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Novel phosphorylation sites of eukaryotic initiation factor-4F and evidence that phosphorylation stabilizes
1Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee.
Abstract:
Only serine phosphorylation of eukaryotic initiation factor-4E (eIF-4E) has been previously reported in intact cells. We found that treatment of HepG2 cells with okadaic acid resulted in as much as 20% of eukaryotic initiation factor (eIF)-4E phosphorylation occurring on threonine residues and that tryptic phosphopeptide maps showed several previously unrecognized phosphopeptides. Analysis of p220 from control and okadaic acid-treated cells demonstrated serine and threonine phosphorylation under both conditions. However, a unique pattern of phosphopeptides in okadaic acid-treated cells was observed. The most notable finding was that hyperphosphorylation of eIF-4E and p220 increased binding of p220 but not eIF-4E to the m7GTP cap structure. We suggest that phosphorylation of eIF-4E is more complicated than previously recognized and that hyperphosphorylation of eIF-4E and p220 recruits more p220 into the protein complex that associates with mRNA caps. A better understanding of these protein-protein and protein-mRNA interactions may aid the design of anti-sense directed chemistries that disrupt such interactions for a specific target mRNA (Baker, B.F., Miraglia, L., and Hagedorn, C. H. (1992) J. Biol. Chem. 267, 11495-11499).
Insights
Previously, only serine phosphorylation of eukaryotic initiation factor-4E (eIF-4E) was known. This study reveals threonine phosphorylation of eIF-4E and p220, impacting mRNA cap binding.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- Previously, only serine phosphorylation of eukaryotic initiation factor-4E (eIF-4E) was documented in intact cells.
- The role of eIF-4E phosphorylation in protein synthesis regulation is critical.
- Understanding post-translational modifications of translation factors is key to cellular control.
Purpose of the Study:
- To investigate novel phosphorylation sites on eukaryotic initiation factor-4E (eIF-4E) beyond serine.
- To analyze the impact of threonine phosphorylation on eIF-4E and p220.
- To determine how altered phosphorylation affects the binding of these proteins to the mRNA cap structure.
Main Methods:
- Treatment of HepG2 cells with okadaic acid to induce hyperphosphorylation.
- Analysis of tryptic phosphopeptide maps to identify novel phosphorylation sites.
- Assessment of m7GTP cap structure binding affinity for eIF-4E and p220.
Main Results:
- Okadaic acid treatment led to up to 20% threonine phosphorylation of eIF-4E, alongside novel phosphopeptides.
- Both serine and threonine phosphorylation were observed in p220 under control and treated conditions.
- Hyperphosphorylation of eIF-4E and p220 enhanced the binding of p220, but not eIF-4E, to the m7GTP cap structure.
Conclusions:
- eIF-4E phosphorylation is more complex than previously understood, involving threonine residues.
- Hyperphosphorylation of eIF-4E and p220 promotes the recruitment of p220 into mRNA cap-binding complexes.
- Further research into these interactions could inform the development of targeted anti-sense therapies.