1505

Differentiation of skeletal muscle cells involves three major steps, namely, withdrawal of myoblasts from the cell cycle, subsequent expression of myotube-specific genes, and formation of multinucleated myotubes. Previous study has shown that proliferation is mediated by the Raf/MEK/ERK pathway, whereas differentiation is mediated by the PI3K/AKT/mTOR pathway. However, exactly why mTOR signaling is essential in myogenic differentiation remains obscure though rapamycin, a very specific inhibitor of mTOR signaling, inhibits myogenic differentiation in C2C12 myoblasts. In cells mTOR is thought to exist in a large complex (> 1 megaDa) with several proteins including Raptor and GβL, and possibly other proteins. Previous studies reported that Raptor binds to mTOR though HEAT regions and binds to 4EBP1 through C terminal TOS motif of 4EBP1 to regulate the mTOR pathway. However, it is unknown whether mTOR complexes are regulated during myogenic differentiation. In this study we have analyzed complexes associated with eIF4E under conditions of growth (growth medium, GM) or differentiation (differentiation medium, DM). Under both conditions eIF4E binds to 7-methyl-GTP-sepharose-4B resin, and can be readily isolated. Using this ‘pull-down’ technique we have analyzed proteins bound to eIF4E under GM and DM conditions. Raptor and 4EBP1 were associated with eIF4E in both GM and DM. Rapamycin increased the association 4EBP1 as anticipated, but not raptor. Interestingly, when C2C12 cells were cultured in GM, 4EBP1 and Raptor were associated with eIF4E, but mTOR was rarely detected in the complex with antibody 26E3. When C2C12 cells were cultured in DM for 24 hrs, mTOR was strongly detected in the eIF4E complex.The results suggest a conformational change during early differentiation that yields tighter binding of mTOR to Raptor. Under nutrient restriction (low amino acids), raptor binds more tightly to mTOR and inhibits mTOR-dependent signaling (Kim et al. Cell 110:163, 2002). To determine whether the mTOR-Raptor complex induced under DM conditions also inhibited signaling, we examined the phosphorylation of two downstream targets of mTOR, p70S6K (Thr389) and 4EBP1 (Thr37). In contrast to the effect of amino acid deficiency, mTOR signaling is not inhibited in differentiating myoblasts. The results suggest that the affinity of mTOR-Raptor-4EBP1-eIF4E interactions increases during myogenic differentiation, and may be be essential for terminal differentiation. Based on these data, we propose that in C2C12 myoblasts there is a dynamic change in mTOR-Raptor-4EBP1-eIF4E complexes associated with cell cycle withdrawal. Other proteins may be involved in the regulation, such as g-actin, which was found associated with the complex in rapamycin-treated cells. The exact relation between the dynamics of these complexes and regulation of myogenesis is under further study. Supported by CA7776, CA96696 and ALSAC

[Proc Amer Assoc Cancer Res, Volume 46, 2005]