Phosphorylation of eIF4E attenuates its interaction with mRNA 5′ cap analogs by electrostatic repulsion: Intein-mediated protein ligation strategy to obtain phosphorylated protein
Open Access
- 1 January 2003
- journal article
- Published by Cold Spring Harbor Laboratory in RNA
- Vol. 9 (1) , 52-61
- https://doi.org/10.1261/rna.2133403
Abstract
Phosphorylation of the eukaryotic initiation factor eIF4E in response to mitogenic stimuli and cytokines is implicated in the regulation of the initiation step of translation. It still remains unclear how the phosphorylation of eIF4E regulates the translation. To address this problem, we applied a unique technique in protein engineering, intein-mediated protein ligation, to synthesize eIF4E, which is selectively phosphorylated at Ser 209. Using selectively chosen synthetic cap analogs, we compared quantitatively the cap affinity for phosphorylated and unphosphorylated eIF4E by a fluorometric time-synchronized titration method. A 1.5- to 4.5-fold reduction of the cap affinity for phosphorylated eIF4E was observed, depending on the negative charge of the 5′-to-5′ phosphate chains as well as the presence of a longer tetraribonucleotide strand. Possible implications for understanding the regulation of eIF4E functioning, cap complex formation, and stability, are discussed.Keywords
This publication has 39 references indexed in Scilit:
- Biophysical Studies of eIF4E Cap-binding Protein: Recognition of mRNA 5′ Cap Structure and Synthetic Fragments of eIF4G and 4E-BP1 ProteinsJournal of Molecular Biology, 2002
- The Mitogen-Activated Protein Kinase Signal-Integrating Kinase Mnk2 Is a Eukaryotic Initiation Factor 4E Kinase with High Levels of Basal Activity in Mammalian CellsMolecular and Cellular Biology, 2001
- Efficient Synthesis of 13C,15N-Labeled RNA Containing the Cap Structure m7GpppAJournal of the American Chemical Society, 2000
- Quantitative Assessment of mRNA Cap Analogues as Inhibitors of in Vitro TranslationBiochemistry, 1999
- Mapping of Functional Domains in Eukaryotic Protein Synthesis Initiation Factor 4G (eIF4G) with Picornaviral ProteasesJournal of Biological Chemistry, 1995
- Synthesis, Conformation and Hydrolytic Stability of p1,p3−Dinucleoside Triphosphates Related to mRNA 5′-cap, and Comparative Kinetic Studies on their Nucleoside and Nucleoside Monophosphate AnalogsNucleosides, Nucleotides and Nucleic Acids, 1990
- A spectroscopic study of the binding of N-7-substituted cap analogs to human protein synthesis initiation factor 4EBiochemistry, 1990
- A spectroscopic study of the binding of m7GTP and m7GpppG to human protein synthesis initiation factor 4EBiochemistry, 1989
- High-level synthesis in Escherichia coli of functional cap-binding eukaryotic initiation factor eIF-4E and affinity purification using a simplified cap-analog resinGene, 1988
- Translational recognition of messenger ribonucleic acid caps as a function of pHBiochemistry, 1983