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Phosphorylation of eIF-4E on serine 209 by protein kinase C is inhibited by the translational repressors, 4E-binding
S G Whalen1, A C Gingras, L Amankwa
1Department of Biochemistry and McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Abstract:
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.
Insights
Protein kinase C phosphorylates eukaryotic initiation factor 4E (eIF4E) at Ser209, a key step for cell growth. Translational repressors 4E-BP1/2 inhibit this eIF4E phosphorylation by protein kinase C.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Translation initiation in eukaryotes relies on the mRNA 5' cap structure binding to eukaryotic initiation factor 4F (eIF4F).
- eIF4E, a subunit of eIF4F, is a phosphoprotein; its phosphorylation correlates with cell growth.
- Protein kinase C is implicated in eIF4E phosphorylation, both in vitro and potentially in vivo.
Purpose of the Study:
- To identify the specific site of eIF4E phosphorylation by protein kinase C.
- To investigate the effect of mutations on eIF4E phosphorylation.
- To determine the role of translational repressors 4E-BP1 and 4E-BP2 in regulating eIF4E phosphorylation.
Main Methods:
- Recombinant eIF4E was incubated with purified protein kinase C.
- Solid-phase phosphopeptide sequencing and HPLC-MS were used to identify phosphorylation sites.
- Site-directed mutagenesis (S209A) was employed to assess the functional significance of Ser209.
- The impact of 4E-BP1 and 4E-BP2 on eIF4E phosphorylation was examined.
Main Results:
- Serine 209 (Ser209) was identified as the major site of eIF4E phosphorylation by protein kinase C in vitro.
- A S209A mutation significantly reduced eIF4E phosphorylation both in vitro and in vivo.
- The S209A mutant showed compensatory phosphorylation on threonine (likely Thr210) in vivo.
- The presence of 4E-BP1 or 4E-BP2 markedly reduced eIF4E phosphorylation by protein kinase C.
Conclusions:
- Ser209 is the primary site for protein kinase C-mediated phosphorylation of eIF4E.
- This phosphorylation event is crucial for eIF4E function and cell growth.
- Translational repressors 4E-BP1/2 inhibit eIF4E phosphorylation, suggesting a regulatory mechanism.
- A two-step model for eIF4E activation is proposed: dissociation from 4E-BPs followed by phosphorylation.