The C terminus of initiation factor 4E-binding protein 1 contains multiple regulatory features that influence its function and phosphorylation

Eukaryotic initiation factor 4E (eIF4E) binds the mRNA cap structure and forms eIF4F complexes that recruit 40S subunits to the mRNA. Formation of eIF4F is blocked by eIF4E-binding proteins such as 4E-BP1, which interacts with eIF4E via a motif in the center of its 118-residue sequence. 4E-BP1 plays key roles in cell proliferation, growth, and survival. Binding of 4E-BP1 to eIF4E is regulated by hierarchical multisite phosphorylation. Here we demonstrate that three different features in the C terminus of 4E-BP1 play distinct roles in regulating its phosphorylation and function. Firstly, we identify a new phosphorylation site in its C terminus (S101). A serine or glutamate at this position is required for efficient phosphorylation at Ser65. A second C-terminal site, S112, directly affects binding of 4E-BP1 to eIF4E without influencing phosphorylation of other sites. Thirdly, a conserved C-terminal motif influences phosphorylation of multiple residues, including rapamycin-insensitive sites. These relatively long-range effects are surprising given the reportedly unstructured nature of 4E-BP1 and may imply that phosphorylation of 4E-BP1 and/or binding to eIF4E induces a more-ordered structure. 4E-BP2 and -3 lack phosphorylatable residues corresponding to both S101 and S112. However, in 4E-BP3, replacement of the alanine at the position corresponding to S112 by serine or glutamate did not confer the ability to be released from eIF4E in response to insulin.     

Phosphorylation dynamics of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) is discordant with its potential to interact with eukaryotic initiation factor 4E (eIF4E)

Mammalian target of rapamycin (mTOR) controls cellular growth and proliferation by virtue of its ability to regulate protein translation. Eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) — a key mTOR substrate, binds and sequesters eIF4E to impede translation initiation that is supposedly overcome upon 4E-BP1 phosphorylation by mTOR. Ambiguity surrounding the precise identity of mTOR regulated sites in 4E-BP1 and their invariable resistance to mTOR inactivation raises concerns about phospho-regulated model proposed for 4E:4E-BP1 interaction. Our attempt to mimic dephosphorylation associated with rapamycin response by introducing phospho deficient mutants for sites implicated in regulating 4E:4E-BP1 interaction individually or globally highlighted no obvious difference in the quantum of their association with CAP bound 4E when compared with their phosphomimicked counterparts or the wild type 4E-BP1. TOS or RAIP motif deletion variants compromised for raptor binding and resultant phosphodeficiency did little to influence their association with CAP bound 4E. Interestingly ectopic expression of ribosomal protein S6 kinase 1 (S6K1) that restored 4E-BP1 sensitivity to rapamycin/Torin reflected by instant loss of 4E-BP1 phosphorylation, failed to bring about any obvious change in 4E:4E-BP1 stoichiometry. Our data clearly demonstrate a potential disconnect between rapamycin response of 4E-BP1 and its association with CAP bound 4E.     

Mutually cooperative binding of eukaryotic translation initiation factor (eIF) 3 and eIF4A to human eIF4G-1

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. The central region of eIF4G binds the ATP-dependent RNA helicase eIF4A, the 40 S binding factor eIF3, and RNA. In the present work, we have further characterized the binding properties of the central region of human eIF4G. Both titration and competition experiments were consistent with a 1:1 stoichiometry for eIF3 binding. Surface plasmon resonance studies showed that three recombinant eIF4G fragments corresponding to amino acids 642-1560, 613-1078, and 975-1078 bound eIF3 with similar kinetics. A dissociation equilibrium constant of approximately 42 nm was derived from an association rate constant of 3.9 x 10(4) m(-1) s(-1) and dissociation rate constant of 1.5 x 10(-3) s(-1). Thus, the eIF3-binding region is included within amino acid residues 975-1078. This region does not overlap with the RNA-binding site, which suggests that eIF3 binds eIF4G directly and not through an RNA bridge, or the central eIF4A-binding site. Surprisingly, the binding of eIF3 and eIF4A to the central region was mutually cooperative; eIF3 binding to eIF4G increased 4-fold in the presence of eIF4A, and conversely, eIF4A binding to the central (but not COOH-terminal) region of eIF4G increased 2.4-fold in the presence of eIF3.     

Assignment of two of the translation initiation factor-4E (EIF4EL1 and EIF4EL2) genes to human chromosomes 4 and 20

The eukaryotic translation initiation factor (eIF-4E) has recently been cloned from human, mouse, and yeast. This polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis. We have designed oligonucleotide primers to the 3' untranslated region of the gene encoding eIF-4E and specifically amplified the human gene in human/rodent somatic cell hybrids using the polymerase chain reaction. By this method, one of the human eIF-4E genes (EIF4EL1, eukaryotic translation initiation factor 4E-like 1) has been mapped to human chromosome 4qter-4p15. In addition, we have localized a second eIF-4E gene (EIF4EL2, eukaryotic translation initiation factor 4E-like 2) to human chromosome 20 by Southern blot analysis of mapping panels established from human/rodent somatic cell hybrids.     

FebA: a gene for eukaryotic translation initiation factor 4E-binding protein (4E-BP) in Dictyostelium discoideum

We have identified a gene encoding a eukaryotic initiation factor 4E-binding protein (4E-BP) in the EST database of the Dictyostelium cDNA project. The Dictyostelium 4E-BP, designated febA (four e-binding), showed significant similarity to mammalian 4E-BPs. Northern blot analysis revealed that febA was expressed at a high level in the vegetative growth phase but the level of expression decreased during late development. The gene was shown to be non-essential since disruption of the gene had no severe effect; the null mutant proliferated normally and formed normal fruiting bodies. However, strains overexpressing the gene could not be established, suggesting that an excess of FebA protein may have a lethal effect on the cells.     

The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5' secondary structure

Eukaryotic initiation factor (elF) 4A functions as a subunit of the initiation factor complex elF4F, which mediates the binding of mRNA to the ribosome. elF4A possesses ATPase and RNA helicase activities and is the prototype for a large family of putative RNA helicases (the DEAD box family). It is thought that the function of elF4A during translation initiation is to unwind the mRNA secondary structure in the 5' UTR to facilitate ribosome binding. However, the evidence to support this hypothesis is rather indirect, and it was reported that elF4A is also required for the translation of mRNAs possessing minimal 5' UTR secondary structure. Were this hypothesis correct, the requirement for elF4A should correlate with the degree of mRNA secondary structure. To test this hypothesis, the effect of a dominant-negative mutant of mammalian elF4A on translation of mRNAs with various degrees of secondary structure was studied in vitro. Here, we show that mRNAs containing stable secondary structure in the 5' untranslated region are more susceptible to inhibition by the elF4A mutant. The mutant protein also strongly inhibits translation from several picornavirus internal ribosome entry sites (IRES), although to different extents. UV crosslinking of elF4F subunits and elF4B to the mRNA cap structure is dramatically reduced by the elF4A mutant and RNA secondary structure. Finally, the elF4A mutant forms a more stable complex with elF4G, as compared to the wild-type elF4A, thus explaining the mechanism by which substoichiometric amounts of mutant elF4A inhibit translation.     

Evidence that eukaryotic initiation factor (eIF) 2 is a cap-binding protein that stimulates cap recognition by eIF-4B and eIF-4F

We studied the mRNA-binding properties of eukaryotic initiation factor (eIF) 2. This Met-tRNA-binding factor interacts with the cap structure of reoviral mRNA in an ATP-independent manner. Both the beta- and gamma-subunit of eIF-2 are involved in the UV-induced cross-linking of eIF-2 to the cap. The interaction of eIF-2 with a messenger is sensitive to the cap analogue 7-methyl-guanosine 5'-triphosphate as measured by cross-linking and by mRNA retention on nitrocellulose filters. The cap-binding property of eIF-2 does not conflict with the current mRNA-binding model of initiation factors eIF-4A, -4B, and -4F: cross-linking of eIF-4E and of eIF-4B is stimulated by eIF-2. The eIF-2-mediated increase of eIF-4E interaction results in a decrease of the cross-linking of the beta- and gamma-subunits of eIF-2. The presence of GTP in the cross-linking assay interferes with the interaction of eIF-2 with the cap structure but does not inhibit the eIF-2 stimulated eIF-4E and -4B cross-linking. These observations indicate a role for eIF-2 in the mRNA recognition.     

Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E.

Human eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA cap structure and interacts with eIF4G, which serves as a scaffold protein for the assembly of eIF4E and eIF4A to form the eIF4F complex. eIF4E is an important modulator of cell growth and proliferation. It is the least abundant component of the translation initiation machinery and its activity is modulated by phosphorylation and interaction with eIF4E-binding proteins (4E-BPs). One strong candidate for the eIF4E kinase is the recently cloned MAPK-activated protein kinase, Mnk1, which phosphorylates eIF4E on its physiological site Ser209 in vitro. Here we report that Mnk1 is associated with the eIF4F complex via its interaction with the C-terminal region of eIF4G. Moreover, the phosphorylation of an eIF4E mutant lacking eIF4G-binding capability is severely impaired in cells. We propose a model whereby, in addition to its role in eIF4F assembly, eIF4G provides a docking site for Mnk1 to phosphorylate eIF4E. We also show that Mnk1 interacts with the C-terminal region of the translational inhibitor p97, an eIF4G-related protein that does not bind eIF4E, raising the possibility that p97 can block phosphorylation of eIF4E by sequestering Mnk1.     

The human platelet-activating factor receptor gene (PTAFR) contains no introns and maps to chromosome 1.

Platelet-activating factor (PAF), a phospholipid, exhibits a variety of potent inflammatory bioactivities that are mediated by a specific cell surface receptor. The gene for the human PAF receptor (PTAFR) has been isolated by hybridization with a guinea pig probe. The coding sequence contains no intervening sequences. The encoded protein is highly homologous to the guinea pig PAF receptor (82% identity) and contains seven putative transmembrane domains. The PAF receptor therefore appears to be a member of the G protein coupled family of receptors and exhibits significant similarity to many members of the family. Analysis of somatic cell hybrids suggests that the PAF receptor is encoded by a single gene on human chromosome 1.     

Cap-binding protein (eukaryotic initiation factor 4E) and 4E-inactivating protein BP-1 independently regulate cap-dependent translation.

Cap-dependent protein synthesis in animal cells is inhibited by heat shock, serum deprivation, metaphase arrest, and infection with certain viruses such as adenovirus (Ad). At a mechanistic level, translation of capped mRNAs is inhibited by dephosphorylation of eukaryotic initiation factor 4E (eIF-4E) (cap-binding protein) and its physical sequestration with the translation repressor protein BP-1 (PHAS-I). Dephosphorylation of BP-I blocks cap-dependent translation by promoting sequestration of eIF-4E. Here we show that heat shock inhibits translation of capped mRNAs by simultaneously inducing dephosphorylation of eIF-4E and BP-1, suggesting that cells might coordinately regulate translation of capped mRNAs by impairing both the activity and the availability of eIF-4E. Like heat shock, late Ad infection is shown to induce dephosphorylation of eIF-4E. However, in contrast to heat shock, Ad also induces phosphorylation of BP-1 and release of eIF-4E. BP-1 and eIF-4E can therefore act on cap-dependent translation in either a mutually antagonistic or cooperative manner. Three sets of experiments further underscore this point: (i) rapamycin is shown to block phosphorylation of BP-1 without inhibiting dephosphorylation of eIF-4E induced by heat shock or Ad infection, (ii) eIF-4E is efficiently dephosphorylated during heat shock or Ad infection regardless of whether it is in a complex with BP-1, and (iii) BP-1 is associated with eIF-4E in vivo regardless of the state of eIF-4E phosphorylation. These and other studies establish that inhibition of cap-dependent translation does not obligatorily involve sequestration of eIF-4E by BP-1. Rather, translation is independently regulated by the phosphorylation states of eIF-4E and the 4E-binding protein, BP-1. In addition, these results demonstrate that BP-1 and eIF-4E can act either in concert or in opposition to independently regulate cap-dependent translation. We suggest that independent regulation of eIF-4E and BP-1 might finely regulate the efficiency of translation initiation or possibly control cap-dependent translation for fundamentally different purposes.     

Crystallization and preliminary crystallographic analysis of human eukaryotic translation initiation factor 5A (eIF-5A)

Eukaryotic translation initiation factor 5A (eIF-5A) is universally found in all eukaryotic cells. It is the only protein in nature known to contain the unusual amino acid hypusine, a post-translationally modified lysine. Recombinant human eIF-5A was crystallized by the hanging-drop vapor diffusion method. Crystals were grown at 291 K using (NH4)2SO4 as precipitant. Diffraction data were obtained to a resolution of 2.7 A from a single frozen crystal belonging to space group C2, with unit-cell parameters a = 147.1 A, b = 60.4 A, c = 76.4 A, beta = 92.4 degrees. There are more than three molecules per asymmetric unit.