PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ

PA28γ is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics, and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28γ promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However, its exact mechanisms of action are unclear and likely involve additional partners that remain to be identified. Here we report the identification of a cofactor of PA28γ, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28γ. Its recruitment to proteasome-containing complexes depends on PA28γ and its expression increases the association of PA28γ with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28γ-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28γ in Cajal bodies by inhibition of its association with the key Cajal body component coilin. Taken together, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including the 20S proteasome, demonstrating that it is an important regulator of PA28γ in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus

14-3-3 proteins and growth control

International audienceThe 14-3-3 proteins constitute a family that is highly conserved in a wide range of organisms, including higher eukaryotes, invertebrates and plants. Variants of 14-3-3 proteins assembled in homo- and heterodimers were found to interact with diverse cellular proteins. Until recently, the biological role of 14-3-3 members was still poorly understood. However, the results of an increasing number of studies on their structure and function are converging to define 14-3-3 proteins as a novel type of adaptor that modulates interactions between components involved in signal transduction pathway and in cell cycle control

Etude de la régulation de la proteolyse de la phosphatase CDC25B1

Les phosphatases à double spécificité de la famille CDC25 jouent un rôle prépondérant à différents points du cycle cellulaire en activant les complexes CDK/Cyclines. Afin de restreindre l'activation de ces complexes, les CDC25s, au nombre de trois (A, B et C) dans les cellules de mammifères, sont finement régulées au cours du cycle cellulaire tant au niveau de leur activité, de leur localisation que de leur stabilité. La phosphatase CDC25B, en activant initialement le complexe CDK1/Cycline B au niveau des centrosomes, est considérée comme le starter de la mitose. Bien que dégradée par le protéasome, peu d'informations sur la régulation de sa dégradation étaient connues. Le but de ce travail a donc été de caractériser le(s) mécanisme(s) impliqué(s), ainsi que les déterminants moléculaires présents sur CDC25B, régulant sa stabilité au cours du cycle cellulaire. Une étude in cellulo de différents mutants ponctuels de la protéine nous a permis d'identifier le motif DDGFVD comme étant nécessaire à l'interaction de CDC25B avec la protéine F-Box TrCP. La perte de cette interaction entraîne une stabilisation de CDC25B à la transition métaphase-anaphase. Cette stabilisation anormale de CDC25B engendre un retard de sortie de mitose accompagné de défauts cellulaires caractéristiques d'une instabilité génétique accrue et d'une fragmentation du matériel péricentriolaire. Par ailleurs, par vidéomicroscopie nous avons observé que les cellules exprimant le mutant stabilisé de CDC25B présentent des caractéristiques morphologiques différentes des cellules exprimant la protéine sauvage, avec une vitesse de déplacement plus importante. Sachant que la surexpression de CDC25B est détectée dans de nombreux cancers généralement très agressifs, et que, comme nous l'avons montrée, une stabilisation de CDC25B en mitose induit une instabilité génétique, nous pouvons supposer que dans certains cancers cette surexpression pourrait résulter d'un défaut de dégradation de la phosphatase. Aussi, une meilleure compréhension des mécanismes de dégradation de CDC25B permettrait d'envisager le développement d'approches thérapeutiques ayant pour but d'agir sur sa stabilitéCDC25 proteins are highly conserved dual specificity phosphatases that play an essential role by activating the CDK/Cyclin complexes all along the cell cycle. To restrain CDK/Cyclin activities, these phosphatases must be tightly regulated in terms of activity, localization and stability. One of the three mammalian members, CDC25B, is considered as the starter of the mitosis through the activation of CDK1/Cyclin B complexes at the centrosomes, at the G2-M transition. This protein is known to be degraded by the proteasome but the exact mechanisms involved in this process are still unclear. To obtain a deeper insight into the regulation of CDC25B stability, we have investigated the molecular determinants and the exact mechanisms involved in CDC25B degradation in vitro as well as in cellulo. Analysis of various mutants of CDC25B led us to identify the DDGFVD motif as a motif required for the interaction of CDC25B with the F-box protein TrCP. The lack of interaction causes a stabilisation of the phosphatase in metaphase-anaphase transition. This stabilisation entails a delay in mitotic exit and several cellular defects related to genetic instability, and the fragmentation of pericentriolar matrix during mitosis. Videomicroscopy's observations indicate that cells expressing the stabilized mutant of the CDC25B exhibit an increased mobility compared to cells expressing wild type protein. Since CDC25B is frequently overexpressed in many cancers cells and that a stabilisation of the protein entails genetic instability, we propose that in some cancers this overexpression could be a consequence of a lack of CDC25B degradation. A better understanding of mechanisms regulating CDC25B degradation could lead to new therapeutical approaches focused on the control of CDC25B stabilityMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

La Phosphatase CDC25B (bases moléculaires de sa localisation intra-cellulaire et recherche de nouveaux partenaires)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

L’étiquette de la mort

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Prix Nobel de Chimie 2004 : Aaron Ciechanover, Avram Hershko et Irwin Rose. L’étiquette de la mort

Aaron Ciechanover, 57 ans, Israélien, né le 1er octobre 1947 à Haïfa, Israël, obtient en 1974 son Doctorat en médecine à l’Hebrew University-Hadassah Medical School de Jérusalem puis, en 1981, son PhD à l’Institut de technologie d’Israël (Technion) de Haïfa. Il y est professeur de biochimie et dirige l’Institut Rappaport de recherche médicale. Membre du Conseil de l’EMBO (European molecular biology organization), de l’Académie européenne des arts et des sciences, il a reçu en 2000 le Prix Albert-Lasker (conjointement avec les professeurs Avram Hershko et Alexander Varshavsky).Avram Hershko, 67 ans, Israëlien, né le 31 décembre 1937 à Karcag, Hongrie, émigre en Israël en 1950 et obtient en 1965 son Doctorat en médecine puis, en 1969, son PhD à l’Hebrew University-Hadassah Medical School de Jérusalem. Professeur de biochimie depuis 1972 à l’Institut de technologie d’Israël (Technion) de Haïfa, il y est depuis 1998 Professeur émérite. Membre de l’EMBO (European molecular biology organization) et de l’Académie des sciences d’Israël, Foreign associate de la National Academy of Sciences américaine, il a reçu en 2000 le Prix Albert-Lasker (conjointement avec les professeurs Aaron Ciechanover et Alexander Varshavsky).Irwin Rose, Américain, né le 16 juillet 1926 à New York, obtient en 1952 son Doctorat en médecine et son PhD à l’Université de Chicago. Professeur de biochimie au Fox Chase Cancer Center de Philadelphia où il effectue toute sa carrière, il est encore, à 78 ans, rattaché, au titre de Professeur émérite, au Département de physiologie et de biophysique du College of Medicine de l’Université de Californie, à Irvine

Subcellular localisation of human wee1 kinase is regulated during the cell cycle.

International audienceWee1 kinase-dependent phosphorylation of cdc2 maintains the cdc2/cyclin B complex in an inert form until it is activated by the cdc25 tyrosine phosphatase at the end of G2. As described for cdc25, cell cycle-linked changes in the intracellular localisation of wee1 may constitute an important aspect of the temporal regulation of cdc2 activity. Here we report that the subcellular distribution of human wee1 changes during the cell cycle in HeLa and IMR90 cells. During interphase, wee1 is found almost exclusively in the nucleus. When the cell enters mitosis, wee1 is relocalised into the cytoplasm. During cell division, wee1 becomes restricted to the mitotic equator and by the end of mitosis it is found exclusively in association with midbody bridges, a phenomenon that is dependent on microtubule assembly. The relocalisations of wee1 and its association with subcellular structures may play key regulatory roles at different stages of the cell cycle and during mitosis

βTrCP-dependent degradation of CDC25B phosphatase at the metaphase-anaphase transition is a pre-requisite for correct mitotic exit

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Proteasome-dependent degradation of human CDC25B phosphatase.

International audienceThe CDC25 dual specificity phosphatase is a universal cell cycle regulator. The evolutionary conservation of this enzyme from yeast to man bears witness to its major role in the control of cyclin-dependent kinases (CDK) activity that are central regulators of the cell cycle machinery. CDC25 phosphatase both dephosphorylates and activates CDKs. Three human CDC25s have been identified. CDC25A is involved in the control of G1/S, and CDC25C at G2/M throught the activation of CDK 1-cyclin B. The exact function of CDC25B however remains elusive. We have found that CDC25B is degraded by the proteasome pathway in vitro and in vivo. This degradation is dependent upon phosphorylation by the CDK1-cyclin A complex, but not by CDK1-cyclin B. Together with the observations of others made in yeast and mammals, our results suggest that CDC25B might act as a 'mitotic starter' triggering the activation of an auto-amplification loop before being degraded