A potential role for the Rictor-mTOR complex in regulating myogenic differentiation

4247 The target of rapamycin (TOR), a highly conserved Ser/Thr kinase, plays a central role in the control of eukaryotic cell growth. TOR exists in two functionally and structurally distinct complexes, TOR complex 1 with Raptor (TORC1) and TOR complex 2 with Rictor (TORC2). TORC1 controls cell growth via a rapamycin-sensitive signaling branch regulating translation, transcription, nutrient uptake, ribosome biogenesis, and autophagy. TORC2 controls the organization of the actin cytoskeleton through a rapamycin-insensitive signaling. Rapamycin, a very specific inhibitor of mTOR signaling inhibits myogenic differentiation in C2C12 myoblasts. However, exactly why mTOR signaling is essential in myogenic differentiation remains obscure. We have analyzed complexes of mTOR in membranous , cytosolic, and nuclear fractions of C2C12 myoblasts in the presence or absence of rapamycin under conditions of proliferation (growth medium,GM) or conditions that force differentiation (differentiation medium, DM). Immunofluorescence staining and immunoprecipitation/western blot analysis were used to determine the subcellular localization of proteins. Both Rictor and mTOR were detected in all three fractions. Rapamycin decreased the total mTOR and reduced phosphorylation of mTOR(pSer2481) in the membrane fraction. Rictor and mTOR complexes were co-immunoprecipitated from the membrane and cytosol fractions, but only trace amounts of binding Rictor could be detected in the nuclear fraction. Rapamycin decreased the interaction of Rictor-mTOR in all fractions, and the phosphorylation of cytosolic Akt(pSer473) was dramatically decreased. Immunofluorescence staining showed that mTOR and mTOR(pSer2448) were detected in both cytoplasm and nucleus, however, mTOR(pSer2481) was predominantly nuclear. Although Rictor was detected mainly in membranes and in cytoplasm, rapamycin induced nuclear localization of Rictor especially in DM. These results suggest that Rictor-mTOR complex may be necessary for C2C12 myoblasts differentiation and this complex is also (indirectly) rapamycin sensitive. As the Rictor-mTOR complex might be the Akt(pSer473) kinase, we propose that when C2C12 cells are cultured in DM in the presence of rapamycin, the Rictor-mTOR complex is disrupted and dissociated Rictor proteins translocate to the nucleus, and therefore Akt signaling is inhibited. This research is supported by CA77776 and CA23099 and by ALSAC.