Biomedical subjects

Jean-Claude Labbé

Publications and source records attributed to Jean-Claude Labbé.

14 recordsLinked to original sources

PubMed2006

Pin1 stabilizes Emi1 during G2 phase by preventing its association with SCF(betatrcp).

The anaphase-promoting complex (APC) early mitotic inhibitor 1 (Emi1) is required to induce S- and M-phase entries by stimulating the accumulation of cyclin A and cyclin B through APC(Cdh1/cdc20) inhibition. In this report, we show that Emi1 proteolysis can be induced by cyclin A/cdk (cdk for cyclin-dependent kinase). Paradoxically, Emi1 is stable during G2 phase, when cyclin A/cdk, Plx1 and SCF(betatrcp) (SCF for Skp1-Cul1-Fbox protein)--which play a role in its degradation--are active. Here, we identify Pin1 as a new regulator of Emi1 that induces Emi1 stabilization by preventing its association with SCF(betatrcp). We show that Pin1 binds to Emi1 and prevents its association with betatrcp in an isomerization-dependent pathway. We also show that Emi1-Pin1 binding is present in vivo in XL2 cells during G2 phase and that this association protects Emi1 from being degraded during this phase of the cell cycle. We propose that S- and M-phase entries are mediated by the accumulation of cyclin A and cyclin B through a Pin1-dependent stabilization of Emi1 during G2.

Animals↗

PubMed2006

A genomewide screen for suppressors of par-2 uncovers potential regulators of PAR protein-dependent cell polarity in Caenorhabditis elegans.

The PAR proteins play an essential role in establishing and maintaining cell polarity. While their function is conserved across species, little is known about their regulators and effectors. Here we report the identification of 13 potential components of the C. elegans PAR polarity pathway, identified in an RNAi-based, systematic screen to find suppressors of par-2(it5ts) lethality. Most of these genes are conserved in other species. Phenotypic analysis of double-mutant animals revealed that some of the suppressors can suppress lethality associated with the strong loss-of-function allele par-2(lw32), indicating that they might impinge on the PAR pathway independently of the PAR-2 protein. One of these is the gene nos-3, which encodes a homolog of Drosophila Nanos. We find that nos-3 suppresses most of the phenotypes associated with loss of par-2 function, including early cell division defects and maternal-effect sterility. Strikingly, while PAR-1 activity was essential in nos-3; par-2 double mutants, its asymmetric localization at the posterior cortex was not restored, suggesting that the function of PAR-1 is independent of its cortical localization. Taken together, our results identify conserved components that regulate PAR protein function and also suggest a role for NOS-3 in PAR protein-dependent cell polarity.

Animals↗

PubMed2006

Ubiquitin-mediated protein degradation in Xenopus egg extracts.

Events controlling cell division are governed by the degradation of different regulatory proteins by the ubiquitin-dependent pathway. In this pathway, the attachment of a polyubiquitin chain to a substrate by an ubiquitin-ligase targets this substrate for degradation. Xenopus egg extracts present many advantages for the study of the cell cycle, including the availability of a large quantity of material synchronized at a particular phase of the cell cycle. In this chapter, we describe various protocols used in Xenopus egg extracts to study the ubiquitination and degradation of different cell cycle regulators. We first provide the method used to obtain interphase- and metaphase II-arrested egg extracts. Subsequently, we describe the protocol employed in these extracts to test the putative ubiquitination and degradation of a protein. Moreover, we describe a detailed practical procedure to test the role of different regulators in the ubiquitin-dependent degradation pathway of a specific protein. To that, we show how to eliminate some of these regulators from the extracts by immunodepletion and how to activate ectopically their function by the translation of their messenger ribonucleic acid. Finally, the Notes provide a series of practical details that explain the different problems that can occur and the possible solutions used to overcome them.

Animals↗

PubMed2005

The anaphase-promoting complex: a key factor in the regulation of cell cycle.

Anaphase↗

PubMed2004

The forces that position a mitotic spindle asymmetrically are tethered until after the time of spindle assembly.

Regulation of the mitotic spindle's position is important for cells to divide asymmetrically. Here, we use Caenorhabditis elegans embryos to provide the first analysis of the temporal regulation of forces that asymmetrically position a mitotic spindle. We find that asymmetric pulling forces, regulated by cortical PAR proteins, begin to act as early as prophase and prometaphase, even before the spindle forms and shifts to a posterior position. The spindle does not shift asymmetrically during these early phases due to a tethering force, mediated by astral microtubules that reach the anterior cell cortex. We show that this tether is normally released after spindle assembly and independently of anaphase entry. Monitoring microtubule dynamics by photobleaching segments of microtubules during anaphase revealed that spindle microtubules do not undergo significant poleward flux in C. elegans. Together with the known absence of anaphase A, these data suggest that the major forces contributing to chromosome separation during anaphase originate outside the spindle. We propose that the forces positioning the mitotic spindle asymmetrically are tethered until after the time of spindle assembly and that these same forces are used later to drive chromosome segregation at anaphase.

Anaphase↗

PubMed2004

Kinetochore localization of spindle checkpoint proteins: who controls whom?

The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other.

Animals↗

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

Embryonic development: a new SPN on cell fate specification.

The recent identification and characterization of the Caenorhabditis elegans gene spn-4 has shed new light on the mechanisms that link embryonic polarity to the specification of cell fates.

Animals↗

PubMed2002

Initial activation of cyclin-B1-cdc2 kinase requires phosphorylation of cyclin B1.

At the G(2)/M transition of the cell cycle, the cdc25c phosphatase dephosphorylates inhibitory residues of cdc2, and cyclin-B-cdc2 kinase (MPF) is activated. Phosphorylation of cyclin B1 induces its nuclear accumulation, and, since cdc25c is also believed to accumulate and activate shortly before G(2)/M in the nucleus, it has been proposed that this induces cyclin-B1-cdc2 kinase activation. We demonstrate that cyclin B1 phosphorylation has another essential function in vivo: it is required for cdc25c and MPF activation, which does not require nuclear accumulation of cyclin B1, and occurs in the cytoplasm.

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

Using RNA interference to identify genes required for RNA interference.

RNA interference (RNAi) is a phenomenon in which double-stranded RNA (dsRNA) silences endogenous gene expression. By injecting pools of dsRNAs into Caenorhabditis elegans, we identified a dsRNA that acts as a potent suppressor of the RNAi mechanism. We have used coinjection of dsRNAs to identify four additional candidates for genes involved in the RNAi mechanism in C. elegans. Three of the genes are C. elegans mes genes, some of which encode homologs of the Drosophila chromatin-binding Polycomb-group proteins. We have used loss-of-function mutants to confirm a role for mes-3, -4, and -6 in RNAi. Interestingly, introducing very low levels of dsRNA can bypass a requirement for these genes in RNAi. The finding that genes predicted to encode proteins that associate with chromatin are involved in RNAi in C. elegans raises the possibility that chromatin may play a role in RNAi in animals, as it does in plants.

Animals↗

PubMed2002

A new role for Mos in Xenopus oocyte maturation: targeting Myt1 independently of MAPK.

The resumption of meiosis in Xenopus arrested oocytes is triggered by progesterone, which leads to polyadenylation and translation of Mos mRNA, then activation of MAPK pathway. While Mos protein kinase has been reported to be essential for re-entry into meiosis in Xenopus, arrested oocytes can undergo germinal vesicle breakdown (GVBD) independently of MAPK activation, leading us to question what the Mos target might be if Mos is still required. We now demonstrate that Mos is indeed necessary, although is independent of the MAPK cascade, for conversion of inactive pre-MPF into active MPF. We have found that Myt1 is likely to be the Mos target in this process, as Mos interacts with Myt1 in oocyte extracts and Mos triggers Myt1 phosphorylation on some sites in vivo, even in the absence of MAPK activation. We propose that Mos is involved, not only in the MAPK cascade pathway, but also in a mechanism that directly activates MPF in Xenopus oocytes.

Animals↗