Molecular Mechanism of mTOR downstream signaling (2003)

The mammalian target of rapamycin, mTOR, is a Ser/Thr kinase that promotes cell growth and proliferation by activating S6 kinases 1 and 2 (S6K1/2), and by inhibiting the translational repressors, eukaryotic initiation factor 4E (eIF4E) binding proteins 1, -2, and -3 (4E-BP1/2/3). The molecular basis of how mTOR regulates S6K1/2 and 4E-BP1/2/3 remains controversial. This study describes the identification and characterization of the conserved TOR signaling (TOS) motif in the N-terminus of all known S6 kinases and in the C-terminus of the 4E-BPs. The TOS motif is required for mTOR to modulate the activity of these translation regulators. The TOS motif is essential for S6K1 activation by mTOR, as mutations in this motif mimic the effect of rapamycin in that they prevent S6K1 phosphorylation and activation. Furthermore, it was found that mutations in the TOS motif render S6K1 insensitive to amino acid signaling. The data suggest that the TOS motif is required for S6K1 to interact with a common mTOR-dependent regulator of S6K1 and 4E-BP1, as overexpression of S6K1 with an intact, but not mutant, TOS motif impairs 4E- BP1 phosphorylation. The TOS motif seems to be crucial for S6K1 regulation by two distinct TOR-dependent mechanisms: phosphorylation of Thr389 within the hydrophobic motif and suppression of an inhibitory signal that is mediated by the C-terminus of S6K1. A conserved RSPRR motif within the C-terminus of S6K1 may mediate this inhibitory effect, as deletion of this motif within a TOS motif mutant renders S6K1 partially rapamycin-resistant. This study also describes how the TOS motif within 4E-BP1 mediates 4E-BP1 regulation by mTOR signaling. A functional TOS motif is required for 4E-BP1 to bind to raptor (a recently identified mTOR-interacting protein). The TOS motif is necessary for efficient phosphorylation of 4E-BP1 by the mTOR/raptor complex in vitro, and for in vivo 4E-BP1 phosphorylation at all characterized mTOR-regulated sites. mTOR/raptor-mediated phosphorylation of 4E-BP1 is necessary for 4E-BP1 to be efficiently released from the translational initiation factor, eIF4E. Consistently, overexpression of a mutant of 4E-BP1 containing a single amino acid change in the TOS motif (F114A) reduces cell size, demonstrating that mTOR-dependent regulation of cell growth by 4E-BP1 is dependent on a functional TOS motif. The data presented suggests that the TOS motif is a docking site for the raptor/mTOR complex, which is required for multi-site phosphorylation of both 4E-BP1 and S6K1. Recently, S6K2, a close homolog of S6K1, has been identified. Like S6K1, S6K2 is regulated by the phosphatidylinositide 3-kinase (PI3K) and mTOR pathways. However, the specific kinase activity of S6K2 is significantly lower than that of S6K1 upon cell treatments that potently activate S6K1. Exchanging the unique region of the N- and C-termini between S6K1 and S6K2 revealed that both the N- and C-terminus of S6K2 have an inhibitory effect on its kinase activity. In contrast, a proline-rich region located within the C-terminus of S6K2 did not contribute to this inhibitory effect. Both the N-and C-termini of S6K2 may negatively regulate its activity by binding to an inhibitor or affecting the localization of S6K2. Additional studies will have to be carried out to determine how these intrinsic self inhibitory domains of S6K2 are regulated and may provide insights into the physiological function of S6K2.