Distinct signaling events downstream of mTOR cooperate to mediate the effects of amino acids and insulin on initiation factor 4E-binding proteins.
Signaling through the mammalian target of rapamycin (mTOR) controls cell size and growth as well as other functions, and it is a potential therapeutic target for graft rejection, certain cancers, and disorders characterized by inappropriate cell or tissue growth. mTOR signaling is positively regulated by hormones or growth factors and amino acids. mTOR signaling regulates the phosphorylation of several proteins, the best characterized being ones that control mRNA translation. Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) undergoes phosphorylation at multiple sites. Here we show that amino acids regulate the N-terminal phosphorylation sites in 4E-BP1 through the RAIP motif in a rapamycin-insensitive manner. Several criteria indicate this reflects a rapamycin-insensitive output from mTOR. In contrast, the insulin-stimulated phosphorylation of the C-terminal site Ser64/65 is generally sensitive to rapamycin, as is phosphorylation of another well-characterized target for mTOR signaling, S6K1. Our data imply that it is unlikely that mTOR directly phosphorylates Thr69/70 in 4E-BP1. Although 4E-BP1 and S6K1 bind the mTOR partner, raptor, our data indicate that the outputs from mTOR to 4E-BP1 and S6K1 are distinct. In cells, efficient phosphorylation of 4E-BP1 requires it to be able to bind to eIF4E, whereas phosphorylation of 4E-BP1 by mTOR in vitro shows no such preference. These data have important implications for understanding signaling downstream of mTOR and the development of new strategies to impair mTOR signaling.
Wang
Xuemin
X
Division of Molecular Physiology, Faculty of Life Sciences, University of Dundee, Dow St., Dundee DD1 5EH, United Kingdom.
Beugnet
Anne
A
Murakami
Mirei
M
Yamanaka
Shinya
S
Proud
Christopher G
CG
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000494:pharmacology*; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D006224:Cricetinae; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D000596:Amino Acids; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002867:Chromones; C087000:EIF4EBP1 protein, human; C499526:Eif4ebp1 protein, rat; D039561:Eukaryotic Initiation Factor-4E; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines; D010750:Phosphoproteins; D013912:Threonine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012694:Serine; D011494:Protein Kinases; C546842:MTOR protein, human; C546845:mTOR protein, rat; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus