APCcdh1 Mediates Degradation of the Oncogenic Rho-GEF Ect2 after Mitosis

Background: Besides regulation of actin cytoskeleton-dependent functions, Rho GTPase pathways are essential to cell cycle progression and cell division. Rho, Rac and Cdc42 regulate G1 to S phase progression and are involved in cytokinesis. RhoA GDP/GTP cycling is required for normal cytokinesis and recent reports have shown that the exchange factor Ect2 and the GTPase activating protein MgcRacGAP regulate RhoA activity during mitosis. We previously showed that the transcription factors E2F1 and CUX1 regulate expression of MgcRacGAP and Ect2 as cells enter S-phase. Methodology/Principal Findings: We now report that Ect2 is subject to proteasomal degradation after mitosis, following ubiquitination by the APC/C complex and its co-activator Cdh1. A proper nuclear localization of Ect2 is necessary for its degradation. APC-Cdh1 assembles K11-linked poly-ubiquitin chains on Ect2, depending upon a stretch of,25 amino acid residues that contain a bi-partite NLS, a conventional D-box and two TEK-like boxes. Site-directed mutagenesis of target sequences generated stabilized Ect2 proteins. Furthermore, such degradation-resistant mutants of Ect2 were found to activate RhoA and subsequent signalling pathways and are able to transform NIH3T3 cells. Conclusions/Significance: Our results identify Ect2 as a bona fide cell cycle-regulated protein and suggest that its ubiquitination-dependent degradation may play an important role in RhoA regulation at the time of mitosis. Our findings raise the possibility that the overexpression of Ect2 that has been reported in some human tumors might result not only from deregulated transcription, but also from impaired degradation

Rôle physiopathologique de la protéine RhoGEF Ect2 (un régulateur des RhoGTPases)

CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Ect2 MgcRacGAP (Deux régulateurs des RhoGTPases dont l'expression dépend du cycle cellulaire)

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

CUX1 and E2F1 Regulate Coordinated Expression of the Mitotic Complex Genes Ect2, MgcRacGAP, and MKLP1 in S Phase ▿ †

Rho GTPases are critical for mitosis progression and completion of cytokinesis. During mitosis, the GDP/GTP cycle of Rho GTPases is regulated by the exchange factor Ect2 and the GTPase activating protein MgcRacGAP which associates with the kinesin MKLP1 in the centralspindlin complex. We report here that expression of Ect2, MgcRacGAP, and MKLP1 is tightly regulated during cell cycle progression. These three genes share similar cell cycle-related signatures within their promoter regions: (i) cell cycle gene homology region (CHR) sites located at −20 to +40 nucleotides of their transcription start sites that are required for repression in G1, (ii) E2F binding elements, and (iii) tandem repeats of target sequences for the CUX1 transcription factor. CUX1 and E2F1 bind these three promoters upon S-phase entry, as demonstrated by chromatin immunoprecipitation, and regulate transcription of these genes, as established using promoter-luciferase reporter constructs and expression of activated or dominant negative transcription factors. Overexpression of either E2F1 or CUX1 increased the levels of the endogenous proteins whereas small interfering RNA knockdown of E2F1 or use of a dominant negative E2F1 reduced their expression levels. Thus, CUX1, E2F, and CHR elements provide the transcriptional controls that coordinate induction of Ect2, MgcRacGAP, and MKLP1 in S phase, leading to peak expression of these interacting proteins in G2/M, at the time they are required to regulate cytokinesis

Regulation of Rho signaling pathways in interleukin-2-stimulated human T-lymphocytes.

Rho GTPases are key regulators of many cellular functions, including cytoskeleton organization which is important for cell morphology and mobility, gene expression, cell cycle progression, and cytokinesis. In addition, it has recently been recognized that Rho GTPase activity is required for development of the immune system, as well as for the specialized functions of the peripheral cells that act in the immune response such as antigen presenting cells and lymphocytes. Stimulation of T lymphocytes with interleukin-2 (IL-2) induces clonal expansion of antigen-specific populations and provides a model to study cell cycle entry and cell cycle progression. We have performed gene expression analysis in a model of human T lymphocytes, which proliferate in response to IL-2. In addition to changes in genes relevant to cell cycling and to the antiapoptotic effects of IL-2, we have analyzed expression and variations of more than 300 genes involved in Rho GTPase signaling pathways. We report here that IL-2 regulates the expression of a number of proteins, which participate in the Rho GTPase pathways, including some of the GTPases themselves, GDP/GTP exchange factors, GTPase activating proteins, as well as GDIs and effectors. Our results suggest that regulation of expression of components of the Rho GTPase pathways may be an important mechanism in assembling specific signal transduction cascades that need to be active at certain times during the cell cycle. Some of our findings may also be relevant to the roles of Rho GTPases in T lymphocyte functions and proliferation

Ciliogenesis is regulated by a huntingtin-HAP1-PCM1 pathway and is altered in Huntington disease

Huntington disease (HD) is a devastating autosomal-dominant neurodegenerative disorder. It is caused by expansion of a CAG repeat in the first exon of the huntingtin (HTT) gene that encodes a mutant HTT protein with a polyglutamine (polyQ) expansion at the amino terminus. Here, we demonstrate that WT HTT regulates ciliogenesis by interacting through huntingtin-associated protein 1 (HAP1) with pericentriolar material 1 protein (PCM1). Loss of Htt in mouse cells impaired the retrograde trafficking of PCM1 and thereby reduced primary cilia formation. In mice, deletion of Htt in ependymal cells led to PCM1 mislocalization, alteration of the cilia layer, and hydrocephalus. Pathogenic polyQ expansion led to centrosomal accumulation of PCM1 and abnormally long primary cilia in mouse striatal cells. PCM1 accumulation in ependymal cells was associated with longer cilia and disorganized cilia layers in a mouse model of HD and in HD patients. Longer cilia resulted in alteration of the cerebrospinal fluid flow. Thus, our data indicate that WT HTT is essential for protein trafficking to the centrosome and normal ciliogenesis. In HD, hypermorphic ciliogenesis may affect signaling and neuroblast migration so as to dysregulate brain homeostasis and exacerbate disease progression

The C-Terminal Domain of LRRK2 with the G2019S Substitution Increases Mutant A53T α-Synuclein Toxicity in Dopaminergic Neurons In Vivo

International audienceAlpha-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) play crucial roles in Parkinson’s disease (PD). They may functionally interact to induce the degeneration of dopaminergic (DA) neurons via mechanisms that are not yet fully understood. We previously showed that the C-terminal portion of LRRK2 (ΔLRRK2) with the G2019S mutation (ΔLRRK2G2019S) was sufficient to induce neurodegeneration of DA neurons in vivo, suggesting that mutated LRRK2 induces neurotoxicity through mechanisms that are (i) independent of the N-terminal domains and (ii) “cell-autonomous”. Here, we explored whether ΔLRRK2G2019S could modify α-syn toxicity through these two mechanisms. We used a co-transduction approach in rats with AAV vectors encoding ΔLRRK2G2019S or its “dead” kinase form, ΔLRRK2DK, and human α-syn with the A53T mutation (AAV-α-synA53T). Behavioral and histological evaluations were performed at 6- and 15-weeks post-injection. Results showed that neither form of ΔLRRK2 alone induced the degeneration of neurons at these post-injection time points. By contrast, injection of AAV-α-synA53T alone resulted in motor signs and degeneration of DA neurons. Co-injection of AAV-α-synA53T with AAV-ΔLRRK2G2019S induced DA neuron degeneration that was significantly higher than that induced by AAV-α-synA53T alone or with AAV-ΔLRRK2DK. Thus, mutated α-syn neurotoxicity can be enhanced by the C-terminal domain of LRRK2G2019 alone, through cell-autonomous mechanisms

Entre les murs / Hors les murs

Depuis plus de trois décennies, les musées ont connu de profondes évolutions dans le regard porté sur le public. Celui-ci se décline aujourd’hui au pluriel, est analysé au travers de ses attentes, de ses motivations, de ses traits spécifiques. Les institutions ont pourtant depuis longtemps conscience qu’une part importante de la population ne les fréquente pas et n’envisage pas de les fréquenter. Depuis la dénonciation des « non-publics » en 1968 jusqu’à l’attention récente pour les publics les plus éloignés de la culture institutionnelle (publics éloignés, empêchés, en difficulté, défavorisés, etc.), une dialectique complexe, à propos de ces publics atypiques, se redéfinit sans cesse sur l’art comme écart et la dimension inclusive de l’action culturelle, sur les lieux de l’action culturelle, entre les murs et hors les murs. Ces murs sont ceux de l’institution mais aussi ceux, subis et intériorisés, qui rendent impossible l’exercice d’un droit fondamental : l’accès à la culture. Ce numéro est consacré aux publics dits « empêchés », notamment aux nouvelles cibles de l’action culturelle, dans les prisons, les centres fermés et les hôpitaux. Comment ces publics sont-ils qualifiés, construits par les actions artistiques et culturelles pro­posées par les institutions ? Quels sont les enjeux de ces actions ? Comment la recherche peut-elle s’en saisir pour repenser les logiques de démocratisation et de démocratie culturelles ? For over three decades, museums have undergone a profound transformation in its attitude towards its audience. These days, this term is used in the plural and analyzed according to their expectations, motivations and specific traits. Institutions have nonetheless been long aware that an important section of the population is not amongst its visitors and has no plans to be. From the naming of “non-audiences” in 1968 to the recent attention given to audiences the most far removed from cultural institutions (geographically distant, impeded or hindered in their visit, disabled visitors, etc.), a complex reasoning concerning these atypical audiences is constantly being redefined in terms of art as difference and the inclusive nature of cultural action on institutions, both on site and off site. These are both the sites of institutions, but also, in a figurative sense, those, for reasons imposed or internalized, that prevent audiences from exercising their fundamental right to have access to culture. This issue is devoted to audiences referred to as “hindered”, especially those that are the targets of cultural action, in prisons, closed centers and hospitals. How are these audiences defined and constructed by the cultural and artistic initiatives undertaken by institutions? What issues are being tackled in these initiatives? In what ways can research take hold of these initiatives in order to rethink certain mechanisms of the democratization, and democracy, of culture