Biomedical subjects

Thierry Lorca

Publications and source records attributed to Thierry Lorca.

8 recordsLinked to original sources

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

Alterations of anaphase-promoting complex genes in human colon cancer cells.

Ubiquitin-mediated proteolysis of cell cycle regulators is a major element of the cell cycle control. The anaphase-promoting complex (APC/C) is a large multisubunit ubiquitin-protein ligase required for the ubiquitination and degradation of G1 and mitotic checkpoint regulators. APC/C-dependent proteolysis regulates cyclin levels in G1, and triggers the separation of sister chromatids at the metaphase-anaphase transition and the destruction of mitotic cyclins at the end of mitosis. Furthermore, it was recently shown that APC/C regulates the degradation of crucial regulators of signal transduction pathways. We report here gene alterations in several components of this complex in human colon cancer cells, including APC6/CDC16 and APC8/CDC23 which are known to be key function elements. The experimental expression of a truncation mutant of APC8/CDC23 subunit (CDC23DeltaTPR) leads to abnormal levels of APC/C targets such as cyclin B1 and disturbs the cell cycle progression of colon epithelial cells through mitosis. Overall, these data support the hypothesis of a deleterious role of these mutations during colorectal carcinogenesis.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2003

[Mitosis under control].

The mitotic checkpoint is essential to ensure accurate chromosome segregation by allowing a mitotic delay in response to a spindle defect. This checkpoint postpones the onset of anaphase until all the chromosomes are attached and correctly aligned onto the mitotic spindle. The checkpoint functions by preventing an ubiquitin ligase called the anaphase-promoting complex (APC) from ubiquitinylating proteins whose degradation is required for anaphase onset. Loss of this checkpoint results in chromosome missegregation in higher eukaryotes and may contribute to the genomic instability observed in most of the tumour cells.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2003

Xkid is degraded in a D-box, KEN-box, and A-box-independent pathway.

During mitosis, the Xenopus chromokinesin Kid (Xkid) provides the polar ejection forces needed at metaphase for chromosome congression, and its degradation is required at anaphase to induce chromosome segregation. Despite the fact that the degradation of Xkid at anaphase seems to be a key regulatory factor to induce chromosome movement to the poles, little is known about the mechanisms controlling this proteolysis. We investigated here the degradation pathway of Xkid. We demonstrate that Xkid is degraded both in vitro and in vivo by APC/Cdc20 and APC/Cdh1. We show that, despite the presence of five putative D-box motifs in its sequence, Xkid is proteolyzed in a D-box-independent manner. We identify a domain within the C terminus of this chromokinesin, with sequence GxEN, whose mutation completely stabilizes this protein by both APC/Cdc20 and APC/Cdh1. Moreover, we show that this degradation sequence acts as a transposable motif and induces the proteolysis of a GST-GXEN fusion protein. Finally, we demonstrate that both a D-box and a GXEN-containing peptides completely block APC-dependent degradation of cyclin B and Xkid, indicating that the GXEN domain might mediate the recognition and association of Xkid with the APC.

Amino Acid Motifs↗

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

Involvement of Aurora A kinase during meiosis I-II transition in Xenopus oocytes.

The Aurora kinase family has been involved both in vivo and in vitro in the stability of the metaphase plate and chromosome segregation. However, to date only one member of this family, the protein kinase Aurora B, has been implicated in the regulation of meiotic division in Caenorhabditis elegans. In this species, disruption of Aurora B results in the failure of polar body extrusion. To investigate whether Aurora A is also required in meiosis, we microinjected highly specific alpha-Aurora A antibodies in Xenopus oocytes. We demonstrated that microinjected oocytes fail to extrude the first polar body and are arrested with condensed chromosomes on a typical metaphase I plate, which has not performed its normal 90 degrees rotation. We additionally found that, although the failure of first polar body extrusion observed in alpha-Aurora A-microinjected oocytes is likely mediated by Eg5, the impairment of the metaphase plate rotation does not involve this kinesin-like protein. Surprisingly, although chromosomes remain condensed at a metaphase I stage in alpha-Aurora A-microinjected oocytes, the cytoplasmic cell cycle events progress normally through meiosis until metaphase II arrest. Moreover, these oocytes are able to undergo parthenogenetic activation. We conclude that Aurora A and Eg5 are involved in meiosis I to meiosis II transition in Xenopus oocytes.

Animals↗

PubMed2002

Interaction between the antiapoptotic protein Nr-13 and cytochrome c. Antagonistic effect of the BH3 domain of Bax.

Mitochondria act as a focal point for upstream apoptosis signals by releasing cytochrome c into the cytosol, leading to the activation of caspases and subsequent cell death. Members of the Bcl-2 protein family regulate this phenomenon by heterodimerization via the BH3 domain of proapoptotic members opposing their pro- and antiapoptotic functions. The mechanism of cytochrome c release from mitochondria and of its regulation remains controversial. In vitro binding studies of purified and biologically active proteins should help in understanding the molecular mechanism of interactions and protein functions. In this work, the Bcl-2-related antiapoptotic chicken protein Nr-13 was overexpressed as a highly soluble recombinant protein which showed correct folding as judged by circular dichroism and fluorescence spectroscopy. Purified Nr-13 inhibits caspase-3 activation in a Xenopus egg-derived cell-free system, and neutralizes the proapoptotic activity of a synthetic peptide containing the BH3 domain of Bax. The latter effect correlates with the high-affinity binding of the BH3 peptide to Nr-13 as monitored by the intrinsic tryptophan fluorescence. On the basis of the structural similarity with Bcl-x(L), putative residues involved in this interaction were identified. Nr-13 exhibits a high-affinity interaction with cytochrome c which is prevented by preincubation with the BH3-Bax peptide. These findings are discussed with respect to a model for the regulation of apoptosis in which a direct interaction between the antiapoptotic protein and cytochrome c may prevent the apoptosis.

Amino Acid Sequence↗

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↗