Chromatographic resolution of in vivo phosphorylated and nonphosphorylated eukaryotic translation initiation factor eIF-4E: increased cap affinity of the phosphorylated form.

Abstract

Eukaryotic translation initiation factor eIF-4E plays a central role in the recognition of the 7-methylguanosine-containing cap structure of mRNA and the formation of initiation complexes during protein synthesis. eIF-4E exists in both phosphorylated and nonphosphorylated forms, and the primary site of phosphorylation has been identified. Previous studies have suggested that eIF-4E phosphorylation facilitates its participation in protein synthesis. However, the biochemical basis for the functional difference between the two forms of eIF-4E is unknown. To address this directly, we have developed a method for the separation of phosphorylated and nonphosphorylated eIF-4E from rabbit reticulocytes by chromatography on rRNA-Sepharose. Using the resultant purified forms, we have studied the protein's interaction with the cap analogs m7GTP and m7GpppG and with the cap of globin mRNA by fluorescence quenching of tryptophan residues. It was found that phosphorylated eIF-4E had 3-to 4-fold greater affinity for cap analogs and mRNA than nonphosphorylated eIF-4E. The equilibrium binding constants (x 105, expressed as M-1) for the interaction of phosphorylated eIF-4E with m7GTP, m7GpppG, and globin mRNA were 20.0 ± 0.1, 16.4 ± 0.1, and 31.0 ± 0.1, respectively, whereas those for the nonphosphorylated form were 5.5 ± 0.4, 4.3 ± 0.4, and 10.0 ± 0.1, respectively. Treatment with potato acid phosphatase converted the phosphorylated form to the nonphosphorylated form and decreased the binding constant for m7GTP by a factor of3. The increased affinity for mRNA caps may account for the in vivo and in vitro correlations between eIF-4E phosphorylation and accelerated protein synthesis and cell growth.Except in unusual circumstances such as virus infection, cellular stress, or deprivation of nutrients, the overall rate of protein synthesis in eukaryotic cells is thought to be determined by the rate of formation of 48S initiation complexes. This is based on the observations that 48S complexes are present at very low steady-state levels compared with other initiation complexes (1), usually being detected only in the presence of inhibitors (2), and that the ATP-dependent step ofinitiation is rate-limiting (3). Formation ofthe 48S initiation complex involves the binding of mRNA to the 43S initiation complex and is catalyzed by group 4 initiation factors (reviewed in refs. 4-6). The four polypeptides of the eIF-4 group-eIF-4A, eIF-4B, eIF-4E, and eIF-41-collectively bind to the m7GTP-containing cap, unwind mRNA secondary structure at the expense of ATP, and facilitate the movement of the 40S ribosomal subunit along the mRNA until the initiation codon is reached. Various complexes of the eIF-4 polypeptides have been isolated, the best studied of which is termed eIF-4F. eIF-4F isolated from mammalian cells was originally shown to contain the polypeptides eIF-4A, eIF-4E, and eIF-4y (7, 8), but other purifications yield a twocomponent factor consisting of eIF-4E and eIF-4y (9-11). Similarly, eIF-4F and eIF(iso)-4F from plants (12) and ...

Selective Modification of Eukaryotic Initiation Factor 4F (eIF4F) at the Onset of Cell Differentiation: Recruitment of eIF4GII and Long-Lasting Phosphorylation of eIF4E

“…These data are quite surprising in light of previous reports where phosphorylation of Ser209 was most often correlated with cell growth in mammalian cells and was also shown to be critical for growth in Drosophila in toto (19). Although it was first reported that phosphorylation of eIF4E enhanced its affinity for the cap (25), reconstitution experiments of eIF4F complexes with purified molecules indicated that phosphorylation of eIF4E was not a prerequisite for complex formation with eIF4G (28), it was not required for translation (27), and it even reduced cap binding in vitro (43). Nevertheless, phosphorylation of Ser209 may still modulate a number of other protein-protein interactions that can affect the multiprotein 43S or 48S preinitiation complex, since the binding of eIF4E to eIF4G per se is known to change eIF4GI conformation, rendering it accessible to proteolytic cleavage (13,28).…”

The effect of p-4E-BP1 and p-eIF4E on cell proliferation in a breast cancer model

“…Previously it was reported that p-eIF4E binds more efficiently to capped mRNAs (17), suggesting that the affinity of phosphorylated eIF4E for 4E-BP1 is decreased. Moreover, Chen et al (19) reported that p-eIF4E binds less well to 4E-BP1 than the unphosphorylated form.…”

“…Although eIF4E phosphorylation on Ser209 was discovered some time ago, it remains unclear whether phosphorylation increases or decreases eIF4E affinity for the cap-structure. Early data indicated that eIF4E phosphorylation increased the affinity for capped mRNAs, whereas other studies have shown that eIF4E phosphorylation actually decreases affinity for the cap-structure and capped oligoribonucleotides (15,(17)(18)(19)(20).…”

Starting the protein synthesis machine: eukaryotic translation initiation

“…It has long been thought that phosphorylation increases the capbinding affinity of eIF4E. (49) Recent studies have now challenged this view (50,51) (discussed in Ref. 48).…”