Phosphorylation of Eukaryotic Initiation Factor 4E Markedly Reduces Its Affinity for Capped mRNA

Abstract

In eukaryotes, a key step in the initiation of translation is the binding of the eukaryotic initiation factor 4E (eIF4E) to the cap structure of the mRNA. Subsequent recruitment of several components, including the small ribosomal subunit, is thought to allow migration of initiation complexes and recognition of the initiation codon. Mitogens and cytokines stimulate the phosphorylation of eIF4E at Ser 209 , but the functional consequences of this modification have remained a major unresolved question. Using fluorescence spectroscopy and surface plasmon resonance techniques, we show that phosphorylation of eIF4E markedly reduces its affinity for capped RNA, primarily due to an increased rate of dissociation. Variant eIF4E proteins harboring negatively charged acidic residues at position 209 also showed decreased binding to capped RNA. Furthermore, a basic residue at position 159 was shown to be essential for cap binding. Although eIF4E-binding protein 1 greatly stabilized binding of phosphorylated eIF4E to capped RNA, in the presence of eIF4E-binding protein 1 the phosphorylated form still dissociated faster compared with nonphopshorylated eIF4E. The implications of our findings for the mechanism of translation initiation are discussed.In eukaryotic cells, all nucleus-encoded mRNAs possess a so-called "cap structure" at their 5Ј-end. This cap consists of a methylated 5Ј-5Ј bound guanosine triphosphate (m 7 GTP) moiety. The cap plays a key role in the translation of the mRNA by permitting recruitment of the eukaryotic initiation factors (eIFs) 1 required for the attachment of the ribosome and correct initiation of translation. The protein that interacts directly with the cap is eIF4E, which forms a complex with the scaffolding protein eIF4G, which in turn recruits other factors. These include eIF4A and eIF4B, which are involved in unwinding secondary structure in the 5Ј-untranslated region of the mRNA, and the poly(A)-binding protein, which by interacting with the 3Ј-tail of the mRNA circularizes it (1, 2). eIF4E also binds to small regulatory proteins termed eIF4E-binding proteins (4E-BPs). These compete with eIF4G for binding to overlapping sites on eIF4E and thus inhibit formation of initiation complexes (3, 4).It has long been known that eIF4E undergoes regulated phosphorylation, and the site has been identified as Ser 209 (5, 6). Phosphorylation of eIF4E is increased by mitogenic stimuli that activate translation (reviewed in Ref. 7) and by cytokines (8, 9). Recently, two kinases were identified that phosphorylate Ser 209 in eIF4E and are targets for the mitogen-activated extracellular signal-regulated kinase and stress/cytokine-activated p38 mitogen-activated protein kinase pathways (9 -13). These enzymes (Mnk1 and Mnk2) also associate with eIF4G in vivo (11,12,14). Despite these advances and the large number of studies on the changes of eIF4E phosphorylation in vivo, the key issue of the effect of phosphorylation on the function of eIF4E has received little attention and has remained a major question in the ...

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).…”

Selective translation of mRNAs in the left ventricular myocardium of the mouse in response to acute pressure overload

“…This process is controlled by eIF4F, a complex composed of three core proteins: 1) eIF4E, which binds to the 7-methylguanosine cap on the 5′-end of mRNA, 2) eIF4A, which functions as an ATP-dependent RNA helicase, and 3) eIF4G, which contains binding sites for eIF4E and eIF4A, and functions as a molecular platform for assembling the eIF4F complex [27]. Affinity of eIF4F for the 7-methylguanosine cap can be modified by phosphorylation of eIF4E on Ser-209, which reduces its cap binding affinity as demonstrated in vitro [28]. Although the precise effects of eIF4E phosphorylation on translational efficiency of specific mRNAs have not been elucidated in cardiac muscle, we have shown in previous studies that eIF4E phosphorylation is increased in response to pressure overload hypertrophy [7,29].…”

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).…”