Miranda Kleijn

The Mitogen-Activated Protein Kinase Signal-Integrating Kinase Mnk2 Is a Eukaryotic Initiation Factor 4E Kinase with High Levels of Basal Activity in Mammalian Cells

ABSTRACT 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), p38MAPKα, and p38MAPKβ. 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.

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 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.

Glucose and amino acids modulate translation factor activation by growth factors in PC12 cells.

In PC12 phaeochromocytoma cells, protein synthesis is activated by epidermal and nerve growth factors (EGF and NGF). EGF and NGF also regulate a number of components of the translational machinery in these cells. Here we show that the ability of EGF and NGF to induce the phosphorylation of the 70 kDa ribosomal protein, S6 kinase, and the eukaryotic initiation factor (eIF), 4E-binding protein 1, is dependent upon the presence of amino acids (but not glucose) in the medium. This resembles the regulation of these proteins by insulin, which also requires amino acids. Glucose, but not amino acids, is required for the activation of eIF2B by EGF and NGF. In contrast, EGF and NGF can still activate protein synthesis in the absence of nutrients, suggesting that other regulatory events are important in this. In nutrient-deprived cells, an increase in the phosphorylation of eIF4E, and the assembly of the eIF4F complex by EGF and NGF, coincided with the activation of protein synthesis. In serum-starved cells, activation of protein synthesis, phosphorylation of eIF4E, and formation of the eIF4F complex, were blocked by inhibition of MEK, a component of the extracellular regulated kinase (ERK) signalling pathway. Thus the ERK pathway plays a key role in the regulation of protein synthesis in PC12 cells.

The activation of eukaryotic initiation factor (eIF)2B by growth factors in PC12 cells requires MEK/ERK signalling.

Epidermal and nerve growth factors (EGF and NGF) activate protein synthesis and initiation factor eIF2B in rat phaeochromocytoma (PC12) cells. The activation of protein synthesis by EGF or NGF depends upon extracellular regulated kinase kinase (MEK)/extracellular regulated kinase signalling. Here we show that PD98059, an inhibitor of MEK activation, blocks the activation of eIF2B by EGF or NGF. It is known that eIF2B activity can be inhibited by phosphorylation at Ser535 in its ϵ‐subunit by glycogen synthase kinase (GSK)‐3. We find that inactivation of GSK‐3 by EGF or NGF is blocked by PD98059. However, neither EGF nor NGF caused a detectable change in phosphorylation of Ser535 of eIF2Bϵ. Thus, the EGF‐ and NGF‐induced activation of eIF2B in PC12 cells involves regulatory mechanisms distinct from dephosphorylation of the GSK‐3 site.

Localisation and regulation of the eIF4E-binding protein 4E-BP3

The cap‐binding protein eIF4E‐binding protein 3 (4E‐BP3) was identified some years ago, but its properties have not been investigated in detail. In this report, we investigated the regulation and localisation of 4E‐BP3. We show that 4E‐BP3 is present in the nucleus as well as in the cytoplasm in primary T cells, HEK293 cells and HeLa cells. 4E‐BP3 was associated with eIF4E in both cell compartments. Furthermore, 4E‐BP3/eIF4E association in the cytoplasm was regulated by serum or interleukin‐2 starvation in the different cell types. Rapamycin did not affect the association of eIF4E with 4E‐BP3 in the cytoplasm or in the nucleus.

Phosphorylation of the eIF4E‐binding protein PHAS‐I after exposure of PC12 cells to EGF and NGF

PHAS‐I or the eIF4E‐binding protein 1 regulates the cap‐binding activity of eIF4E by sequestering eIF4E. Binding of eIF4E to PHAS‐I is regulated by phosphorylation of PHAS‐I. PC12 cells were used to study the signal transduction pathway leading to phosphorylation of PHAS‐I. Both EGF and NGF induced phosphorylation of PHAS‐I. Wortmannin, a PI‐3 kinase inhibitor, staurosporine, a PKC inhibitor, and rapamycin, a FRAP inhibitor all blocked the phosphorylation of PHAS‐I. Of the three inhibitors, only wortmannin was able to inhibit MAPK phosphorylation. This excludes a role for MAPK in NGF‐ and EGF‐induced PHAS‐I phosphorylation in PC12 cells. Apparently, PHAS‐I was phosphorylated in a PI‐3 kinase‐, PKC‐, and FRAP‐dependent manner after EGF or NGF stimulation. Only PI‐3 kinase and FRAP are involved in the regulation of the basal level of PHAS‐I phosphorylation.

The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes

BackgroundActivation of human resting T lymphocytes results in an immediate increase in protein synthesis. The increase in protein synthesis after 16–24 h has been linked to the increased protein levels of translation initiation factors. However, the regulation of protein synthesis during the early onset of T cell activation has not been studied in great detail. We studied the regulation of protein synthesis after 1 h of activation using αCD3 antibody to stimulate the T cell receptor and αCD28 antibody to provide the co-stimulus.ResultsActivation of the T cells with both antibodies led to a sustained increase in the rate of protein synthesis. The activities and/or phosphorylation states of several translation factors were studied during the first hour of stimulation with αCD3 and αCD28 to explore the mechanism underlying the activation of protein synthesis. The initial increase in protein synthesis was accompanied by activation of the guanine nucleotide exchange factor, eukaryotic initiation factor (eIF) 2B, and of p70 S6 kinase and by dephosphorylation of eukaryotic elongation factor (eEF) 2. Similar signal transduction pathways, as assessed using signal transduction inhibitors, are involved in the regulation of protein synthesis, eIF2B activity and p70 S6 kinase activity. A new finding was that the p38 MAPK α/β pathway was involved in the regulation of overall protein synthesis in primary T cells. Unexpectedly, no changes were detected in the phosphorylation state of the cap-binding protein eIF4E and the eIF4E-binding protein 4E-BP1, or the formation of the cap-binding complex eIF4F.ConclusionsBoth eIF2B and p70 S6 kinase play important roles in the regulation of protein synthesis during the early onset of T cell activation.

Regulation of Protein Synthesis by Insulin Through IRS-1

Most regulatory mechanisms for the overall rate of protein synthesis described to date involve the initiation phase, which is catalyzed by the initiation factors (eIF1; Merrick and Hershey 1996; Rhoads 1999). Protein synthesis occurs in a series of discrete steps. A ternary complex of eIF2•GTP•Met-tRNAi binds to a complex of the 40 S ribosomal subunit and eIF3 to form the 43 S initiation complex. The eIF4 factors plus the poly(A)-binding protein collectively recognize the 5′-terminal cap or 3′-terminal poly(A) tract of mRNA, unwind its secondary structure, and transfer it to the 43 S initiation complex, resulting in the 48 S initiation complex. Scanning for the first initiation codon in good sequence context requires ATP hydrolysis by eIF4A and the presence of eIF1 and eIF1A (Pestova et al. 1998). Then, eIF5 stimulates GTP hydrolysis by eIF2, after which the initiation factors are replaced by the 60 S subunit to form the 80 S initiation complex. The released eIF2-GDP is recycled to eIF2-GTP by the guanine nucleotide exchange factor eIF2B. The first elongator aminoacyl-tRNA is brought to the A-site by eEF1, followed by synthesis of the first peptide bond and translocation catalyzed by eEF2