ABSTRACT
The interaction between recombinant human eukaryotic initiation factor (eIF)4E and recombinant glutathione S-transferase (GST)-fused human 4E-binding protein (BP)1 was analyzed by using the surface plasmon resonance method. The association rate of 4E-BP1 with eIF4E increased by about two orders of magnitude in the presence of m7GTP (a model compound of mRNA cap structure), but the dissociation rate was scarcely affected, indicating the cap-dependent binding of 4E-BP1 to eIF4E. On the other hand, phosphorylation of 4E-BP1 weakened its interaction with eIF4E whether m7GTP was present or not. However, phosphorylation of 4E-BP1 already associated with eIF4E or its m7GTP complex did not cause any change of the association, probably because of incomplete phosphorylation. This suggests that the regulation of eIF4E by 4E-BP phosphorylation is performed at its free state, not when it is in the associated state with eIF4E. Given these results, we discuss how 4E-BP regulates eIF4E at the first step of translation initiation.
Subject(s)
Carrier Proteins , Peptide Initiation Factors/metabolism , Phosphoproteins/metabolism , Surface Plasmon Resonance/methods , Adaptor Proteins, Signal Transducing , Cell Cycle Proteins , Eukaryotic Initiation Factor-4E , Glutathione Transferase/metabolism , Guanosine Triphosphate/metabolism , Humans , Models, Biological , Phosphorylation , Protein Binding , Recombinant Fusion Proteins/metabolism , Time FactorsABSTRACT
A translation initiation factor, eIF4E, of Xenopus laevis was purified by affinity column chromatography after the gene expression as a full-length protein in a baculovirus-insect cell system. Interaction between X. laevis eIF4E and 4E-BP2 was analyzed by affinity column chromatography, gel permeation chromatography (GPC), and surface plasmon resonance (SPR). It was found that the interaction of eIF4E with an mRNA cap-analogue enhanced the binding activity of eIF4E with 4E-BP2. Furthermore, the SPR analysis showed that the eIF4E-cap-analogue interaction was very weak regardless of complex formation of 4E-BP2 with eIF4E; the dissociation constant of eIF4E for the cap-analogue was estimated to be 10(-2)-10(-4) M. These results suggest that the participation of another initiation factor is required for eIF4E to recognize the cap structure in vivo. The results reported in this paper support "the performed complex model" of Lee et al., in which eIF4E binds to the mRNA cap structure after the initiation factors have formed the initiation complex eIF4F.
Subject(s)
Eukaryotic Initiation Factors , Peptide Initiation Factors/metabolism , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Eukaryotic Initiation Factor-4E , Humans , In Vitro Techniques , Molecular Sequence Data , Peptide Chain Initiation, Translational , Peptide Initiation Factors/genetics , Peptide Initiation Factors/isolation & purification , RNA Caps/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Xenopus laevisABSTRACT
An artificial gene coding for the human initiation factor (eIF) 4E-binding protein 1 (4E-BP1) was chemically synthesized and cloned. Although the expression of the 4E-BP1 gene alone has not yet been accomplished, the gene was expressed in Escherichia coli [BL21(DE3)] as a fusion gene with the glutathione-S-transferase (GST) gene using a prokaryotic gene fusion vector (pGEX-4T-2), which contains a gene sequence coding the cleavage site for a specific protease, alpha-thrombin. The fusion gene product was purified to homogeneity by glutathione Sepharose-4B affinity column chromatography. It was shown by m7GTP- and glutathione-affinity chromatography that the binding ability of 4E-BP1 to eIF-4E is nearly the same as that to the eIF-4E x m7GTP complex, implying different binding sites of eIF-4E and its nonallosteric obligation for 4E-BP1 and mRNA cap structure. In contrast with the binding of eIF-4E to the mRNA cap structure, where some functional amino acids play an important role in the binding, the binding to 4E-BP1 was suggested to occur via multiple nonspecific interactions.
