Myogenic differentiation is dependent on both the kinase function and the N-terminal sequence of mammalian target of rapamycin.
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.
Shu
Lili
L
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-2794, USA.
Zhang
Xiongwen
X
Houghton
Peter J
PJ
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D016193:G1 Phase; D017353:Gene Deletion; D051379:Mice; D008856:Microscopy, Fluorescence; D010448:Peptide Initiation Factors / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases; D013997:Time Factors
D048868:Adaptor Proteins, Signal Transducing; D000903:Antibiotics, Antineoplastic; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C499527:Eif4ebp1 protein, mouse; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D011494:Protein Kinases; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus