SUMOylation regulates nucleo-cytoplasmic shuttling of Elk-1

The transcription factor Elk-1 is a nuclear target of mitogen-activated protein kinases and regulates immediate early gene activation by extracellular signals. We show that Elk-1 is also conjugated to SUMO on either lysines 230, 249, or 254. Mutation of all three sites is necessary to fully block SUMOylation in vitro and in vivo. This Elk-1 mutant, Elk-1(3R), shuttles more rapidly to nuclei of Balb/C cells fused to transfected HeLa cells. Coexpression of SUMO-1 or -2 strongly reduces shuttling by Elk-1 without affecting that of Elk-1(3R), indicating that SUMOylation regulates nuclear retention of Elk-1. Accordingly, overexpression of Elk-1(3R) in PC12 cells, where cytoplasmic relocalization of Elk-1 has been linked to differentiation, enhances neurite extension relative to Elk-1. The effect of Elk-1, but not of the 3R mutant, was blocked upon cotransfection with SUMO-1 or -2 and enhanced by coexpression with mutant Ubc-9. Thus, SUMO conjugation is a novel regulator of Elk-1 function through the control of its nuclear-cytoplasmic shuttling

Ubiquitin-independent degradation of proteins by the proteasome.

International audienceThe proteasome is the main proteolytic machinery of the cell and constitutes a recognized drugable target, in particular for treating cancer. It is involved in the elimination of misfolded, altered or aged proteins as well as in the generation of antigenic peptides presented by MHC class I molecules. It is also responsible for the proteolytic maturation of diverse polypeptide precursors and for the spatial and temporal regulation of the degradation of many key cell regulators whose destruction is necessary for progression through essential processes, such as cell division, differentiation and, more generally, adaptation to environmental signals. It is generally believed that proteins must undergo prior modification by polyubiquitin chains to be addressed to, and recognized by, the proteasome. In reality, however, there is accumulating evidence that ubiquitin-independent proteasomal degradation may have been largely underestimated. In particular, a number of proto-oncoproteins and oncosuppressive proteins are privileged ubiquitin-independent proteasomal substrates, the altered degradation of which may have tumorigenic consequences. The identification of ubiquitin-independent mechanisms for proteasomal degradation also poses the paramount question of the multiplicity of catabolic pathways targeting each protein substrate. As this may help design novel therapeutic strategies, the underlying mechanisms are critically reviewed here

Ubiquitin-Independent Proteasomal Degradation of Fra-1 Is Antagonized by Erk1/2 Pathway-Mediated Phosphorylation of a Unique C-Terminal Destabilizer▿

Fra-1, a transcription factor that is phylogenetically and functionally related to the proto-oncoprotein c-Fos, controls many essential cell functions. It is expressed in many cell types, albeit with differing kinetics and abundances. In cells reentering the cell cycle, Fra-1 expression is transiently stimulated albeit later than that of c-Fos and for a longer time. Moreover, Fra-1 overexpression is found in cancer cells displaying high Erk1/2 activity and has been linked to tumorigenesis. One crucial point of regulation of Fra-1 levels is controlled protein degradation, the mechanism of which remains poorly characterized. Here, we have combined genetic, pharmacological, and signaling studies to investigate this process in nontransformed cells and to elucidate how it is altered in cancer cells. We report that the intrinsic instability of Fra-1 depends on a single destabilizer contained within the C-terminal 30 to 40 amino acids. Two serines therein, S252 and S265, are phosphorylated by kinases of the Erk1/2 pathway, which compromises protein destruction upon both normal physiological induction and tumorigenic constitutive activation of this cascade. Our data also indicate that Fra-1, like c-Fos, belongs to a small group of proteins that may, under certain circumstances, undergo ubiquitin-independent degradation by the proteasome. Our work reveals both similitudes and differences between Fra-1 and c-Fos degradation mechanisms. In particular, the presence of a single destabilizer within Fra-1, instead of two that are differentially regulated in c-Fos, explains the much faster turnover of the latter when cells traverse the G0/G1-to-S-phase transition. Finally, our study offers further insights into the signaling-regulated expression of the other Fos family proteins

La dégradation protéasomique : De l’adressage des protéins aux nouvelles perspectives thérapeutiques

Le protéasome est la principale machinerie protéolytique de la cellule. Il est impliqué dans toutes les grandes fonctions et décisions cellulaires. On a longtemps pensé que presque tous ses substrats devaient préalablement être ubiquitinylés. On a aussi longtemps considéré que l’ubiquitinylation et la dégradation étaient deux mécanismes découplés, et que le recrutement des conjugués ubiquitine s’effectuait directement par des sous-unités spécialisées du protéasome. La littérature récente remet en cause cette vue simplifiée. Elle suggère ainsi que la fraction des protéines hydrolysées par le protéasome, indépendamment de toute ubiquitinylation, a largement été sous-estimée, et que la reconnaissance des protéines ubiquitinylées fait intervenir des systèmes d’adressage complexes. Par ailleurs, elle indique un ordre d’organisation supérieur pour la voie ubiquitine/protéasome, une fraction du protéasome et des enzyme d’ubiquitinylation étant engagée dans des complexes supramoléculaires. Enfin, la dégradation protéasomique est altérée dans de nombreuses situations pathologiques. Elle constitue donc une cible thérapeutique dont les premières applications commencent à émerger

The AP-1 transcriptional complex: Local switch or remote command?

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La dégradation protéasomique : De l’adressage des protéines aux nouvelles perspectives thérapeutiques

Le protéasome est la principale machinerie protéolytique de la cellule. Il est impliqué dans toutes les grandes fonctions et décisions cellulaires. On a longtemps pensé que presque tous ses substrats devaient préalablement être ubiquitinylés. On a aussi longtemps considéré que l’ubiquitinylation et la dégradation étaient deux mécanismes découplés, et que le recrutement des conjugués ubiquitine s’effectuait directement par des sous-unités spécialisées du protéasome. La littérature récente remet en cause cette vue simplifiée. Elle suggère ainsi que la fraction des protéines hydrolysées par le protéasome, indépendamment de toute ubiquitinylation, a largement été sous-estimée, et que la reconnaissance des protéines ubiquitinylées fait intervenir des systèmes d’adressage complexes. Par ailleurs, elle indique un ordre d’organisation supérieur pour la voie ubiquitine/protéasome, une fraction du protéasome et des enzyme d’ubiquitinylation étant engagée dans des complexes supramoléculaires. Enfin, la dégradation protéasomique est altérée dans de nombreuses situations pathologiques. Elle constitue donc une cible thérapeutique dont les premières applications commencent à émerger.The proteasome is the main intracellular proteolytic machinery. It is involved in all major cellular functions and decisions. It has long been thought that prior ubiquitinylation of almost all of its substrates was necessary for degradation. It has also long been considered that ubiquitinylation and degradation were two uncoupled mechanisms and that the recruitment of ubiquitinylated species was only performed by specialized subunits of the proteasome. The recent literature questions this simplified view. It also suggests that, on the one hand, the fraction of proteins hydrolyzed by the proteasome independently of their ubiquitinylation has largely been underestimated and, on the other hand, that the recognition of ubiquitinylated proteins involves complex addressing systems. Furthermore, it indicates a higher order structuration of the ubiquitin/proteasome pathway, a fraction of the proteasome and of ubiquitinylation enzymes being engaged in supramolecular complexes. Finally, proteasomal degradation is altered in a number of pathological situations. It, thus, constitutes a therapeutic target and the first applications are emerging

c-Fos Proto-Oncoprotein Is Degraded by the Proteasome Independently of Its Own Ubiquitinylation In Vivo

Prior ubiquitinylation of the unstable c-Fos proto-oncoprotein is thought to be required for recognition and degradation by the proteasome. Contradicting this view, we report that, although c-Fos can form conjugates with ubiquitin in vivo, nonubiquitinylatable c-Fos mutants show regulated degradation identical to that of the wild-type protein in living cells under two classical conditions of study: transient c-fos gene expression during the G(0)/G(1) phase transition upon stimulation by mitogens and constitutive expression during asynchronous growth. Moreover, c-Fos destruction during the G(0)/G(1) phase transition is unusual because it depends on two distinct but cumulative mechanisms. We report here that one mechanism involves a C-terminal destabilizer which does not need an active ubiquitin cycle, whereas the other involves an N-terminal destabilizer dependent on ubiquitinylation of an upstream c-Fos breakdown effector. In addition to providing new insights into the mechanisms of c-Fos protein destruction, an important consequence of our work is that ubiquitinylation-dependent proteasomal degradation claimed for a number of proteins should be reassessed on a new experimental basis

Ubiquitin-independent- versus ubiquitin-dependent proteasomal degradation of the c-Fos and Fra-1 transcription factors: Is there a unique answer?

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