Abstract
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 raptor-independent regulation of downstream signaling is important for myogenic differentiation.
Highlights
We have shown that myogenic differentiation is remained in a hyperphosphorylated form.[26]. To examine this furassociated with increased rates of translation, biphasic activation ther we have explored the phosphorylation of 4E-BP1 in more of p70S6K, and the phosphorylation of both eIF4E and 4E- detail and compared the effects of RAD001 with kinase inhibitors BP1.26 Paradoxically, treatment of C2C12 myoblasts with an of mTOR, such as Torin 113 and KU0063794,31 and downstream inhibitor of mTORC1 (RAD00127), delayed differentiation but signaling following ablation of rictor or raptor using siRNA
The differentiation of skeletal muscle cells involves their exit from the cells cycle, changes in activity of an array of signaling pathways, altered gene expression, and cell fusion to form multinucleated myotubes.[1,2]
Current models suggest that hyperphosphorylated 4E-BP1 is released from eIF4E to allow for cap-dependent translation.[5,14]. This would promote the regulated recruitment of specific mRNAs into active eIF4F complexes, disassembly of stored mRNA from granules and the release of small, inhibitory RNAs which prevent translation of the associated mRNAs.[5,17,22,23,24,25]
Summary
The differentiation of skeletal muscle cells involves the exit of mononucleated myoblasts from the cell cycle, changes in activity of an array of signaling pathways, altered gene expression, and cell fusion to form multinucleated myotubes.[1,2] It is likely that this process reflects the induction of IGF-II3,4 which signals via the Insulin-like growth factor 1 (IGF-1) receptor to activate phosphatidylinositol-3 kinase (PI3-K), protein kinase B (Akt), the Tuberous Sclerosis Complex (TSC1/2), and mammalian target of rapamycin (mTORC1) signaling.[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]. We show that as cells exit the cell cycle, depletion of raptor and rpS6 (lane 3) To see if this was true for other mTORC does not affect p70S6K or 4E-BP1 phosphorylation, but pro- inhibitors, we used KU0063794 which inhibits the kinase activmoted an increase in mTORC2 activity (as evidenced by ity of mTOR.[31] Figure 1A shows that KU0063794 inhibits the increased Akt Ser[473] phosphorylation). Ablation of rictor (lane 10 vs lane 6) led to increased p70S6K activity possibly reflecting increased expression of raptor To investigate this further, cells were incubated with a scrambled siRNA or depleted of either raptor, rictor or both proteins for 24 hours and cells subsequently allowed to differentiate for 48 hours in the absence or presence of KU0063794 (Fig. 5). Depletion of rictor using siRNA (lane 5 vs lanes 2 and 12) had no effect on p70S6K activity, S6
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