Phosphorylation of eIF4E attenuates its interaction with mRNA 5′ cap analogs by electrostatic repulsion: Intein-mediated protein ligation strategy to obtain phosphorylated protein
- JOANNA ZUBEREK1,
- ALEKSANDRA WYSLOUCH-CIESZYNSKA2,
- ANNA NIEDZWIECKA1,
- MICHAL DADLEZ1,2,
- JANUSZ STEPINSKI1,
- WOJCIECH AUGUSTYNIAK3,
- ANNE-CLAUDE GINGRAS4,
- ZHIBO ZHANG5,
- STEPHEN K. BURLEY6,
- NAHUM SONENBERG4,
- RYSZARD STOLARSKI1, and
- EDWARD DARZYNKIEWICZ1
- 1Department of Biophysics, Institute of Experimental Physics, Warsaw University, 02-089 Warszawa, Poland
- 2Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02–106 Warszawa, Poland
- 3Faculty of Chemistry, Warsaw University, 02-093 Warszawa, Poland
- 4Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec, H3G 1Y6, Canada
- 5Institute of Organic Chemistry, University of Turku, Turku, Finland
- 6Laboratories of Molecular Biophysics, The Rockefeller University, New York, New York 10021, USA
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
- expressed protein ligation
- fluorescence
- intein
- mRNA 5′ cap
- translation initiation
- CBD, chitin binding domain from Bacillus circulans
- CPG, controlled pore glass
- IPTG, isopropyl-β-D-thiogalactopyranoside
- eIF, eukaryotic initiation factor
- 4E-BP, 4E binding protein
- LCAA, long chain alkylamine
- MESNA, 2-mercaptoethanesulfonic acid
- Mxe GryA intein, intein from Mycobacterium xenopi gryA gene
- m7GMP, 7-methylguanosine 5′-monophosphate
- m7GDP, 7-methylguanosine 5′-diphosphate
- m7GTP, 7-methylguanosine 5′-triphosphate
- m7GpppG, P1-7-methylguanosine-5′ P3-guanosine-5′ triphosphate
- TCEP, tris-(2-carboxyethyl)phosphine
- TST, time-synchronized titration
Footnotes
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Article and publication are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2133403.
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- Accepted October 7, 2002.
- Received September 4, 2002.
- Copyright 2003 by RNA Society
