Serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI.
The eukaryotic translation initiation factor 4G (eIF4G) proteins play a critical role in the recruitment of the translational machinery to mRNA. The eIF4Gs are phosphoproteins. However, the location of the phosphorylation sites, how phosphorylation of these proteins is modulated and the identity of the intracellular signaling pathways regulating eIF4G phosphorylation have not been established. In this report, two-dimensional phosphopeptide mapping demonstrates that the phosphorylation state of specific eIF4GI residues is altered by serum and mitogens. Phosphopeptides resolved by this method were mapped to the C-terminal one-third of the protein. Mass spectrometry and mutational analyses identified the serum-stimulated phosphorylation sites in this region as serines 1108, 1148 and 1192. Phosphoinositide-3-kinase (PI3K) inhibitors and rapamycin, an inhibitor of the kinase FRAP/mTOR (FKBP12-rapamycin-associated protein/mammalian target of rapamycin), prevent the serum-induced phosphorylation of these residues. Finally, the phosphorylation state of N-terminally truncated eIF4GI proteins acquires resistance to kinase inhibitor treatment. These data suggest that the kinases phosphorylating serines 1108, 1148 and 1192 are not directly downstream of PI3K and FRAP/mTOR, but that the accessibility of the C-terminus to kinases is modulated by this pathway(s).
Raught
B
B
Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec H3G 1Y6, Canada.
Gingras
A C
AC
Gygi
S P
SP
Imataka
H
H
Morino
S
S
Gradi
A
A
Aebersold
R
R
Sonenberg
N
N
D000595:Amino Acid Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D039603:Eukaryotic Initiation Factor-4G; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000235:genetics; D010449:Peptide Mapping; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D014176:Protein Biosynthesis; D011494:Protein Kinases; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases
D004791:Enzyme Inhibitors; D039603:Eukaryotic Initiation Factor-4G; D010448:Peptide Initiation Factors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010748:Phosphopeptides; D012333:RNA, Messenger; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus