By preventing the phosphorylation of specific residues on p70 S6k and 4E-BP1, rapamycin inhibits mitogen-stimulated activation of p70 S6k and phosphorylation of S6 ( 8, 9), and disassociation of 4E-BP1 from eIF-4E ( 6, 7).ĬDNAs for rat p70 S6k or regions comprising residues 332–506, 332–415, 415–502, and 66–235 of p70 S6k were amplified from p85 S6k in Pmt2 using PCR with appropriate primers. Growth stimuli activate phosphorylation of 4E-BP1, which decreases its affinity for eIF-4E and releases the block on cap-dependent translation ( 3). In quiescent cells, 4E-BP1 competes with eIF-4G for binding to eIF-4E and represses translation by displacing the initiation factor 4F from the mRNA. Initiation factor 4F complexes with these mRNAs through the interaction of its eIF-4G subunit with eIF-4E, the cap-binding protein that recognizes the N 7-methyl-GpppN structure of the 5′ end of all nonorganellar mRNAs. Phosphorylation of 4E-BP1 controls cap-dependent translation of mRNAs with extensive secondary structure ( 3). Phosphorylation by p70 S6k of the S6 protein of the small ribosomal subunit permits, through unknown mechanisms, efficient translation of mRNAs containing oligopyrimidine tracts in their 5′ untranslated regions ( 12, 13). Ribosomal S6 and 4E-BP1 regulate the initiation of translation of distinct classes of mRNAs. This raises the possibility that, in vivo, an unidentified kinase analogous to p70 S6k is activated by RAFT1 phosphorylation and acts at the rapamycin-sensitive phosphorylation sites of 4E-BP1. RAFT1 phosphorylates p70 S6k much more effectively than 4E-BP1, and the phosphorylation sites on the two proteins show little homology. RAFT1 phosphorylation of 4E-BP1 on Thr-36 and Thr-45 blocks its association with the cap-binding protein, eIF-4E, in vitro, and phosphorylation of Thr-45 seems to be the major regulator of the 4E-BP1–eIF-4E interaction in vivo. RAFT1 phosphorylates p70 S6k on Thr-389, a residue whose phosphorylation is rapamycin-sensitive in vivo and necessary for S6 kinase activity.

Here, we show that RAFT1 directly phosphorylates p70 S6k, 4E-BP1, and 4E-BP2 and that serum stimulates RAFT1 kinase activity with kinetics similar to those of p70 S6k and 4E-BP1 phosphorylation. The function of RAFT1 in the rapamycin-sensitive pathway and its connection to downstream components of the pathway, such as p70 S6 kinase and 4E-BP1, are poorly understood. The complex of rapamycin with its intracellular receptor, FKBP12, interacts with RAFT1/FRAP/mTOR, the in vivo rapamycin-sensitive target and a member of the ataxia telangiectasia mutated (ATM)-related family of kinases that share homology with the catalytic domain of phosphatidylinositol 3-kinase.

Raft People by M.L. Katz