AGC kinases regulate phosphorylation and activation of eukaryotic translation initiation factor 4B.
Eukaryotic translation initiation factor 4B (eIF4B) plays a critical role during the initiation of protein synthesis and its activity can be regulated by multiple phosphorylation events. In a search for novel protein kinase B (PKB/c-akt) substrates, we identified eIF4B as a potential target. Using an in vitro kinase assay, we found that PKB can directly phosphorylate eIF4B on serine 422 (ser422). Activation of a conditional PKB mutant, interleukin-3 (IL-3) or insulin stimulation resulted in PKB-dependent phosphorylation of this residue in vivo. This was prevented by pretreatment of cells with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or pharmacological inhibition of PKB. Pretreatment of cells with rapamycin, inhibiting mTOR or U0126 to inhibit MEK, had little effect on eIF4B ser422 phosphorylation. In contrast, following amino-acid refeeding, eIF4B ser422 phosphorylation was found to be mammalian target of rapamycin (mTOR)-dependent. We further identified eIF4B ser406 as a novel mitogen-regulated phosphorylation site. Insulin-induced phosphorylation of eIF4B ser406 was dependent on both MEK and mTOR activity. Utilizing a novel translational control luciferase assay, we could further demonstrate that phosphorylation of ser406 or ser422 is essential for optimal translational activity of eIF4B. These data provide novel insights into complex multikinase regulation of eIF4B phosphorylation and reveal an important mechanism by which PKB can regulate translation, potentially critical for the transforming capacity of this AGC kinase family member.
Molecular Immunology Laboratory, Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.
A G M
AG
van Gorp
K E
KE
van der Vos
A B
AB
Brenkman
A
A
Bremer
N
N
van den Broek
F
F
Zwartkruis
J W
JW
Hershey
B M T
BM
Burgering
C F
CF
Calkhoven
P J
PJ
Coffer
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism*; D002867:Chromones / Q000494:pharmacology; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D010442:Peptide Chain Initiation, Translational; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D058570:TOR Serine-Threonine Kinases
D002867:Chromones; D039642:Eukaryotic Initiation Factors; D007328:Insulin; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; C031799:eIF-4B; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012694:Serine; D011494:Protein Kinases; C546843:mTOR protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases