aurora-B is not degraded by this ubiquitin ligase. We have constructed a chimera Aurora-A/B with the N-terminus of Aurora-A and the C-terminus of Aurora-B and we have examined its degradation by APC/Fizzy-Related. We demonstrate that the N-terminus of Aurora-A confers degradation capacity on the C-terminus of Aurora-B and that this feature is blocked by mutation of the conserved D-Box sequence. We characterize the minimal degradation signal at the N-terminus of Aurora-A and demonstrate that its deletion blocks the degradation of this protein by APC/Fizzy-Related. Thus, we conclude that two different degradation signals are required for proteolysis of Aurora-A. The first one, which we designated D-Box-activating domain, within the N-terminal domain of Aurora-A confers the functionality to the second, a silent D-Box, present within the C-terminus of the kinase.

Animals↗
PubMed2002

A Method for Analyzing the Ubiquitination and Degradation of Aurora-A.

The cell cycle machinery consists of regulatory proteins that control the progression through the cell cycle ensuring that DNA replication alternates with DNA segregation in mitosis to maintain cell integrity. Some of these key regulators have to be degraded at each cell cycle to prevent cellular dysfunction. Mitotic exit requires the inactivation of cyclin dependent kinase1 (cdk1) and it is the degradation of the cyclin subunit that inactivates the kinase. Cyclin degradation has been well characterized and it was shown that it is ubiquitin proteasome pathway that leads to the elimination of cyclins. By now, many other regulatory proteins were shown to be degraded by the same pathway, among them members of the aurora kinase family, degraded many other regulatory proteins. Aurora kinases are involved in mitotic spindle formation as well as in cytokinesis. The abundance and activity of the kinase is precisely regulated during the cell cycle. To understand how proteolysis regulates transitions through the cell cycle we describe two assays for ubiquitination and degradation of xenopus aurora kinase A using extracts from xenopus eggs or somatic cell lines.

Journal Article↗
PubMed2002

On the role of aurora-A in centrosome function.

Mammalian aurora-A belongs to a multigenic family of mitotic serine/threonine kinases comprising two other members: aurora-B and aurora-C. In this review we will focus on aurora-A that starts to localize to centrosomes only in S phase as soon as centrioles have been duplicated, the protein is then degraded in early G1. Works in various organisms have revealed that the kinase is involved in centrosome separation, duplication and maturation as well as in bipolar spindle assembly and stability. Aurora kinases are found in all organisms in which their function has been conserved throughout evolution, namely the control of chromosome segregation. In human, aurora-A has focused a lot of attention, since its overexpression has been found to be correlated with the grade of various solid tumours. Ectopic kinase overexpression in any culture cell line leads to polyploidy and centrosome amplification. However, overexpression of aurora-A in particular cell lines such as NIH3T3 is sufficient to induce growth on soft agar. Those transformed cells form tumours when implanted in immunodeficient mice, indicating that the kinase is an oncogene.

Animals↗
PubMed2002

APC/Fizzy-Related targets Aurora-A kinase for proteolysis.

Aurora-A kinase is a mitotic spindle-pole-associated protein that has been implicated in duplication and separation of centrosomes and in spindle assembly. The proper timing and amplitude of Aurora-A expression seems to be important, as elevated levels of this protein have been associated with centrosome abnormalities and aneuploidy in mammalian cells. We show that Aurora-A increases at the G2-M transistion and disappears completely at G1 in XL2 cells. Using Xenopus oocyte extracts, we demonstrate that degradation of Aurora-A is mediated by the anaphase-promoting complex (APC) and is regulated by Fizzy-Related but not by Fizzy. Degradation of Aurora-A depends on a D-Box, but not on its KEN-Box motif, as mutation of its C-terminal D-Box sequence induces stabilization of the protein. Accordingly, addition into the extracts of a cyclin B-type D-Box-motif-containing peptide completely suppresses its degradation. Furthermore, APC/Fizzy-Related ubiquitylates the wild type but not a D-Box mutant form of Aurora-A in vitro. Consistent with these data, ectopic expression of Fizzy-Related in Xenopus oocytes induces complete degradation of endogenous Aurora-A. Aurora-A is thus the first protein, at least in our assay system, that undergoes a D-Box-dependent degradation mediated by APC/Fizzy-Related but not by APC/Fizzy.

Amino Acid Motifs↗
PubMed2002

Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules.

Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC. We show that a subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. We propose that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein may be recruited, and thus modulate the behavior of astral microtubules.

Adenosine Triphosphate↗
PubMed2002

Centrosome separation: respective role of microtubules and actin filaments.

In mammalian cells, the separation of centrosomes is a prerequisite for bipolar mitotic spindle assembly. We have investigated the respective contribution of the two cytoskeleton components, microtubules and actin filaments, in this process. Distances between centrosomes have been measured during cell cycle progression in Xenopus laevis XL2 cultured cells in the presence or absence of either network. We considered two stages in centrosome separation: the splitting stage, when centrosomes start to move apart (minimum distance of 1 microm), and the elongation stage (from 1 to 7 microm). In interphase, depolymerisation of microtubules by nocodazole significantly inhibited the splitting stage, while the elongation stage was, on the contrary, facilitated. In mitosis, while nocodazole treatment completely blocked spindle assembly, in prophase, we observed that 55% of the centrosomes separated, versus 94% in the control. Upon actin depolymerisation by latrunculin, splitting of the interphase centrosome was blocked, and cells entered mitosis with unseparated centrosomes. Cells compensated for this separation delay by increasing the length of both prophase and prometaphase stages to allow for centrosome separation until a minimal distance was reached. Then the cells passed through anaphase, performing proper chromosome separation, but cytokinesis did not occur, and binuclear cells were formed. Our results clearly show that the actin microfilaments participate in centrosome separation at the G2/M transition and work in synergy with the microtubules to accelerate centrosome separation during mitosis.

Actin Cytoskeleton↗