Dual roles of mTORC1-dependent activation of the ubiquitin-proteasome system in muscle proteostasis.

Muscle size is controlled by the PI3K-PKB/Akt-mTORC1-FoxO pathway, which integrates signals from growth factors, energy and amino acids to activate protein synthesis and inhibit protein breakdown. While mTORC1 activity is necessary for PKB/Akt-induced muscle hypertrophy, its constant activation alone induces muscle atrophy. Here we show that this paradox is based on mTORC1 activity promoting protein breakdown through the ubiquitin-proteasome system (UPS) by simultaneously inducing ubiquitin E3 ligase expression via feedback inhibition of PKB/Akt and proteasome biogenesis via Nuclear Factor Erythroid 2-Like 1 (Nrf1). Muscle growth was restored by reactivation of PKB/Akt, but not by Nrf1 knockdown, implicating ubiquitination as the limiting step. However, both PKB/Akt activation and proteasome depletion by Nrf1 knockdown led to an immediate disruption of proteome integrity with rapid accumulation of damaged material. These data highlight the physiological importance of mTORC1-mediated PKB/Akt inhibition and point to juxtaposed roles of the UPS in atrophy and proteome integrity.

mTORC1 signalling is not essential for the maintenance of muscle mass and function in adult sedentary mice.

BACKGROUND: The balance between protein synthesis and degradation (proteostasis) is a determining factor for muscle size and function. Signalling via the mammalian target of rapamycin complex 1 (mTORC1) regulates proteostasis in skeletal muscle by affecting protein synthesis and autophagosomal protein degradation. Indeed, genetic inactivation of mTORC1 in developing and growing muscle causes atrophy resulting in a lethal myopathy. However, systemic dampening of mTORC1 signalling by its allosteric inhibitor rapamycin is beneficial at the organismal level and increases lifespan. Whether the beneficial effect of rapamycin comes at the expense of muscle mass and function is yet to be established. METHODS: We conditionally ablated the gene coding for the mTORC1-essential component raptor in muscle fibres of adult mice [inducible raptor muscle-specific knockout (iRAmKO)]. We performed detailed phenotypic and biochemical analyses of iRAmKO mice and compared them with muscle-specific raptor knockout (RAmKO) mice, which lack raptor in developing muscle fibres. We also used polysome profiling and proteomics to assess protein translation and associated signalling in skeletal muscle of iRAmKO mice. RESULTS: Analysis at different time points reveal that, as in RAmKO mice, the proportion of oxidative fibres decreases, but slow-type fibres increase in iRAmKO mice. Nevertheless, no significant decrease in body and muscle mass or muscle fibre area was detected up to 5 months post-raptor depletion. Similarly, ex vivo muscle force was not significantly reduced in iRAmKO mice. Despite stable muscle size and function, inducible raptor depletion significantly reduced the expression of key components of the translation machinery and overall translation rates. CONCLUSIONS: Raptor depletion and hence complete inhibition of mTORC1 signalling in fully grown muscle leads to metabolic and morphological changes without inducing muscle atrophy even after 5 months. Together, our data indicate that maintenance of muscle size does not require mTORC1 signalling, suggesting that rapamycin treatment is unlikely to negatively affect muscle mass and function.

Activation of mTORC1 in skeletal muscle regulates whole-body metabolism through FGF21.

Skeletal muscle is the largest organ, comprising 40% of the total body lean mass, and affects whole-body metabolism in multiple ways. We investigated the signaling pathways involved in this process using TSCmKO mice, which have a skeletal muscle-specific depletion of TSC1 (tuberous sclerosis complex 1). This deficiency results in the constitutive activation of mammalian target of rapamycin complex 1 (mTORC1), which enhances cell growth by promoting protein synthesis. TSCmKO mice were lean, with increased insulin sensitivity, as well as changes in white and brown adipose tissue and liver indicative of increased fatty acid oxidation. These differences were due to increased plasma concentrations of fibroblast growth factor 21 (FGF21), a hormone that stimulates glucose uptake and fatty acid oxidation. The skeletal muscle of TSCmKO mice released FGF21 because of mTORC1-triggered endoplasmic reticulum (ER) stress and activation of a pathway involving PERK (protein kinase RNA-like ER kinase), eIF2α (eukaryotic translation initiation factor 2α), and ATF4 (activating transcription factor 4). Treatment of TSCmKO mice with a chemical chaperone that alleviates ER stress reduced FGF21 production in muscle and increased body weight. Moreover, injection of function-blocking antibodies directed against FGF21 largely normalized the metabolic phenotype of the mice. Thus, sustained activation of mTORC1 signaling in skeletal muscle regulated whole-body metabolism through the induction of FGF21, which, over the long term, caused severe lipodystrophy.

Sustained activation of mTORC1 in skeletal muscle inhibits constitutive and starvation-induced autophagy and causes a severe, late-onset myopathy.

Autophagy is a catabolic process that ensures homeostatic cell clearance and is deregulated in a growing number of myopathological conditions. Although FoxO3 was shown to promote the expression of autophagy-related genes in skeletal muscle, the mechanisms triggering autophagy are unclear. We show that TSC1-deficient mice (TSCmKO), characterized by sustained activation of mTORC1, develop a late-onset myopathy related to impaired autophagy. In young TSCmKO mice, constitutive and starvation-induced autophagy is blocked at the induction steps via mTORC1-mediated inhibition of Ulk1, despite FoxO3 activation. Rapamycin is sufficient to restore autophagy in TSCmKO mice and improves the muscle phenotype of old mutant mice. Inversely, abrogation of mTORC1 signaling by depletion of raptor induces autophagy regardless of FoxO inhibition. Thus, mTORC1 is the dominant regulator of autophagy induction in skeletal muscle and ensures a tight coordination of metabolic pathways. These findings may open interesting avenues for therapeutic strategies directed toward autophagy-related muscle diseases.

Carboxy-Terminal Modulator Protein (CTMP) is a mitochondrial protein that sensitizes cells to apoptosis.

The Carboxy-Terminal Modulator Protein (CTMP) protein was identified as a PKB inhibitor that binds to its hydrophobic motif. Here, we report mitochondrial localization of endogenous and exogenous CTMP. CTMP exhibits a dual sub-mitochondrial localization as a membrane-bound pool and a free pool of mature CTMP in the inter-membrane space. CTMP is released from the mitochondria into the cytosol early upon apoptosis. CTMP overexpression is associated with an increase in mitochondrial membrane depolarization and caspase-3 and polyADP-ribose polymerase (PARP) cleavage. In contrast, CTMP knock-down results in a marked reduction in the loss of mitochondrial membrane potential as well as a decrease in caspase-3 and PARP activation. Mutant CTMP retained in the mitochondria loses its capacity to sensitize cells to apoptosis. Thus, proper maturation of CTMP is essential for its pro-apoptotic function. Finally, we demonstrate that CTMP delays PKB phosphorylation following cell death induction, suggesting that CTMP regulates apoptosis via inhibition of PKB.

eIF3-f function in skeletal muscles: to stand at the crossroads of atrophy and hypertrophy.

The control of muscle cell size is a physiological process balanced by a fine tuning between protein synthesis and protein degradation. MAFbx/Atrogin-1 is a muscle specific E3 ubiquitin ligase upregulated during disuse, immobilization and fasting or systemic diseases such as diabetes, cancer, AIDS and renal failure. This response is necessary to induce a rapid and functional atrophy. To date, the targets of MAFbx/Atrogin-1 in skeletal muscle remain to be identified. We have recently presented evidence that eIF3-f, a regulatory subunit of the eukaryotic translation factor eIF3 is a key target that accounts for MAFbx/Atrogin-1 function in muscle atrophy. More importantly, we showed that eIF3-f acts as a "translational enhancer" that increases the efficiency of the structural muscle proteins synthesis leading to both in vitro and in vivo muscle hypertrophy. We propose that eIF3-f subunit, a mTOR/S6K1 scaffolding protein in the IGF-1/Akt/mTOR dependent control of protein translation, is a positive actor essential to the translation of specific mRNAs probably implicated in muscle hypertrophy. The central role of eIF3-f in both the atrophic and hypertrophic pathways will be discussed in the light of its promising potential in muscle wasting therapy.

The initiation factor eIF3-f is a major target for atrogin1/MAFbx function in skeletal muscle atrophy.

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.

PKB and the mitochondria: AKTing on apoptosis.

Cellular homeostasis depends upon the strict regulation of responses to external stimuli, such as signalling cascades triggered by nutrients and growth factors, and upon cellular metabolism. One of the major molecules coordinating complex signalling pathways is protein kinase B (PKB), a serine/threonine kinase also known as Akt. The number of substrates known to be phosphorylated by PKB and its interacting partners, as well as our broad understanding of how PKB is implicated in responses to growth factors, metabolic pathways, proliferation, and cell death via apoptosis is constantly increasing. Activated by the insulin/growth factor-phosphatidylinositol 3-kinase (PI3K) cascade, PKB triggers events that promote cell survival and prevent apoptosis. It is also now widely accepted that mitochondria are not just suppliers of ATP, but that they participate in regulatory and signalling events, responding to multiple physiological inputs and genetic stresses, and regulate both cell proliferation and death. Thus, mitochondria are recognized as important players in apoptotic events and it is logical to predict some form of interplay with PKB. In this review, we will summarize mechanisms by which PKB mediates its anti-apoptotic activities in cells and survey recent developments in understanding mitochondrial dynamics and their role during apoptosis.

Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the HIV-1 envelope.

Syncytia arising from the fusion of cells expressing the HIV-1-encoded Env gene with cells expressing the CD4/CXCR4 complex undergo apoptosis following the nuclear translocation of mammalian target of rapamycin (mTOR), mTOR-mediated phosphorylation of p53 on Ser15 (p53(S15)), p53-dependent upregulation of Bax and activation of the mitochondrial death pathway. p53(S15) phosphorylation is only detected in syncytia in which nuclear fusion (karyogamy) has occurred. Karyogamy is secondary to a transient upregulation of cyclin B and a mitotic prophase-like dismantling of the nuclear envelope. Inhibition of cyclin-dependent kinase-1 (Cdk1) prevents karyogamy, mTOR activation, p53(S15) phosphorylation and apoptosis. Neutralization of p53 fails to prevent karyogamy, yet suppresses apoptosis. Peripheral blood mononuclear cells from HIV-1-infected patients exhibit an increase in cyclin B and mTOR expression, correlating with p53(S15) phosphorylation and viral load. Cdk1 inhibition prevents the death of syncytia elicited by HIV-1 infection of primary CD4 lymphoblasts. Thus, HIV-1 elicits a pro-apoptotic signal transduction pathway relying on the sequential action of cyclin B-Cdk1, mTOR and p53.

Dephosphorylation and subcellular compartment change of the mitotic Bloom's syndrome DNA helicase in response to ionizing radiation.

Bloom's syndrome is a rare human autosomal recessive disorder that combines a marked genetic instability and an increased risk of developing all types of cancers and which results from mutations in both copies of the BLM gene encoding a RecQ 3'-5' DNA helicase. We recently showed that BLM is phosphorylated and excluded from the nuclear matrix during mitosis. We now show that the phosphorylated mitotic BLM protein is associated with a 3'-5' DNA helicase activity and interacts with topoisomerase III alpha. We demonstrate that in mitosis-arrested cells, ionizing radiation and roscovitine treatment both result in the reversion of BLM phosphorylation, suggesting that BLM could be dephosphorylated through the inhibition of cdc2 kinase. This was supported further by our data showing that cdc2 kinase activity is inhibited in gamma-irradiated mitotic cells. Finally we show that after ionizing radiation, BLM is not involved in the establishment of the mitotic DNA damage checkpoint but is subjected to a subcellular compartment change. These findings lead us to propose that BLM may be phosphorylated during mitosis, probably through the cdc2 pathway, to form a pool of rapidly available active protein. Inhibition of cdc2 kinase after ionizing radiation would lead to BLM dephosphorylation and possibly to BLM recruitment to some specific sites for repair.