"Research of crosstalk between nutrient sensing mTOR signaling pathway and energy metabolic sensing AMPK"

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1.The inhibition of mTOR signaling pathway by AMPKWe isolated a portion of the γ1 subunit of AMPK, which played a role as an energy metabolic sensor, by yeast two-hybrid screening using mTOR kinase domain as a bait. This result suggests the possibility of the linkage between the nutrient sensing mTOR signaling pathway and the cellular energy metabolic system. Actually the treatments of mammalian cells with AMPK activators inhibit phosphorylations of p70 S6 kinase (p70 S6k) and eukaryotic translational initiation factor 4E binding protein (4EBP1) which function as downstream effectors of mTOR, however a rapamycin-resistant p70 S6k variant is insensitive to AMPK activators. In addition, overexpression of the constitutively active form of AMPK in mammalian cells inhibits phosphorylations of both p70 S6k and 4EBP1. On the other hand, in the dominant negative form of AMPK-over-expressing cells, these inhibitions are restored. These results suggest a possible linkage between AMPK and mTOR signaling.2.The analysis of a role of mTOR interacting protein raptor as a scaffold proteinBoth p70 S6k and 4EBP1 have evolutionally conserved TOS (TOR signaling) motif. The mutants of this motif are no longer pbosphorylated by mitogens in cells. We showed that the TOS motif was a binding site of mTOR binding protein raptor. The mutants of the TOS motif cannot bind to raptor and these mutants can be no longer phosphorylated by mTOR in the presence of both mTOR and raptor.