Phosphorylation of eIF4E attenuates its interaction with mRNA 5' cap analogs by electrostatic repulsion: intein-mediated protein ligation strategy to obtain phosphorylated protein.

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.

[1]  Anne-Claude Gingras,et al.  Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins. , 2002, Journal of molecular biology.

[2]  K. Tomoo,et al.  Research Communication , 1971, PS: Political Science & Politics.

[3]  N. Sonenberg,et al.  Phosphorylation of Eukaryotic Translation Initiation Factor 4E Is Critical for Growth , 2002, Molecular and Cellular Biology.

[4]  T. E. Dever,et al.  Gene-Specific Regulation by General Translation Factors , 2002, Cell.

[5]  G. Scheper,et al.  Phosphorylation of Eukaryotic Initiation Factor 4E Markedly Reduces Its Affinity for Capped mRNA* , 2002, The Journal of Biological Chemistry.

[6]  S K Burley,et al.  Hierarchical phosphorylation of the translation inhibitor 4E-BP1. , 2001, Genes & development.

[7]  T. C. Evans,et al.  Intein-mediated ligation and cyclization of expressed proteins. , 2001, Methods.

[8]  G. Scheper,et al.  The Mitogen-Activated Protein Kinase Signal-Integrating Kinase Mnk2 Is a Eukaryotic Initiation Factor 4E Kinase with High Levels of Basal Activity in Mammalian Cells , 2001, Molecular and Cellular Biology.

[9]  M. Sekine,et al.  Efficient Synthesis of 13C,15N-Labeled RNA Containing the Cap Structure m7GpppA , 2000 .

[10]  N. Sonenberg,et al.  Translational control of gene expression , 2000 .

[11]  R. Rhoads,et al.  Quantitative assessment of mRNA cap analogues as inhibitors of in vitro translation. , 1999, Biochemistry.

[12]  Jonathan A. Cooper,et al.  Phosphorylation of the Cap-Binding Protein Eukaryotic Translation Initiation Factor 4E by Protein Kinase Mnk1 In Vivo , 1999, Molecular and Cellular Biology.

[13]  A. Gingras,et al.  Human eukaryotic translation initiation factor 4G (eIF4G) recruits Mnk1 to phosphorylate eIF4E , 1999, The EMBO journal.

[14]  T. Muir,et al.  Introduction of unnatural amino acids into proteins using expressed protein ligation. , 1999, Biopolymers.

[15]  A. Gingras,et al.  eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. , 1999, Annual review of biochemistry.

[16]  N. Sonenberg,et al.  A newly identified N‐terminal amino acid sequence of human eIF4G binds poly(A)‐binding protein and functions in poly(A)‐dependent translation , 1998, The EMBO journal.

[17]  T. Muir,et al.  Adding 'splice' to protein engineering. , 1998, Structure.

[18]  T. Muir,et al.  Expressed Protein Ligation, a Novel Method for Studying Protein-Protein Interactions in Transcription* , 1998, The Journal of Biological Chemistry.

[19]  G. Scheper,et al.  Regulation of translation initiation factors by signal transduction. , 1998, European journal of biochemistry.

[20]  Ming-Qun Xu,et al.  Modulation of Protein Splicing of the Saccharomyces cerevisiae Vacuolar Membrane ATPase Intein* , 1998, The Journal of Biological Chemistry.

[21]  F. Perler,et al.  Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. , 1997, Gene.

[22]  A. Gingras,et al.  Cocrystal Structure of the Messenger RNA 5′ Cap-Binding Protein (eIF4E) Bound to 7-methyl-GDP , 1997, Cell.

[23]  G. Dreyfuss,et al.  General RNA binding proteins render translation cap dependent. , 1996, The EMBO journal.

[24]  F. Perler,et al.  The mechanism of protein splicing and its modulation by mutation. , 1996, The EMBO journal.

[25]  C. Proud,et al.  Serine 209, Not Serine 53, Is the Major Site of Phosphorylation in Initiation Factor eIF-4E in Serum-treated Chinese Hamster Ovary Cells (*) , 1995, The Journal of Biological Chemistry.

[26]  R. Rhoads,et al.  Mapping of Functional Domains in Eukaryotic Protein Synthesis Initiation Factor 4G (eIF4G) with Picornaviral Proteases , 1995, The Journal of Biological Chemistry.

[27]  R. Rhoads,et al.  Phosphorylation of Eukaryotic Protein Synthesis Initiation Factor 4E at Ser-209 (*) , 1995, The Journal of Biological Chemistry.

[28]  Z. Wieczorek,et al.  Fluorescence and absorption spectroscopic properties of RNA 5'-cap analogues derived from 7-methyl-, N2,7-dimethyl- and N2,N2,7-trimethyl-guanosines. , 1995, Journal of photochemistry and photobiology. B, Biology.

[29]  B. Dunn,et al.  Peptide Synthesis Protocols , 1994 .

[30]  R. Rhoads,et al.  Chromatographic resolution of in vivo phosphorylated and nonphosphorylated eukaryotic translation initiation factor eIF-4E: increased cap affinity of the phosphorylated form. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[31]  S. Kusumoto,et al.  An Efficient Procedure for Solid-Phase Synthesis of Phosphopeptides by the Fmoc Strategy , 1994 .

[32]  T. Tanaka,et al.  Expression of a synthetic gene for human cap binding protein (human IF-4E) in Escherichia coli and fluorescence studies on interaction with mRNA cap structure analogues. , 1991, Journal of biochemistry.

[33]  I. Ekiel,et al.  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 Analogs , 1990 .

[34]  R. Panniers,et al.  Cap binding protein complex that restores protein synthesis in heat-shocked Ehrlich cell lysates contains highly phosphorylated eIF-4E. , 1990, The Journal of biological chemistry.

[35]  R. Rhoads,et al.  A spectroscopic study of the binding of N-7-substituted cap analogues to human protein synthesis initiation factor 4E. , 1990, Biochemistry.

[36]  W. Rychlik,et al.  Alteration of the major phosphorylation site of eukaryotic protein synthesis initiation factor 4E prevents its association with the 48 S initiation complex. , 1990, The Journal of biological chemistry.

[37]  R. Rhoads,et al.  A spectroscopic study of the binding of m7GTP and m7GpppG to human protein synthesis initiation factor 4E. , 1989, Biochemistry.

[38]  N. Sonenberg,et al.  High-level synthesis in Escherichia coli of functional cap-binding eukaryotic initiation factor eIF-4E and affinity purification using a simplified cap-analog resin. , 1988, Gene.

[39]  J. Hershey,et al.  Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in translational control. Heat shock effects on eIF-4F. , 1987, The Journal of biological chemistry.

[40]  W. H. Elliott,et al.  Data for Biochemical Research , 1986 .

[41]  A. Shatkin,et al.  Chemical synthesis and characterization of 7-methylguanosine cap analogs , 1985 .

[42]  J. Ebel,et al.  Translational recognition of messenger ribonucleic acid caps as a function of pH. , 1983, Biochemistry.

[43]  M. Sekine,et al.  Efficient Synthesis of 13 C , 15 N-Labeled RNA Containing the Cap Structure m 7 GpppA , 2022 .