Phosphorylation of eIF-4E on serine 209 by protein kinase C is inhibited by the translational repressors, 4E-binding proteins.
Translation initiation in eukaryotes is facilitated by the mRNA 5' cap structure (m7GpppX, where X is any nucleotide) that binds the multisubunit initiation factor eIF4F through one of its subunits, eIF4E. eIF4E is a phosphoprotein whose phosphorylation state positively correlates with cell growth. Protein kinase C phosphorylates eIF4E in vitro, and possibly in vivo. Using recombinant eIF4E incubated in vitro with purified protein kinase C and analyzed by solid-phase phosphopeptide sequencing in combination with high performance liquid chromatography coupled to mass spectrometry, we demonstrated that the third amino acid of the peptide SGSTTK (Ser209) is the major site of phosphorylation. This finding is consistent with the newly assigned in vivo phosphorylation site of eIF4E (Joshi, B., Cai, A. L., Keiper, B. D., Minich, W. B., Mendez, R., Beach, C. M., Stepinski, J., Stolarski, R., Darzynkiewicz, E., and Rhoads, R. E. (1995) J. Biol. Chem. 270, 14597-14603). A S209A mutation resulted in dramatically reduced phosphorylation, both in vitro and in vivo. Furthermore, the mutant protein was phosphorylated on threonine (most probably threonine 210) in vivo. Here we show that in the presence of the recently characterized translational repressors 4E-BP1 or 4E-BP2, phosphorylation of eIF4E by protein kinase C is strongly reduced. This suggests a two-step model for the phosphorylation (and activation) of eIF4E by growth factors and hormones: first, dissociation of eIF4E from 4E-BPs, followed by eIF4E phosphorylation.
Whalen
S G
SG
Department of Biochemistry and McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Gingras
A C
AC
Amankwa
L
L
Mader
S
S
Branton
P E
PE
Aebersold
R
R
Sonenberg
N
N
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D051379:Mice; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010750:Phosphoproteins / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D012097:Repressor Proteins / Q000502:physiology*; D012694:Serine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C499527:Eif4ebp1 protein, mouse; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D012097:Repressor Proteins; D012694:Serine; D011493:Protein Kinase C