Signal-induced ubiquitination of p57Kip2 is independent of the C-terminal consensus Cdk phosphorylation site

AbstractThe cyclin-dependent kinase inhibitor p57Kip2 is required for normal mouse embryonic development. p57Kip2 consists of four structurally distinct domains in which the conserved C-terminal nuclear targeting domain contains a putative Cdk phosphorylation site (Thr342) that shares a great similitude in the adjacent sequences with p27Kip1 but not with p21Cip1. Phosphorylation on Thr187 has been shown to promote degradation of p27Kip1. Although there is sequence homology between the C-terminal part of p27Kip1 and p57Kip2, we show that the ubiquitination and degradation of p57Kip2 are independent of Thr342. In contrast a destabilizing element located in the N-terminal is implicated in p57Kip2 destabilization

The Translation Regulatory Subunit eIF3f Controls the Kinase-Dependent mTOR Signaling Required for Muscle Differentiation and Hypertrophy in Mouse

The mTORC1 pathway is required for both the terminal muscle differentiation and hypertrophy by controlling the mammalian translational machinery via phosphorylation of S6K1 and 4E-BP1. mTOR and S6K1 are connected by interacting with the eIF3 initiation complex. The regulatory subunit eIF3f plays a major role in muscle hypertrophy and is a key target that accounts for MAFbx function during atrophy. Here we present evidence that in MAFbx-induced atrophy the degradation of eIF3f suppresses S6K1 activation by mTOR, whereas an eIF3f mutant insensitive to MAFbx polyubiquitination maintained persistent phosphorylation of S6K1 and rpS6. During terminal muscle differentiation a conserved TOS motif in eIF3f connects mTOR/raptor complex, which phosphorylates S6K1 and regulates downstream effectors of mTOR and Cap-dependent translation initiation. Thus eIF3f plays a major role for proper activity of mTORC1 to regulate skeletal muscle size

Inhibition of Atrogin-1/MAFbx Mediated MyoD Proteolysis Prevents Skeletal Muscle Atrophy In Vivo

Ubiquitin ligase Atrogin1/Muscle Atrophy F-box (MAFbx) up-regulation is required for skeletal muscle atrophy but substrates and function during the atrophic process are poorly known. The transcription factor MyoD controls myogenic stem cell function and differentiation, and seems necessary to maintain the differentiated phenotype of adult fast skeletal muscle fibres. We previously showed that MAFbx mediates MyoD proteolysis in vitro. Here we present evidence that MAFbx targets MyoD for degradation in several models of skeletal muscle atrophy. In cultured myotubes undergoing atrophy, MAFbx expression increases, leading to a cytoplasmic-nuclear shuttling of MAFbx and a selective suppression of MyoD. Conversely, transfection of myotubes with sh-RNA-mediated MAFbx gene silencing (shRNAi) inhibited MyoD proteolysis linked to atrophy. Furthermore, overexpression of a mutant MyoDK133R lacking MAFbx-mediated ubiquitination prevents atrophy of mouse primary myotubes and skeletal muscle fibres in vivo. Regarding the complex role of MyoD in adult skeletal muscle plasticity and homeostasis, its rapid suppression by MAFbx seems to be a major event leading to skeletal muscle wasting. Our results point out MyoD as the second MAFbx skeletal muscle target by which powerful therapies could be developed

Rôle pivot du facteur d'initiation eIF3f dans l'antagonisme atrophie/hypertrophie du muscle squelettique

Le contrôle de la masse musculaire est déterminé par un équilibre entre les processus anaboliques et cataboliques. L'hypertrophie musculaire se caractérise par une augmentation de la synthèse protéique par activation de la voie IGF1/Akt/mTORC1. L'atrophie est le résultat d'une augmentation de la dégradation des protéines, dont le système ubiquitine-protéasome joue un rôle majeur. MAFbx est une E3 ubiquitine ligase dont l'expression est fortement augmentée lors de l'induction de l'atrophie. Les substrats ciblés par MAFbx restent à déterminer. Nous avons identifié le facteur d'initiation eIF3f comme cible du complexe SCFMAFbx lors de l'atrophie. MAFbx induit la poly-ubiquitination sur six residus lysines d'eIF3f conduisant ainsi sa dégradation par le protéasome. Nous montrons le rôle clé d'eIF3f dans l'induction de l'hypertrophie, caractérisée par une augmentation de l'activité de la voie mTORC1. De plus, la sur-expression d'un mutant d'eIF3f résistant à l'ubiquitination est associée à une protection effective contre l'atrophie. eIF3f apparaît être une cible clé de la fonction de MAFbx lors de l'atrophie du muscle et joue un rôle majeur dans l'hypertrophieSkeletal muscle (SM) mass depends upon a dynamic balance between anabolic and catabolic processes. SM hypertrophy is characterized by increased protein synthesis mainly by activation of the IGF1/Akt/mTORC1 pathway. SM atrophy is the results of increased protein breakdown, in which the ubiquitin-proteasome pathway plays a major role. MAFbx is an E3 ligase whose expression is upregulated during muscle atrophy. However the precise function of the SCFMAFbx ubiquitin-ligase in muscle wasting remains unknown. Here, we characterized eIF3f as a target of the SCFMAFbx complex during muscle atrophy. MAFbx induces the poly-ubiquitination of eIF3f in six lyisine residues leading to its proteasomal degradation. We demonstrate that eIF3f plays a key role in hypertrophic by increasing the activity of the mTORC1 pathway. In addition, the overexpression of an eIF3f mutant resistant to poly-ubiquitination is associated with protection against muscle atrophy. Taken together, the specific targeting of eIF3f by MAFbx may account for the decreased protein synthesis observed in atrophy, and eIF3f plays an important role as a mediator of SM hypertrophyMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Identification of essential sequences for cellular localization in the muscle-specific ubiquitin E3 ligase MAFbx/Atrogin 1

Contact: [email protected] audienceIn skeletal muscle atrophy, upregulation and nuclear accumulation of the Ubiquitin E3 ligase MAFbx is essential for accelerated muscle protein loss, but the nuclear/cytoplasmic shuttling of MAFbx is undefined. Here we found that MAFbx contains two functional nuclear localization signals (NLS). Mutation or deletion of only one NLS induced cytoplasmic localization of MAFbx. We identified a non-classical NES located in the leucine charged domain (LCD) of MAFbx, which is leptomycin B insensitive. We demonstrated that mutation (L169Q) in LLXXL motif of LCD suppressed cytoplasmic retention of MAFbx. Nucleocytoplasmic shuttling of MAFbx represents a novel mechanism for targeting its substrates and its cytosolic partners in muscle atrophy

MyoD et eIF3f : cibles majeures du complexe ubiquitine-ligase SCFMAFbx au cours de l'atrophie musculaire

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

Contrôle des fonctions du facteur de transcription musculaire myod par modifications post-traductionnelles

MyoD est un facteur de transcription spécifique du muscle squelettique (MRFs) régulateur de l'antagonisme prolifération/différenciation au cours de la myogénèse, et essentiel à la régénération musculaire, et dont les fonctions dépendent de son taux d'expression. Dans cette étude nous montrons que la voie ubiquitine/protéasome contrôle post-traductionnellement la dégradation de MyoD, par deux mécanismes d'adressage indépendants. Le premier requiert la phosphorylation spécifique de MyoD au cours de la phase G1 du cycle et permet le maintient des myoblastes en prolifération. Le second est médié par l'activité ubiquitine ligase de la protéine MAFbx/Atrogine-1, surexprimée au cours de l'atrophie musculaire, dont la surexpression active la dégradation de MyoD et inhibe la différenciation terminale des myoblastes. Enfin, MyoD exerce un contrôle sur la transition G2/M, son inactivation et sa dégradation phosphorylation-dépendante à la transition G2/M conditionnent le passage en mitose.The transcription factor MyoD, member of the myogenic regulators family (MRFs), induces differentiation in precursor cells and satellites cells by regulating proliferation/differenciation antagonism and this function seems to be closely link to the level of MyoD protein. Here we show that ubiquitin-proteasom pathway regulates MyoD turn-over by two independant mechanisms. The first requires specific phosphorylation of MyoD at the end of G1 phase of cell cycle and induced MyoD degradation and progression in S phase. The second is mediated by Atrogin-1/MAFbx, an E3 ubiquitin ligase required for skeletal muscle atrophy. MAFbx mediates MyoD ubiquitination in proliferating and differentiating myoblasts and it's over-expression prevent myoblasts differentiation. We finally show that MyoD regulates G2/M transition. MyoD protein's levels increase in G2, its inactivation and destabilization is mediated by specific phosphorylation and trigger mitotic entry.PARIS5-BU Saints-Pères (751062109) / SudocSudocFranceF

The translational factor eIF3f: the ambivalent eIF3 subunit.

International audienceThe regulation of the protein synthesis has a crucial role in governing the eukaryotic cell growth. Subtle changes of proteins involved in the translation process may alter the rate of the protein synthesis and modify the cell fate by shifting the balance from normal status into a tumoral or apoptotic one. The largest eukaryotic initiation factor involved in translation regulation is eIF3. Amongst the 13 factors constituting eIF3, the f subunit finely regulates this balance in a cell-type-specific manner. Loss of this factor causes malignancy in several cells, and atrophy in normal muscle cells. The intracellular interacting partners which influence its physiological significance in both cancer and muscle cells are detailed in this review. By delineating the global interaction network of this factor and by clarifying its intracellular role, it becomes apparent that the f subunit represents a promising candidate molecule to use for biotherapeutic applications