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mTORCing about myogenic differentiation

The C2C12 myoblast cell line has been extensively studied as it has the potential to differentiate from serum-starved confluent monolayer cultures into myotubes, (Fig. 1, upper panel) which can further align and form muscle fibers. Furthermore, C2C12 myoblasts can differentiate into adipose and bone-type lineages, depending on the conditions present. As such the system has to undergo huge changes in both transcription and translation to reflect the different proteomes required for each lineage. Therefore, there must be a great degree of control exerted to ensure the expression of genes involved in the commitment to and progression through myogenesis, to ensure that their temporal and spatial expression is properly regulated. 
 
 
 
Figure 1. 
 
C2C12 myoblasts grown to confluency and starved undergo myogenic differentiation. Allosteric inhibitors of mTORC1 inhibit this process. Availability of eIF4E for mRNA cap-binding in translation is determined by interaction with eIF4E binding proteins ... 
 
 
 
In their previous work, the Morley group confirmed that the mTORC1 pathway is activated during differentiation in C2C12 cells and that a delay in myogenic differentiation can be caused by allosteric inhibitors of mTORC1 activity, namely rapamycin and RAD001 (everolimus), a rapamycin derivative.1 This rapamycin-sensitive inhibition of myogenesis has been extensively reviewed elsewhere.2 
 
The mTOR kinase is present in the cell within 2 complexes, the rapamycin sensitive mTORC1 which contains Raptor (Fig. 1, lower panel), and the rapamycin insensitive mTORC2 complex which contains Rictor (for a recent review of this vast subject see3). Among many targets, mTORC1 is a master regulator of cap-dependent translation and controls the phosphorylation and the activation of the targets 4E-BP1 (Fig. 1, lower panel) and S6K1. These components are involved in the control of protein synthesis by modulating the availability of the cap-binding protein eIF4E and the activity of the ribosome respectively. In their previous work, the Morley group observed a high level of 4E-BP1 phosphorylation during C2C12 myogenic differentiation, even in the presence of Rad001.1 This contradicts the usual models of eIF4E availability which show that 4E-BP1 phosphorylation is usually downregulated upon mTORC1 inhibition. 
 
In their current paper,4 Pollard, Willett and Morley sought to further investigate the role of mTOR kinase signaling pathway during myogenic differentiation, and in particular, the kinases responsible for the phosphorylation of 4E-BP1. The authors show that the phosphorylation is maintained during myogenic differentiation in the presence of RAD001 but is abolished when using direct inhibitors of the mTOR kinase, KU0063794 and Torin1. The ATM kinase, which is thought to phosphorylate 4E-BP1 on other sites was not implicated in this work. In order to clarify the role of the mTORC complexes during myogenic differentiation they used siRNA to knock down the components that are responsible for target specificity. Their targets were either Raptor, a component of the mTORC1 complex, or Rictor from the mTORC2 complex. The separate or combined knockdowns demonstrate that the depletion of raptor or rictor still does not affect the phosphorylation of 4E-BP1. 
 
Together, these findings suggest that at least in this system, phosphorylation of 4E-BP1 can be due to mTOR signaling that is not part of the mTORC1 or mTORC2 complexes. While this is unusual, work from the Meyuhas group5 suggests that it is not unprecedented that mTOR may be able to act alone, or in an as yet uncharacterised “mTORC3” complex, both of which would be immune to the presence of rapamycin, but not the direct mTOR kinase inhibitors. 
 
The sustained phosphorylation of 4E-BP1 is key to allowing translation to be maintained during the serum starvation required for myogenic differentiation, which would otherwise be inhibitory to the recruitment of eIF4E to the rest of the translation initiation machinery. A secondary effect may also be to help contribute to the selective translation of the subset of mRNAs involved in the differentiation process. The recent advent of ribosomal profiling technologies to examine the translating pool of mRNAs raises the exciting possibility that we may soon be able to understand such key molecular details in this well-studied, but still not fully understood model system.