Abstract
The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase known to control initiation of translation through two downstream pathways: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)/eukaryotic initiation factor 4E and ribosomal p70 S6 kinase (S6K1). We previously showed in C2C12 murine myoblasts that rapamycin arrests cells in G(1) phase and completely inhibits terminal myogenesis. To elucidate the pathways that regulate myogenesis, we established stable C2C12 cell lines that express rapamycin-resistant mTOR mutants (mTORrr; S2035I) that have N-terminal deletions (Delta10 or Delta91) or are full-length kinase-dead mTORrr proteins. Additional clones expressing a constitutively active S6K1 were also studied. Our results show that Delta10mTORrr signals 4E-BP1 and permits rapamycin-treated myoblasts to differentiate, confirming the mTOR dependence of the inhibition of myogenesis by rapamycin. C2C12 cells expressing either Delta91mTORrr or kinase-dead mTORrr(D2338A) could not phosphorylate 4E-BP1 in the presence of rapamycin and could not abrogate the inhibition of myogenesis. Taken together, our results indicate that both the kinase function of mTOR and the N terminus (residues 11-91, containing part of the first HEAT domain) are essential for myogenic differentiation. In contrast, constitutive activation of S6K1 does not abrogate rapamycin inhibition of either proliferation or myogenic differentiation.
Highlights
Myogenic differentiation entails a cascade of intracellular events that coordinate muscle-specific gene expression, induce withdrawal from the cell cycle, and generate terminally differentiated myotubes [1]
Rapamycin Inhibits Myogenic Differentiation of C2C12 Myoblasts but not ⌬10mTORrr-expressing C2C12 Cells— the inhibition of C2C12 differentiation by rapamycin has been reported previously, relatively high concentrations of rapamycin were used in those studies, and myotube formation was only partially inhibited
The results showed that C2C12 cells differentiated to myotubes when shifted to differentiation medium (DM) for 3 days and that rapamycin (10 ng/ml) completely inhibited myotube formation
Summary
Cell Line and Cultures—Mouse C2C12 myoblasts were purchased from American Type Culture Collection (Manassas, VA) and routinely grown in antibiotic-free Dulbecco’s modified Eagle’s medium with 15% fetal calf serum (growth medium (GM)). Protein expression was evaluated by immunoprecipitation with anti-Myc antibody and by Western blot detection with anti-S6K1. Individual clones were screened for expression of mutant proteins by immunoprecipitation with M2 anti-FLAG or anti-Au1 antibodies and by Western blot with anti-mTOR (26E3) mouse monoclonal antibody. Cells were rinsed thoroughly, incubated with mouse monoclonal anti-myosin heavy chain antibody (Sigma; 15 g/ml in 1% swine serum-PBS) for 2 h, rinsed with PBS, and incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Inc.; 4 g/ml in 1% swine serum-PBS) for 2 h. Nonfluorescent immunohistochemical detection of myosin heavy chain was performed as described previously [31]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
