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.
Zuberek
Joanna
J
Department of Biophysics, Institute of Experimental Physics, Warsaw University, Poland.
Wyslouch-Cieszynska
Aleksandra
A
Niedzwiecka
Anna
A
Dadlez
Michal
M
Stepinski
Janusz
J
Augustyniak
Wojciech
W
Gingras
Anne-Claude
AC
Zhang
Zhibo
Z
Burley
Stephen K
SK
Sonenberg
Nahum
N
Stolarski
Ryszard
R
Darzynkiewicz
Edward
E
D039561:Eukaryotic Initiation Factor-4E / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D012315:RNA Caps; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D055672:Static Electricity
D039561:Eukaryotic Initiation Factor-4E; D012315:RNA Caps; D012333:RNA, Messenger