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In response to cancer, AIDS, sepsis and other systemic diseases inducing muscle atrophy, the E3 ubiquitin ligase Atrogin1/MAFbx (MAFbx) is dramatically upregulated and this response is necessary for rapid atrophy. However, the precise function of MAFbx in muscle wasting has been questioned. Here, we present evidence that during muscle atrophy MAFbx targets the eukaryotic initiation factor 3 subunit 5 (eIF3‐f) for ubiquitination and degradation by the proteasome. Ectopic expression of MAFbx in myotubes induces atrophy and degradation of eIF3‐f. Conversely, blockade of MAFbx expression by small hairpin RNA interference prevents eIF3‐f degradation in myotubes undergoing atrophy. Furthermore, genetic activation of eIF3‐f is sufficient to cause hypertrophy and to block atrophy in myotubes, whereas genetic blockade of eIF3‐f expression induces atrophy in myotubes. Finally, eIF3‐f induces increasing expression of muscle structural proteins and hypertrophy in both myotubes and mouse skeletal muscle. We conclude that eIF3‐f is a key target that accounts for MAFbx function during muscle atrophy and has a major role in skeletal muscle hypertrophy. Thus, eIF3‐f seems to be an attractive therapeutic target.

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.

We show that expression of p57(Kip2), a potent tight-binding inhibitor of several G(1) cyclin-cyclin-dependent kinase (Cdk) complexes, increases markedly during C2C12 myoblast differentiation. We examined the effect of p57(Kip2) on the activity of the transcription factor MyoD. In transient transfection assays, transcriptional transactivation of the mouse muscle creatine kinase promoter by MyoD was enhanced by the Cdk inhibitors. In addition, p57(Kip2), p21(Cip1), and p27(Kip1) but not p16(Ink4a) induced an increased level of MyoD protein, and we show that MyoD, an unstable nuclear protein, was stabilized by p57(Kip2). Forced expression of p57(Kip2) correlated with hypophosphorylation of MyoD in C2C12 myoblasts. A dominant-negative Cdk2 mutant arrested cells at the G(1) phase transition and induced hypophosphorylation of MyoD. Furthermore, phosphorylation of MyoD by purified cyclin E-Cdk2 complexes was inhibited by p57(Kip2). In addition, the NH2 domain of p57(Kip2) necessary for inhibition of cyclin E-Cdk2 activity was sufficient to inhibit MyoD phosphorylation and to stabilize it, leading to its accumulation in proliferative myoblasts. Taken together, our data suggest that repression of cyclin E-Cdk2-mediated phosphorylation of MyoD by p57(Kip2) could play an important role in the accumulation of MyoD at the onset of myoblast differentiation.

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.

Kip2 , which is conserved in the Cip/Kip proteins, is implicated in protein-protein interaction and confers a specific regulatory mechanism, outside of their Cdk-inhibitory activity, by which the p57 Kip2 family members positively act on myogenic differentiation.

We recently presented evidence that the subunit eIF3-f of the eukaryotic initiation translation factor eIF3 that interacts with the E3-ligase Atrogin-1/muscle atrophy F-box (MAFbx) for polyubiquitination and proteasome-mediated degradation is a key target that accounts for MAFbx function during muscle atrophy. To understand this process, deletion analysis was used to identify the region of eIF3-f that is required for its proteolysis. Here, we report that the highly conserved C-terminal domain of eIF3-f is implicated for MAFbx-directed polyubiquitination and proteasomal degradation. Site-directed mutagenesis of eIF3-f revealed that the six lysine residues within this domain are required for full polyubiquitination and degradation by the proteasome. In addition, mutation of these six lysines (mutant K 5-10 R) displayed hypertrophic activity in cellulo and in vivo and was able to protect against starvation-induced muscle atrophy. Taken together, our data demonstrate that the C-terminal modifications, believed to be critical for proper eIF3-f regulation, are essential and contribute to a fine-tuning mechanism that plays an important role for eIF3-f function in skeletal muscle.Skeletal muscle is a dynamic tissue that has the capacity to continuously regulate its size in response to a variety of external cues including mechanical load, neural activity, hormones/ growth factors, stress, and nutritional status. In addition, skeletal muscle serves as the most significant repository for protein in the body, a source that is tapped to provide a pool of amino acids for tissue repair and gluconeogenesis under conditions of starvation and other stresses. The maintenance of muscle mass is controlled by a fine balance between catabolic and anabolic processes, which determine the level of muscle proteins and the diameter of muscle fibers. Muscle loss occurs as the result of a number of disparate conditions including cancer, diabetes, AIDS, sepsis, renal failure, aging, cachexia, and other systemic diseases (1). These diverse conditions result in reduced protein synthesis and increased protein breakdown. The process of atrophy is characterized by the activation of the ATP-dependent ubiquitin-proteasome proteolysis pathway (2). Proteins destined for degradation by the ubiquitin-proteasome pathway are marked by covalent linkage with a chain of ubiquitin molecules (Ub) 3 on lysine residue(s) for further degradation into short peptides by the 26 S proteasome. This process requires an Ub-activating enzyme (E1), an Ub-conjugating enzyme (E2), and an Ub ligase (E3) that acts as the last step of the cascade (3). E3 proteins regulate the timing and the substrate specificity in protein degradation. In multiple models of skeletal muscle atrophy, the muscle-specific F-box protein MAFbx/Atrogin-1 (MAFbx) is up-regulated and appears to be essential for accelerated muscle protein loss (4 -6). MAFbx mRNA increases 8 -40-fold in all types of atrophy studied, and this increase precedes the onset of muscle weight loss. Moreover, knock-out animals l...

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