Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1

Park, In Hyun; Erbay, Ebru; Nuzzi, Paul D.; Chen, Jiawen

doi:10.1016/j.yexcr.2005.05.017

Cited by 68 publications

(57 citation statements)

References 42 publications

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“…In both cases, results are shown from a single experiment but similar data were obtained in at least 3 independent experiments. results were seen with extracts from cells allowed to differentiate in the presence of RAD001 (lanes [8][9][10][11][12][13]. Figure 2A shows that lambda phosphatase was able to dephosphorylate 4E-BP1 in extracts prepared at 48 hours ( Fig.…”

Section: Rad001 Inhibits P70s6k But Maintains the Phosphorylation Of mentioning

confidence: 77%

“…1,2 Previous work has shown a role for mTOR in this response [9][10][11][12]26 but the role of 4E-BP1 phosphorylation remains elusive. Current models suggest that hyperphosphorylated 4E-BP1 is released from eIF4E to allow for cap-dependent translation.…”

Section: Discussionmentioning

confidence: 99%

“…[5][6][7] Activation of mTORC1 and phosphorylation of its downstream targets are central to the control of protein synthesis in many cell types, but the role of mTOR activity and its downstream signaling pathways in regulating differentiation in C2C12 myoblasts remains unclear. [8][9][10][11][12][13] mTOR is contained within 2 distinct complexes: mTORC1 and mTORC2. [13][14][15] The former complex includes mTOR, raptor, mLST8, proline-rich Akt substrate 40 kDa (PRAS40), DEPdomain-containing partner of mTOR (DEPTOR), and scaffolds tti1/tel2 7,13,16 mTORC1 regulates the phosphorylation and activity of eIF4E-binding proteins (4E-BP1 5,7,14 ) and the ribosomal protein S6 kinase, p70S6K.…”

Section: Introductionmentioning

confidence: 99%

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mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation

2014

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Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Previously we have shown that the mTORC1 signaling inhibitor, RAD001, decreased protein synthesis rates, delayed C2C12 myoblast differentiation, decreased p70S6K activity but did not affect the hypermodification of 4E-BP1. Here we have further investigated the modification of 4E-BP1 during the early phase of differentiation as cells exit the cell cycle, using inhibitors to target mTOR kinase and siRNAs to ablate the expression of raptor and rictor. As predicted, inhibition of mTOR kinase activity prevented p70S6K, 4E-BP1 phosphorylation and was associated with an inhibition of myogenic differentiation. Surprisingly, extensive depletion of raptor did not affect p70S6K or 4E-BP1 phosphorylation, but promoted an increase in mTORC2 activity (as evidenced by increased Akt Ser473 phosphorylation). These data suggest that an mTOR kinase-dependent, but raptorindependent regulation of downstream signaling is important for myogenic differentiation.

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Section: Rad001 Inhibits P70s6k But Maintains the Phosphorylation Of mentioning

confidence: 77%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation

2014

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“…Serum was added to a final concentration of 20% for 30 min at 37°C. Cells were lysed as described previously (22), and cell lysates were separated by SDS-PAGE. Resolved proteins were transferred to a polyvinylidene difluoride membrane and immunoblotted with a phosphospecific primary antibody against Thr-389 of p70 S6 kinase (catalog number 9205, Cell Signaling Technology, Inc., Danvers, MA).…”

Section: Yeast Strains-yse2mentioning

confidence: 99%

The Rapamycin-binding Domain of the Protein Kinase Mammalian Target of Rapamycin Is a Destabilizing Domain

Edwards

Wandless

2007

Journal of Biological Chemistry

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Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506-and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to 10-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retained the ability to inhibit mTOR, although with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wild-type FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems.A common approach for studying complex biological processes is to perturb a protein of interest and observe subsequent changes to the biological system. The classical approach of disrupting a gene and studying the resultant phenotype is problematic for examining essential genes. As a result, several conditional techniques have been developed including the tetracycline/doxycycline and Cre recombinase/lox P systems, where control is exerted at the transcriptional level (1, 2). At the post-transcriptional level, RNA interference (RNAi) 2 is becoming rapidly used as a method of gene silencing (3), but RNAi lacks tunability and does not affect existing protein levels. An ideal perturbation method would directly affect the protein with a cell-permeable probe that rapidly and reversibly targets a specific protein of interest.Small molecules have been very useful as conditional perturbants; however, the lack of specificity of many of these reagents can make it difficult to unambiguously interpret results. One approach to ensure specificity is to chemically modify a small molecule with a large substituent, and this technique has been widely used to interrogate specific protein tyrosine kinases that possess a complementary cavity-forming mutation (4, 5). An alternative system has recently been developed using a cellperm...

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“…The p42 MAPK has been implicated in the induction of hypertrophy by IGF-1 in rodents although the results are somewhat contradictory (Haddad and Adams, 2003;Rommel et al, 1999;Wu et al, 2000). The role of Akt in muscle hypertrophy is more commonly accepted and Akt has been shown to increase protein synthesis in both in vitro and in vivo studies in mice by activating mTOR (mammalian target of rapamycin)-p70S6K-S6 pathway and by inhibiting GSK-3 (glycogen synthase kinase-3) involved in the regulation of protein translation (Bodine et al, 2001b;Park et al, 2005;Rommel et al, 2001). More recently, IGF-1 has been reported to decrease protein degradation via the inhibition of the transcription factor Foxo, which controls the expression of the muscle-specific ubiquitin ligase F-box protein 32 (Fbxo32, also known as atrogin-1), involved in protein degradation during muscular atrophy (Gomes et al, 2001;Sandri et al, 2004;Stitt et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

IL-13 mediates the recruitment of reserve cells for fusion during IGF-1-induced hypertrophy of human myotubes

Jacquemin

Butler-Browne

Furling

et al. 2007

Journal of Cell Science

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Insulin-like growth factor-1 (IGF-1) has been shown to induce skeletal muscle hypertrophy, to prevent the loss of muscle mass with ageing and to improve the muscle phenotype of dystrophic mice. We previously developed a model of IGF-1-induced hypertrophy of human myotubes, in which hypertrophy was not only characterized by an increase in myotube size and myosin content but also by an increased recruitment of reserve cells for fusion. Here, we describe a new mechanism of IGF-1-induced hypertrophy by demonstrating that IGF-1 signals exclusively to myotubes but not to reserve cells, leading, under the control of the transcription factor NFATc2, to the secretion of IL-13 that will secondly recruit reserve cells for differentiation and fusion. In addition, we show that IGF-1 also signals to myotubes to stimulate protein metabolism via Akt by (1) activating the mTOR-p70S6K-S6 pathway and inhibiting GSK-3␤, both involved in the control of protein translation, and (2) inhibiting the Foxo1-atrogin-1 protein degradation pathway. Journal of Cell Science 671 IL-13-mediated reserve cell recruitment (Abbott et al., 1998;Horsley et al., 2001), the data in the literature concerning the role of calcineurin in skeletal muscle hypertrophy are again often contradictory since some studies in rodent models show that IGF-1-induced hypertrophy can be suppressed using the calcineurin inhibitors cyclosporine A or FK506 Semsarian et al., 1999b), whereas other groups see no effect of these inhibitors on hypertrophy and no increase in calcineurin activity in the presence of IGF-1 (Bodine et al., 2001b;Rommel et al., 2001).The ability of IGF-1 to act as an anabolic factor on skeletal muscle and to counterbalance the signalling pathways of muscle atrophy has led to the proposition that IGF-1 could be used as a therapeutic agent to combat muscle atrophy related to age (sarcopenia) or to various diseases. However all data available until now describing the mechanisms of IGF-1-induced hypertrophy have been obtained in rodent models, and very little is known about the effects and the signalling pathways of IGF-1 in human skeletal muscle. It is becoming increasingly evident that the results obtained in rodent models cannot always be directly transposed to man. For example, whereas a twofold increase was observed in the lifespan of myoblasts from transgenic mice overexpressing IGF-1 in muscle (Chakravarthy et al., 2000), we recently showed in human myoblasts that IGF-1 has no effect on the proliferative lifespan, suggesting a different mechanism of regulation in these two species (Jacquemin et al., 2004).We previously developed an in vitro model of human myotube hypertrophy induced by IGF-1 where cultures were exposed to IGF-1 only 3 days after the induction of differentiation, a time when most of the myoblasts have already fused into myotubes and no more proliferation is observed. This model allows us to distinguish between the different effects of IGF-1 on proliferation, differentiation and hypertrophy (Jacquemin et al., 2004). In these condition...

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Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1

Cited by 68 publications

References 42 publications

mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation

mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation

The Rapamycin-binding Domain of the Protein Kinase Mammalian Target of Rapamycin Is a Destabilizing Domain

IL-13 mediates the recruitment of reserve cells for fusion during IGF-1-induced hypertrophy of human myotubes

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