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.     

Phosphorylation of eIF-4F by protein kinase C or multipotential S6 kinase stimulates protein synthesis at initiation

Eukaryotic initiation factor (eIF) 4F, a multiprotein cap binding complex, has been shown to be phosphorylated in vivo in response to phorbol 12-myristate 13-acetate and insulin (Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 264, 2401-2404; Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 265, 10611-10616). The effect of phosphorylation on the activity of purified eIF-4F, utilizing both protein kinase C and a multifunctional S6 kinase, previously identified as protease activated kinase II, has been examined; these protein kinases modify eIF-4F p25 and p220 and eIF-4F p220, respectively. Studies with an eIF-4F-dependent protein synthesis system showed that phosphorylation of eIF-4F with either protein kinase resulted in a 3-5-fold stimulation of translation relative to the nonphosphorylated control. Chemical cross-linking of eIF-4F to cap-labeled mRNA, showed that phosphorylation increased the interaction of both the p25 and p220 subunits of eIF-4F with the 5' end of mRNA. This effect was manifested by a stimulation of initiation complex formation as measured by an increase in the association of labeled mRNA with 40 S ribosomal subunits in the translation system. Thus, phosphorylation of eIF-4F enhances binding to mRNA, resulting in a stimulation of protein synthesis at initiation.     

Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation.

Eukaryotic translation initiation factor-4A (eIF-4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA-dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA-ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF-4F. To gain further understanding into the mechanism of action of eIF-4A in mRNA-ribosome binding, defective eIF-4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF-4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap-independent internal initiation mechanism. Addition of eIF-4A or eIF-4F relieved inhibition of translation, but eIF-4F was six times more effective than eIF-4A, whereas eIF-4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF-4A is defective in recycling through eIF-4F, thus explaining the dramatic inhibition of translation. Mutant eIF-4A proteins also inhibited eIF-4F-dependent, but not eIF-4A-dependent RNA helicase activity. Taken together these results suggest that eIF-4A functions primarily as a subunit of eIF-4F, and that singular eIF-4A is required to recycle through the complex during translation. Surprisingly, eIF-4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs.     

Interaction of wheat germ protein synthesis initiation factors eIF-3, eIF-(iso)4F, and eIF-4F with mRNA analogues

The interaction of wheat germ eIF-3 with the wheat germ cap-binding proteins eIF-(iso)4F and eIF-4F as a function of pH and ionic strength is described. Direct fluorescence titration experiments are used to measure the equilibrium association constants (Keq) for the binary protein/protein complexes as well as for the interaction of eIF-3 with methylated cap analogues and rabbit alpha-globin mRNA oligonucleotide analogues. The Keq values for ternary eIF-3/eIF-(iso)4F/analogue and eIF-3/eIF-4F/analogue interactions were also measured. The equilibrium binding constants were used to calculate coupling free energies, which provide an estimate of the cooperativity for the interaction of the mRNA analogues, eIF-3, and either eIF-4F or eIF-(iso)4F. These data suggest a mechanism in which the binding of eIF-(iso)4F or eIF-4F to mRNA enhances the subsequent binding of eIF-3 to the message. This may lead to favorable positioning of the complex on the ribosome and thereby enhance translation.     

Phosphorylation of translational initiation factor 3 (eIF-3) by cyclic AMP-regulated protein kinase.

Translational initiation factor 3 (eIF-3) is phosphorylated by the cyclic AMP-regulated protein kinases from rabbit reticulocytes. eIF-3 is a large molecular weight complex which facilitates binding of the ternary complex containing met tRNA_f, GTP and initiation factor 2 to 40S ribosomal subunits. A single polypeptide with a molecular weight of 130,000 is modified. The phosphorylation is dependent upon the presence of cyclic AMP and is inhibited by the inhibitor protein diagnostic for cyclic AMP-regulated protein kinase. Assuming a molecular weight of 700,000 for eIF-3, one mole of phosphate is incorporated per mole of eIF-3. Thus the phosphorylation of two interacting components of the protein synthesizing system, 40S ribosomal subunits and eIF-3, is controlled by cyclic AMP.     

Differential stimulation of phosphorylation of initiation factors eIF-4F, eIF-4B, eIF-3, and ribosomal protein S6 by insulin and phorbol esters

Exposure of quiescent, serum-starved 3T3-L1 cells to insulin promotes phosphorylation of initiation factors eIF-4F, eIF-4B, and eIF-3 p120, as well as ribosomal protein S6. Phosphorylation of both the p25 and p220 subunits of eIF-4F is stimulated typically by 2.5-5-fold, with a 2-4-fold increase in phosphorylation of eIF-4B and eIF-3 p120. Optimal stimulation is observed by 10(-9) M insulin. A similar pattern of stimulation is seen upon treatment of 3T3-L1 cells with 1 x 10(-6) M phorbol 12-myristate 13-acetate (PMA). Two-dimensional phosphopeptide mapping of p25, isolated from quiescent, insulin- or PMA-stimulated cells, results in a single tryptic phosphopeptide, indicating a single phosphorylation site identical to that obtained with protein kinase C. A more complex phosphopeptide map is observed with the p220 subunit. Following PMA-stimulation of 3T3-L1 cells, phosphopeptide mapping of p220 results in a pattern similar to that observed in vitro with Ca2+/phospholipid-dependent protein kinase (protein kinase C). Following insulin stimulation, mapping of p220 results in the appearance of novel peptides. Upon prolonged exposure to PMA, the cells are no longer responsive to this mitogen and no stimulation of phosphorylation of eIF-4F, eIF-4b, eIF-3 p120, or S6 via a protein kinase C-dependent mechanism is observed. Addition of insulin to these down-regulated cells leads to stimulation of phosphorylation of eIF-4F p220, ribosomal protein S6, and to a lesser extent, eIF-4B; little or no stimulation of phosphorylation of eIF-4F p25 and eIF-3 p120 is observed. Thus, eIF-4F p220, eIF-4B and ribosomal protein S6 are phosphorylated via PMA-dependent and insulin-dependent pathways, whereas phosphorylation of eIF-4F p25 and eIF-3 p120 is stimulated only upon activation of protein kinase C. Phosphopeptide maps of eIF-4F p220 and ribosomal protein S6 suggest that protease-activated kinase II is one of the protein kinases involved in the insulin-stimulated response in protein kinase C-depleted cells.     

Separation of protein synthesis initiation factor eIF4A from a p220-associated cap binding complex activity

A cap binding complex activity was purified from HeLa cells by a procedure which does not depend on the use of cap-affinity chromatography. The activity co-purified with a Mr 220000 polypeptide (p220), but not with elF4A. The active complex therefore differs from eIF4F, the complex purified by cap analog-affinity chromatography, in that it lacks the Mr 50000 subunit which is antigenically identical to elF4A. The activities of elF4F, CBP I and the eIF4A free complex purified here were compared in a fractionated system translating capped globin mRNA. Results indicate that the two complexes have similar activities and that they perform a function which cannot be provided by CBP I alone. Cap binding complex activity can be partly separated from eIF4A activity on sucrose gradients, thus eIF4A provides a function that is distinct from cap binding complex activity. The results indicate that eIF4A can be physically separated from the cap binding complex without affecting the ability of the remaining structure to function in an in vitro translation system. They suggest that the eIF4A-free complex may provide a function that is not a property of either CBP I or of eIF4A, but may be a property of p220.     

Thermodynamics of mRNA 5' cap binding by eukaryotic translation initiation factor eIF4E

Translation of mRNA in eukaryotes begins with specific recognition of the 5' cap structure by the highly conserved protein, eIF4E. The thermodynamics of eIF4E interaction with nine chemical cap analogues has been studied by means of emission spectroscopy. High-sensitivity measurements of intrinsic protein fluorescence quenching upon cap binding provided equilibrium association constants in the temperature range of 279 to 314 K. A van't Hoff analysis yielded the negative binding enthalpies for the entire cap analogue series, -16.6 to -81 kJ mol(-1), and the entropies covering the range of +40.3 to -136 J mol(-1) K(-1) at 293 K. The main enthalpic contributions come from interactions of the phosphate chains and positively charged amino acids and the cation-pi stacking of 7-methylguanine with tryptophans. A nontrivial, statistically important isothermal enthalpy-entropy compensation has been detected (T(c) = 399 +/- 24 K), which points to significant fluctuations of apo-eIF4E and indicates that the cap-binding microstate lies 9.66 +/- 1.7 kJ mol(-1) below the mean energy of all available conformational states. For five cap analogues, large and positive heat capacity changes have been found. The values of DeltaC(p) degrees correlate with the free energies of eIF4E binding due to stiffening of the protein upon interaction with cap analogues. At biological temperatures, binding of the natural caps has both favorable enthalpy and favorable entropy. Thermodynamic coupling of cap-eIF4E association to intramolecular self-stacking of dinucleotide cap analogues strongly influences the enthalpies and entropies of the binding, but has a negligible effect on the resultant DeltaG degrees and DeltaC(p) degrees values.     

Phosphorylation of the p220 subunit of eIF-4F by cAMP dependent protein kinase and protein kinase C in vitro

Changes in the extent of phosphorylation of the 25 kDa subunit of eIF-4F occur during several major biological events including mitosis and heat shock in mammalian cells and shortly after fertilization of sea urchin (Lytechinus pictus) eggs. In vitro phosphorylation studies using highly purified protein kinases demonstrated that the 220 kDa subunit of eIF-4F was phosphorylated by cAMP dependent protein kinase, protein kinase C and probably to a lesser extent by cGMP dependent protein kinase. In addition, eIF-4A was readily phosphorylated by cAMP and cGMP dependent protein kinases whereas p48 of eIF-4F was not. The effect of these phosphorylation events on eIF-4F function, its assembly or disassembly, susceptibility to viral initiated proteolysis or the ability of p25 to be phosphorylated at serine-53 remain to be investigated.     

Phosphorylation of eukaryotic translation initiation factor 4E is critical for growth

Eukaryotic translation initiation factor 4E (eIF4E) binds to the cap structure at the 5' end of mRNAs and is a critical target for the control of protein synthesis. eIF4E is phosphorylated in many systems in response to extracellular stimuli, but biochemical evidence to date has been equivocal as to the biological significance of this modification. Here we use a genetic approach to this problem. We show that, in Drosophila melanogaster, homozygous eIF4E mutants arrest growth during larval development. In Drosophila eIF4EI, Ser251 corresponds to Ser209 of mammalian eIF4E, which is phosphorylated in response to extracellular signals. We find that, in vivo, eIF4EI Ser251 mutants cannot incorporate labeled phosphate. Furthermore, transgenic Drosophila organisms expressing eIF4E(Ser251Ala) in an eIF4E mutant background have reduced viability. Escapers develop more slowly than control siblings and are smaller. These genetic data provide evidence that eIF4E phosphorylation is biologically significant and is essential for normal growth and development.     

A comparison of the binding of methylated cap analogues to wheat germ protein synthesis initiation factors 4F and (iso)4F

The binding of the 5'-terminal cap analogues m7GpppG and m7GTP to wheat germ protein synthesis initiation factors eIF-4F and eIF-(iso)4F as a function of pH, ionic strength, and temperature is described. Equilibrium binding data indicate that eIF-4F and eIF-(iso)4F have different mechanisms for interacting with the 5'-cap structure, but the complexes formed between m7GpppG and wheat germ factor eIF-(iso)4F more closely resemble complexes formed between this cap analogue and either mammalian eIF-4E or eIF-4F. The binding of these initiation factors to the hypermethylated cap analogues m2,7GMP, m2,7GpppG, and m2,2,7GpppG is also investigated. The differences in affinity of eIF-4F and eIF-(iso)4F for the hypermethylated 5'-terminal cap structures suggest that these factors may have discriminatory activity.     

Insulin promoted decrease in the phosphorylation of protein synthesis initiation factor eIF-2

Insulin stimulates cellular protein synthesis in calf chondrocytes in suspension culture. This enhanced synthetic activity is seen in association with a decrease in phosphorylation of the α subunit of protein synthesis initiation factor eIF-2. [³²P] associated with the α subunit is reduced approximately 50% by insulin treatment of chondrocytes incubated in [³²P] containing media. Identical or closely located amino acids in the eIF-2 α subunit are phosphorylated by the chondrocyte kinase(s) and the rabbit reticulocyte hemin regulated kinase as indicated by comparative peptide fragment analysis of [³²P] labeled α subunits.     

Translational control by neuroguidin, a eukaryotic initiation factor 4E and CPEB binding protein

CPEB-mediated translation is important in early development and neuronal synaptic plasticity. Here, we describe a new eukaryotic initiation factor 4E (eIF4E) binding protein, Neuroguidin (Ngd), and its interaction with CPEB. In the mammalian nervous system, Ngd is detected as puncta in axons and dendrites and in growth cones and filopodia. Ngd contains three motifs that resemble those present in eIF4G, 4EBP, Cup, and Maskin, all of which are eIF4E binding proteins. Ngd binds eIF4E directly, and all three motifs must be deleted to abrogate the interaction between these two proteins. In injected Xenopus oocytes, Ngd binds CPEB and, most importantly, represses translation in a cytoplasmic polyadenylation element (CPE)-dependent manner. In Xenopus embryos, Ngd is found in both neural tube and neural crest cells. The injection of morpholino-containing antisense oligonucleotides directed against ngd mRNA disrupts neural tube closure and neural crest migration; however, the wild-type phenotype is restored by the injection of a rescuing ngd mRNA. These data suggest that Ngd guides neural development by regulating the translation of CPE-containing mRNAs.     

Purification and characterization of protein synthesis initiation factor eIF-4E from the yeast Saccharomyces cerevisiae

A 24 000-dalton protein [yeast eukaryotic initiation factor 4E (eIF-4E)] was purified from yeast Saccharomyces cerevisiae postribosomal supernatant by m7GDP-agarose affinity chromatography. The protein behaves very similarly to mammalian protein synthesis initiation factor eIF-4E with respect to binding to and elution from m7GDP-agarose columns and cross-linking to oxidized reovirus mRNA cap structures. Yeast eIF-4E is required for translation as shown by the strong and specific inhibition of cell-free translation in a yeast extract by a monoclonal antibody directed against yeast eIF-4E.     

Binding analysis of Xenopus laevis translation initiation factor 4E (eIF4E) in initiation complex formation.

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.     

Association of protein kinase CK2 with eukaryotic translation initiation factor eIF-2 and with grp94/endoplasmin

Protein kinase CK2 forms complexes with some protein substrates what may be relevant for the physiological control of this protein kinase. In previous studies in rat liver cytosol we had detected that the trimeric form of eukaryotic translation initiation factor 2 (eIF-2) co-eluted with protein kinase CK2. We have now observed that the ratio between eIF-2 and cytosolic CK2 contents in testis, liver and brain is quite similar, being eIF-2 levels about 5-fold higher than those of CK2. Furthermore eIF-2 was present in liver samples immunoprecipitated with anti-CK2/ antibodies, confirming the existence of complexes containing both proteins. Nonetheless, these complexes would represent only a fraction of total cytosolic CK2 and eIF-2.We had also observed that rat liver membrane glycoproteins obtained through chromatography on wheat-germ lectin-Sepharose contain CK2 activity which copurifies with grp94/endoplasmin. We have now confirmed that this activity was due to the presence of protein kinase CK2 as evidenced by immunodetection with antibodies against CK2/. The fractions enriched in grp94/endoplasmin and CK2 also contained another 55-kDa polypeptide (p55) phosphorylated by CK2 which has been identified as calreticulin by N-terminal sequencing. Calreticulin and grp94/endoplasmin could be partially resolved from CK2 by chromatography on heparin-agarose and almost completely on ConA-Sepharose. However, phosphorylation of immunoprecipitated grp94/endoplasmin was enhanced by its preincubation with purified CK2 prior to immunoprecipitation, what confirms the easy reassociation between these proteins.The association of protein kinase CK2 with eIF-2 and with grp94/endoplasmin may serve to locate the enzyme in the cellular machinery involved in protein synthesis and folding, and reinforces the possible involvement of CK2 in these processes.