A possible mechanism of competition between Paip2 and eIF4G for PABP binding. Shown is PAM1 motif of Paip2 and the N-terminal segment of eIF4G interacting with an overlapping region in PABP.

Paip2 inhibits the interaction of PABP with eIF4GI. A solution of PABP was prepared with Paip2 WT or its mutants, Paip2 (Δ31–56) or Paip2 (1–111), and the PAM2 peptide (amino acids 112–127 of Paip2) using molar ratios of PABP to Paip2 of 1:1 (lanes 2, 6, 9, and 12), 1:2 (lane 3), 1:4 (lanes 4, 7, 10, and 13), or 1:8 (lanes 5, 8, 11, and 14). The mixtures were then incubated at 0°C for 30 min with recombinant GST-fused eIF4GI-Nt immobilized onto glutathione-Sepharose beads. After incubation, the resin was washed, and bound proteins were eluted with Laemmli buffer. After resolving on an SDS/10% polyacrylamide gel, proteins were analyzed by Western blotting using antibodies against eIF4GI or PABP, as indicated. The signals in different lanes were quantified by using nih image software (version 1.63). The value obtained in the absence of added competitor (lane 1) was set as 100%. Values representing the average from three independent experiments and standard deviations from the mean are shown.

Paip2 negates the stimulatory activity of tethered PABP. (A) Diagram of the components used for the assay of the tethered PABP function. The capped reporter luciferase mRNA (Luc-MS2) was not polyadenylated but contained binding sites for MS2 coat protein within its 3′ UTR. The fusion proteins contained the MS2 coat protein sequence at their N terminus. (B) Tethered functional analysis of PABP. The assay was conducted in a PABP-depleted Krebs-2 cell extract. Translation reactions (10 μl) containing increasing concentrations of MS2–PABP (2.8, 5.5, 8.2, and 11 μg/ml in lanes 2, 3, 4, and 5, respectively) were incubated at 30°C for 1 h with 25 ng of Luc-MS2 mRNA. Translation was measured by luciferase assay and is expressed in relative light units (RLU). As negative controls, MS2 protein, either alone or fused to U1A, or PABP mutant M161A were used (at 82 μg/ml). (C) Inhibition of the tethered PABP function by Paip2. Luc–MS2 mRNA translation was assayed as in B in the presence of 82 ng (1 pmol) of MS2–PABP and increasing amounts of recombinant Paip2 WT or its mutants, Δ31–56 or 1–111 [used at the MS2–PABP to Paip2 molar ratios of 1:1 (lanes 3, 8, and 11), 1:2 (lanes 4, 9, and 12), 1:4 (lanes 5, 10, and 13), 1:8 (lane 6), or 1:16 (lane 7)]. In B and C the levels of luciferase in the presence of buffer (25 mM Hepes-KOH, pH 7.3/50 mM KCl/1.5 mM MgCl₂/1 mM DTT) are shown in lane 1. The data are the average from three experiments with standard deviations from the mean.

Coimmunoprecipitation of eIF4GI with anti-PABP is inhibited by Paip2. Extracts from Krebs-2 cells were incubated with anti-PABP (lanes 2 and 3) or preimmune serum (lane 4) coupled to beads in the absence (lane 2) or presence (lane 3) of Paip2. For the pull-down assay, extracts were incubated with GST–Paip2 (lane 5) that were immobilized on glutathione-Sepharose beads (33). Bound proteins were eluted in Laemmli sample buffer, resolved by SDS/7.5% PAGE, and subjected to Western blotting using anti-eIF4GI and anti-PABP antibodies. One-fiftieth of the extract used for immunoprecipitation or pull-down assay was loaded in lane 1.

SPR analysis of eIF4GI interactions with PABP RRMs 1–4: effect of Paip2 and related proteins on the interactions. (A) Control-corrected sensorgrams corresponding to the interaction of RRMs 1–4 with biosensor surface-immobilized eIF4G. RRMs 1–4 from PABP was injected in duplicate for 60 s at different concentrations over surface-immobilized eIF4GI (400 resonance units). The dissociation of the complexes was followed for up to 400 s. The resulting control-corrected sensorgrams (Upper) were fitted by using a 1-to-1 stoichiometry model. Dotted lines show experimental sensorgrams, and solid lines show calculated fits. The residuals (difference between calculated and experimental data points) are shown in Lower, and the related kinetic and thermodynamic constants determined from the fit are listed in Table 1. (B) SPR in-solution competition experiments. RRMs 1–4 from PABP (25 nM) was injected alone or after preincubation with Paip2 (Left), Paip2 (1–111) (Right), or Paip2 (Δ31–56) (at 50 nM; sensorgram not shown). (C) Control-corrected SPR signal recorded after 55 s of injection (shown in B; triplicates not shown) were expressed as percentages (100% corresponds to RRMs 1–4 binding in the absence of Paip2) and plotted as a function of Paip2 or Paip2 mutant concentrations.