Pollard Hilary J, Willett Mark, Morley Simon J
a Department of Biochemistry, School of Life Sciences ; University of Sussex ; Brighton , UK.
Cell Cycle. 2014;13(16):2517-25. doi: 10.4161/15384101.2014.941747.
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.
在C2C12成肌细胞模型系统中，肌源性分化反映了细胞退出细胞周期后转录和翻译的协同且受控的激活过程。此前我们已经表明，mTORC1信号抑制剂RAD001降低了蛋白质合成速率，延迟了C2C12成肌细胞的分化，降低了p70S6K活性，但不影响4E-BP1的超甲基化。在此，我们使用靶向mTOR激酶的抑制剂和小干扰RNA（siRNA）来消除raptor和rictor的表达，进一步研究了在细胞退出细胞周期的分化早期阶段4E-BP1的修饰情况。正如预期的那样，抑制mTOR激酶活性可阻止p70S6K、4E-BP1磷酸化，并与肌源性分化的抑制相关。令人惊讶的是，raptor的大量缺失并不影响p70S6K或4E-BP1磷酸化，但促进了mTORC2活性的增加（如Akt Ser473磷酸化增加所示）。这些数据表明，下游信号的mTOR激酶依赖性但raptor非依赖性调节对肌源性分化很重要。
