TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function.
BACKGROUND
The mammalian target of rapamycin, mTOR, is a serine/threonine kinase that controls cell growth and proliferation via the translation regulators eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). We recently identified a TOR signaling (TOS) motif in the N terminus of S6K1 and the C terminus of 4E-BP1 and demonstrated that in S6K1, the TOS motif is necessary to facilitate mTOR signaling to phosphorylate and activate S6K1. However, it is unclear how the TOS motif in S6K1 and 4E-BP1 mediates mTOR signaling.


RESULTS
Here, we show that a functional TOS motif is required for 4E-BP1 to bind to raptor (a recently identified mTOR-interacting protein), for 4E-BP1 to be efficiently phosphorylated in vitro by the mTOR/raptor complex, and for 4E-BP1 to be phosphorylated in vivo at all identified mTOR-regulated sites. mTOR/raptor-regulated phosphorylation is necessary for 4E-BP's efficient release 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.


CONCLUSIONS
Our data demonstrate that the TOS motif functions as a docking site for the mTOR/raptor complex, which is required for multisite phosphorylation of 4E-BP1, eIF4E release from 4E-BP1, and cell growth.
The Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Stefanie S
SS
Schalm
Diane C
DC
Fingar
David M
DM
Sabatini
John
J
Blenis
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002460:Cell Line; D045744:Cell Line, Tumor; D048429:Cell Size; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000076223:Regulatory-Associated Protein of mTOR; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D039561:Eukaryotic Initiation Factor-4E; D010750:Phosphoproteins; D011506:Proteins; C463659:RPTOR protein, human; D011993:Recombinant Fusion Proteins; D000076223:Regulatory-Associated Protein of mTOR; D011494:Protein Kinases; C546842:MTOR protein, human; C069329:RPS6KA1 protein, human; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D058570:TOR Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1