MAFbx/Atrogin-1 Controls the Activity of the Initiation Factor eIF3-f in Skeletal Muscle Atrophy by Targeting Multiple C-terminal Lysines

Abstract

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...

Regulators of protein metabolism are affected by cyclical nutritional treatments with diets varying in protein and energy content

“…Interestingly, we observed concomitant changes in atrogin-1 expression and in phosphorylation levels of mTOR targets involved in mRNA translation into proteins (e.g., lower phosphorylation of S6K1, S6 and 4EBP1 in P− vs. P+). These findings may corroborate recent evidence indicating that atrogin-1 is related to muscle protein synthesis and growth through the regulation of downstream effectors of mTOR, and that the activity or inactivity of a common set of molecules controlling specific cellular pathways determines whether the skeletal muscle tissue will respond to defined stimuli with muscle growth or loss [13,43,44]. eIF3f has been characterized as a substrate of atrogin-1 [45].…”

Exercise and the Regulation of Skeletal Muscle Hypertrophy

“…Support for this contention comes from data showing that elevations in the early stage of disuse could be targeting the degradation of translational proteins such as eukaryotic initiation factor 3F. 99,100 The reduction in the content of these proteins subsequently serves to limit the capacity of the translational machinery to mount a sufficient response to anabolic stimulation necessary to sustain positive protein balance. Although to our knowledge, no study has identified if a period of muscle disuse in humans results in the targeted degradation of these proteins and, as such, this hypothesis awaits experimental confirmation.…”

Weighing up the possibilities: Controlling translation by ubiquitylation and sumoylation

“…75 This E3 is upregulated and essential for accelerated muscle protein loss in a number of disorders. 76 Ubiquitylation of eIF3f occurs on multiple (6) lysines in the Cterminus 74 and results in its ubiquitin-mediated proteolysis in myotubes undergoing atrophy. Under these conditions both MAFbnx and eIF3f are detected in the nucleus.…”

“…Its role as a translational enhancer came to light in a study on muscle atrophy. 74 Here, eIF3f is ubiquitylated by the MAFbnx/ Atrogin1 protein which is a muscle-specific F-box protein ubiquitin E3 ligase. 75 This E3 is upregulated and essential for accelerated muscle protein loss in a number of disorders.…”