Gert C. Scheper

Nerve and epidermal growth factor induce protein synthesis and eIF2B activation in PC12 cells.

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 EGF-induced protein synthesis and eIF2B activation were phosphatidylinositol-3′-OH kinase dependent. The EGF-induced stimulation of protein synthesis and activation of eIF2B was dependent upon FK506-binding protein-rapamycin-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.

Inactivation of eIF2B and Phosphorylation of PHAS-I in Heat-shocked Rat Hepatoma Cells

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 43u2009°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.

THE 5' UNTRANSLATED REGION OF ENCEPHALOMYOCARDITIS VIRUS CONTAINS A SEQUENCE FOR VERY EFFICIENT BINDING OF EUKARYOTIC INITIATION FACTOR EIF-2/2B

The mechanism by which internal ribosomal binding on the picornaviral RNA takes place is still not known. An important role has been suggested for eukaryotic initiation factors eIF-4A, eIF-4B, as well as for some not yet defined trans-acting factors like p52 for poliovirus and p58 for encephalomyocarditis virus (EMCV). In this paper we describe the competition between the 5 untranslated region (UTR) of EMCV and globin mRNA for the translational apparatus in rabbit reticulocyte lysates and show that the factor that is competed for is eIF-2/2B. The EMC 5 UTR is a very strong inhibitor of globin synthesis in the rabbit reticulocyte lysate because of a 30-fold higher eIF-2/2B binding capacity. Mutations 100 to 140 nucleotides upstream of the initiation codon led to a decreased efficiency to initiate translation and to a decreased ability to inhibit globin mRNA translation. The results suggest an important role for eIF-2/2B binding in EMC RNA translation and therefore in internal initiation.

Regulation of the activity of eukaryotic initiation factors in stressed cells.

Exposure of cells to elevated temperatures, also known as heat shock, or exposure to other kinds of environmental stress, gives rise to a general inhibition of protein synthesis, on the one hand, and to the increased synthesis of a group of chaperone proteins, on the other hand (Rhoads and Lamphear 1995; Duncan 1996). These chaperone proteins or heat shock proteins (HSPs) function mainly in folding and refolding of denatured or newly synthesized proteins and therefore counteract the toxic effects of denatured proteins on the cell (Wu 1995). The inhibition of protein synthesis is achieved by a reduced initiation of translation through the inactivation of various eukaryotic initiation factors (eIFs; Duncan 1996). Most studies have implicated the inactivation of eIF2, which is involved in the delivery of the initiator methionyl tRNA to the small ribosomal subunit, and the inactivation of eIF4E, a key component in the binding of several initiation factors to the mRNA, as the main causes of the reduced levels of initiation (e.g., Duncan and Hershey 1989; Duncan et al. 1995; Feigenblum and Schneider 1996; Scheper et al. 1997; Wang et al. 1998)

Binding of eukaryotic initiation factor-2 and trans-acting factors to the 5′ untranslated region of encephalomyocarditis virus RNA

The encephalomyocarditis virus 5 untranslated region (EMC 5 UTR) has a binding site for eukaryotic initiation factor eIF-2. Mutations in the 3 end or deletion of the 5 end of the internal ribosomal entry site had a negative effect on the binding of eIF-2 to the EMC 5 UTR. The binding of eIF-2 to the mutant 5 UTRs was completely inhibited by the addition of competitor tRNA. Cross-linking of the EMC 5 UTR with proteins from rabbit reticulocyte lysates showed binding of trans-acting factors p52 and p57. Deletions in the 5 end of the internal ribosomal entry site resulted in a loss of the ability to bind trans-acting factor p57, in accordance with literature data, while p52 binding to these deletion mutants was weak compared to the wildtype EMC 5 UTR. Mutations in the 3 part of the 5 UTR of EMC still resulted in binding of both trans-acting factors, as with wild type RNA, but binding was more sensitive to competitor tRNA when compared to the binding of p52/p57 to the wild type 5 UTR.

An initiation signal in the 5′ untranslated leader sequence of encephalomyocarditis virus RNA

A 593 nucleotide fragment of the 5 leader of encephalomyocarditis virus RNA (EMCV-RNA) was linked to the SP6 promoter and inserted upstream of the reporter gene chloramphenicol acetyltransferase (CAT). The presence of the 5-UTR of EMCV-RNA in the RNA transcripts, made in vitro with the SP6 polymerase, resulted in a strong translational enhancement when tested in the micrococcal nuclease-treated reticulocyte lysate. The transcripts were equally active with or without a 5 methylated capstructure as expected, since EMCV-RNA is one of the mRNAs capable of internal initiation. We searched for a signal in the 5 leader that allows the 43S preinitiation complex to bind internally and localized a hairpin containing a unique nucleotide sequence, CUUUA, present in a domain conserved among cardio- and aphtoviral RNAs. Replacing this sequence into AGCU resulted in a 50% loss of translational activity. A second mutation involving a U-G change in the stem of that hairpin resulted in an almost complete loss of initiation.