The cap-binding translation initiation factor eukaryotic initiation factor 4E (eIF4E) is phosphorylated in vivo at Ser209 in response to a variety of stimuli. In this paper, we show that the mitogen-activated protein kinase (MAPK) signal-integrating kinase Mnk2 phosphorylates eIF4E at this residue. Mnk2 binds to the scaffolding protein eIF4G, and overexpression of Mnk2 results in increased phosphorylation of endogenous eIF4E, showing that it can act as an eIF4E kinase in vivo. We have identified eight phosphorylation sites in Mnk2, of which at least three potential MAPK sites are likely to be essential for Mnk2 activity. In contrast to that of Mnk1, the activity of overexpressed Mnk2 is high under control conditions and could only be reduced substantially by a combination of PD98059 and SB203580, while the activity of endogenous Mnk2 in Swiss 3T3 cells was hardly affected upon treatment with these inhibitors. These compounds did not abolish phosphorylation of eIF4E, implying that Mnk2 may mediate phosphorylation of eIF4E in Swiss 3T3 cells. In vitro phosphorylation studies show that Mnk2 is a significantly better substrate than Mnk1 for extracellular signal-regulated kinase 2 (ERK2), p38MAPK␣, and p38MAPK␤. Therefore, the high levels of activity of Mnk2 under several conditions may be explained by efficient activation of Mnk2 by low levels of activity of the upstream kinases. Interestingly, we found that the association of both Mnk1 and Mnk2 with eIF4G increased upon inhibition of the MAPK pathways while activation of ERK resulted in decreased binding to eIF4G. This might reflect a mechanism to ensure rapid, but transient, phosphorylation of eIF4E upon stimulation of the MAPK pathways.

Regulation of eukaryotic translation initiation is a process that requires collaboration between multiple proteins. The cap-binding factor eukaryotic initiation factor (eIF)4E, its binding protein 4E-BP1, and the guanine-nucleotide-exchange factor eIF2B play important roles in the regulation of the rate of protein synthesis. This review describes the regulation of the activity of these three proteins and the signaltransduction pathways involved therein.Keywords : signal transduction; translation initiation; eukaryotic initiation factor 4E; initiation-factor-4E-binding protein; eukaryotic initiation factor 2B ; mitogen-activated protein kinase; FK506-binding protein/ rapamycin-associated protein; mammalian target of rapamycin ; inositolphospholipid 3-kinase.Signalling between cells is crucial for development of multi-volved therein will be discussed. The picture that arises in this review is that changes in protein synthesis are probably not cellular organisms. Cells communicate in order to organise growth, migration and differentiation. Communication, or signal achieved by changes in the activity of a single initiation factor, but rather by simultaneous activation or inhibition of a number transduction, starts with binding of a ligand, such as hormones or growth factors, to the extracellular part of a receptor. The of initiation factors. signal is conveyed to the intracellular part of the receptor, often by autophosphorylation induced by ligand binding, then the signal is transmitted further to various cellular processes. Abroga-The role of initiation factors in protein synthesis tion of intracellular or extracellular communication can be a Translation initiation. Eukaryotic translation initiation and cause for growth and differentiation abnormalities such as those the factors involved have been reviewed recently [2], and therethat occur during embryo development and in cancer, diabetes fore will be described only briefly. In Fig. 1 the main steps in and disorders of the immune and cardiovascular systems.translation initiation are depicted. The cap-binding protein Over the years it has become evident that changes in signaleIF4E [3,4] is obligatory for the start of cap-dependent translaling pathways affect the rate of protein synthesis and the translation initiation. eIF4E is activated by release from its binding tion of a specific subset of mRNAs. Initiation of protein syntheprotein, 4E-BP1, and by phosphorylation. The 5′ cap structure of sis is regulated by many translation initiation factors (eIF), and an mRNA [7-methylguanosine(5′)triphospho(5′)-ribonucleoside] most of the known initiation factors are phosphoproteins [1].is recognized by eIF4E, and the eIF4F complex is formed, either However, for the majority of the factors the significance of phosin solution or at the cap, by assembly of a trimeric complex phorylation for their activity is not known. In this review the between eIF4E, eIF4A, an ATP-dependent RNA helicase [5Ϫ7], regulation of two initiation factors, eIF4E and eIF2B, and of the and eIF4G. eIF4G...

The regulation of protein synthesis and of eukaryotic initiation factor eIF2B was studied in PC12 cells. An increase in protein synthesis was observed after nerve growth factor (NGF) and epidermal growth factor (EGF) treatment of PC12 cells, and this increase coincided with activation of eIF2B. Growth factor addition in the presence of the phosphatidylinositol-3-OH kinase inhibitor wortmannin showed that both NGF-and EGFinduced protein synthesis and eIF2B activation were phosphatidylinositol-3-OH kinase dependent. The EGFinduced stimulation of protein synthesis and activation of eIF2B was dependent upon FK506-binding proteinrapamycin-associated protein, as shown with the immunosuppressant rapamycin, whereas NGF induction was partially dependent upon FK506-binding protein-rapamycin-associated protein.The activities of two kinases that act on eIF2B, glycogen synthase kinase-3 and casein kinase II, were measured to assess their potential roles in the activation of eIF2B in PC12 cells. Inactivation of glycogen synthase kinase-3 was seen in response to both NGF and EGF and this coincided with activation of eIF2B. However, inactivation of glycogen synthase kinase-3 was not rapamycin sensitive, in contrast to the activation of eIF2B. This indicates the involvement of another protein kinase or regulatory mechanism in the eIF2B activation. Both growth factors activated casein kinase II. However, the time course of its activation and its insensitivity to wortmannin and rapamycin suggest that casein kinase II does not play a major regulatory role in eIF2B activation under these conditions.

Various factors are involved in the heat shock-induced inhibition of protein synthesis. Changes upon heat shock in phosphorylation, leading to inactivation, of eukaryotic initiation factors (eIFs) eIF2 and eIF4E have been shown for several cell types. However, in mammalian cells these changes occur at temperatures of 43°C or higher while protein synthesis is already affected at milder heat shock temperatures. In searching for the cause for the inhibition of protein synthesis, the regulation of eIF2 and eIF4E by additional factors was analyzed. In this respect, the activity of eIF2B was measured during and after heat shock. A very clear correlation was found between the activity of this guanine exchange factor and the levels of protein synthesis, also at mild heat shock conditions. Changes in the phosphorylation of eIF4E and of the eIF4E-binding protein PHAS-I were also analyzed. Surprisingly, in H35 cells as well as in some other cell lines, PHAS-I phosphorylation was increased by heat shock, whereas in others it was decreased. Therefore, decreasing the eIF4E availability under stressful conditions does not seem to be a general mechanism to inhibit protein synthesis by heat shock. Regulation of eIF2B activity appears to be the main mechanism to control translation initiation after heat shock at mild temperatures.Incubating mammalian cells at elevated temperatures inhibits the translation of mRNAs (1-4). The translational lesion in heat-shocked cells principally occurs at the initiation step of translation (1,(5)(6)(7) for review, see Ref. 8). In the initiation process, the 7-methyl guanosine cap at the 5Ј end of the mRNA is bound by a complex of eukaryotic initiation factors (eIFs), 1 including the cap-binding protein eIF4E and the RNA helicase eIF4A. Unwinding of the RNA enables recognition of the AUG initiation codon by the 43 S ribosomal complex and delivery of initiator methionyl tRNA. Then the large ribosomal subunit joins the complex, and peptide synthesis begins (8).Both the concentration and activity of protein synthesis initiation factors and the primary sequence of the 5Ј-untranslated region of an mRNA can affect the rates of eukaryotic translation initiation. With respect to the heat shock-induced modifications of eukaryotic protein synthesis initiation factors, it is of interest that the activity of many initiation factors can be regulated by phosphorylation. It has been shown that changes in the phosphorylation state of various of these eukaryotic initiation factors, such as eIF2, eIF2B, eIF4B, and eIF4E, could coincide with changes in protein synthesis (9, 10).The proteins eIF2 and eIF2B act to bring Met-tRNA to the 40 S ribosomal subunit: eIF2 by binding to Met-tRNA and GTP, and eIF2B by replacing GDP on eIF2 for GTP. Previous studies from several laboratories have focused on heat shock-induced phosphorylation of eIF2␣ (11-13). However, it was found that less severe heat shocks that inhibit protein synthesis by more than 70% do not elicit this phosphorylation. Duncan and Hershey (13) suggested tha...

The eukaryotic translation initiation factor eIF4E, the cap-binding protein, seems to play an essential role in the establishment of the host shut-off after viral infection. Infection with adenovirus and influenza virus caused dephosphorylation of eIF4E and an involvement of a viral protein was suggested. In this report, we studied several other viruses for their ability to change the phosphorylation state of eIF4E, and we looked for the mechanism of eIF4E dephosphorylation. First, it was shown that after encephalomyocarditis virus (EMCV) and poliovirus infection, dephosphorylation of eIF4E occurred. Dephosphorylation of eIF4E was not observed after Semliki Forest virus and reovirus infection. An artificial increase of the level of phosphorylated eIF4E by treating the cells with the phosphatase inhibitor okadaic acid changed neither the kinetics of EMCV and poliovirus infection, nor that of host shut-off. Infections with the uv-treated EMCV showed that the virus binding or entry into the cell initiates eIF4E dephosphorylation. Besides this entry-induced eIF4E dephosphorylation, dephosphorylation was also induced by blocking protein synthesis with the initiation inhibitor pactamycin, or with the elongation inhibitor cycloheximide. We conclude that eIF4E is dephosphorylated by entry of EMCV, and the effect is strengthened by the decrease in cap-dependent translation.