No abstract

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.

Specific recognition of the mRNA 5' cap by eukaryotic initiation factor eIF4E is a rate-limiting step in the translation initiation. Fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry were used to examine the thermodynamics of eIF4E binding to a cap-analogue, 7-methylGpppG. A van't Hoff plot revealed nonlinearity characterized by an unexpected, large positive molar heat capacity change (DeltaC(degree)(p) = +1.92 +/- 0.93 kJ.mol(-1).K(-1)), which was confirmed by direct ITC measurements (DeltaC(degree)(p) = +1.941 +/- 0.059 kJ.mol(-1).K(-1)). This unique result appears to come from an extensive additional hydration upon binding and charge-related interactions within the binding site. As a consequence of the positive DeltaC(degree)(p), the nature of the thermodynamic driving force changes with increasing temperature, from enthalpy-driven and entropy-opposed, through enthalpy- and entropy-driven in the range of biological temperatures, into entropy-driven and enthalpy-opposed. Comparison of the van't Hoff and calorimetric enthalpy values provided proof for the ligand protonation at N(1) upon binding, which is required for tight stabilization of the cap-eIF4E complex. Intramolecular self-stacking of the dinucleotide cap-analogue was analyzed to reveal the influence of this coupled process on the thermodynamic parameters of the eIF4E-mRNA 5' cap interaction. The temperature-dependent change in the conformation of 7-methylGpppG shifts significantly the intrinsic DeltaH(degree)(0) = -72.9 +/- 4.2 kJ.mol(-1) and DeltaS(degree)(0) = -116 +/- 58 J.mol(-1).K(-1) of binding to the less negative resultant values, by DeltaH(degree)(sst) = +9.76 +/- 1.15 kJ.mol(-1) and DeltaS(degree)(sst) = +24.8 +/- 2.1 J.mol(-1).K(-1) (at 293 K), while the corresponding DeltaC(degree)(p)(sst) = -0.0743 +/- 0.0083 kJ.mol(-1).K(-1) is negligible in comparison with the total DeltaC(degree)(p) .

In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5′ end of an mRNA. The eIF4E-binding proteins (4E-BPs) interdict translation initiation by binding to the translation factor eIF4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. We reported previously that phosphorylation of 4E-BP1 on Thr 37 and Thr 46 is relatively insensitive to serum deprivation and rapamycin treatment, and that phosphorylation of these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Here, using mass spectrometry, we identify the serum-responsive, rapamycin-sensitive sites as Ser 65 and Thr 70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS-PAGE and Western blotting with phosphospecific antibodies, we also establish the order of 4E-BP1 phosphorylation in vivo; phosphorylation of Thr 37/Thr 46 is followed by Thr 70 phosphorylation, and Ser 65 is phosphorylated last. Finally, we show that phosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF4E.