From the Department of Biosciences, University of
Kent at Canterbury, Canterbury, CT2 7NJ, United Kingdom and the
Fred Hutchinson Cancer Research Centre,
Seattle, Washington 98109
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ABSTRACT |
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Initiation factor eIF4E binds to the 5'-cap of
eukaryotic mRNAs and plays a key role in the mechanism and
regulation of translation. It may be regulated through its own
phosphorylation and through inhibitory binding proteins (4E-BPs), which
modulate its availability for initiation complex assembly. eIF4E
phosphorylation is enhanced by phorbol esters. We show, using specific
inhibitors, that this involves both the p38 mitogen-activated protein
(MAP) kinase and Erk signaling pathways. Cell stresses such as
arsenite and anisomycin and the cytokines tumor necrosis factor- and
interleukin-1
also cause increased phosphorylation of eIF4E, which
is abolished by the specific p38 MAP kinase inhibitor, SB203580. These
changes in eIF4E phosphorylation parallel the activity of the eIF4E
kinase, Mnk1. However other stresses such as heat shock, sorbitol, and H2O2, which also stimulate p38 MAP kinase
and increase Mnk1 activity, do not increase phosphorylation of eIF4E.
The latter stresses increase the binding of eIF4E to 4E-BP1, and we
show that this blocks the phosphorylation of eIF4E by Mnk1 in
vitro, which may explain the absence of an increase in
eIF4E phosphorylation under these conditions.
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INTRODUCTION |
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Initiation factor eIF4E plays a key role in mRNA translation and its regulation (1, 2). eIF4E binds to the 7-methylguanosine triphosphate ("cap") structure found at the 5'-end of eukaryotic cytoplasmic mRNAs. eIF4E also interacts with eIF4G, a large scaffolding protein, which itself binds to other translation factors including eIF4A, an RNA helicase, and eIF3, a multimeric protein that binds to the 40 S ribosomal subunit (3). The complex of eIF4E, eIF4G, and eIF4A is often termed eIF4F and is believed to be especially important for the translation of mRNAs whose 5'-untranslated regions are rich in secondary structure, because such structures in general inhibit translation but can be unwound by eIF4A (1, 2). Binding of eIF4E to eIF4G can be blocked by regulator proteins termed eIF4E-binding proteins (4E-BPs),1 which interact with the same region of eIF4E that binds eIF4G (4). The best characterized of them is 4E-BP1 (also called PHAS-I, (4)). It is regulated by phosphorylation at multiple sites, in response, e.g. to insulin, which causes its dissociation from eIF4E (4).
eIF4E is a phosphoprotein, and its phosphorylation is generally enhanced by agents that activate translation (reviewed in Refs. 1 and 2). Phosphorylation of eIF4E increases its affinity for the cap and for mRNA and may also favor its entry into initiation complexes (1, 2). Both effects may be important in the activation of translation under conditions that increase eIF4E phosphorylation. The phosphorylation site in eIF4E is Ser209 (5, 6), although the identity of the protein kinase responsible for its phosphorylation in vivo is less clear. We have recently shown that insulin-induced eIF4E phosphorylation requires the MAP kinase signaling pathway (also termed the Erk, extracellular signal-regulated kinase pathway, the term used here) (7). However, eIF4E is not a substrate for the Erks, and we have shown that it is phosphorylated instead at Ser209 by a novel Erk-activated protein kinase, MAP kinase signal-integrating kinase-1 (Mnk1) (8, 9). Mnk1 is also phosphorylated and activated by an additional enzyme related to Erk, p38 MAP kinase, which lies on a distinct signaling pathway activated by cell stresses and cytokines (8-10).
Here we show that the increased phosphorylation of eIF4E brought about by the phorbol ester tetradecanoylphorbol 13-acetate (TPA), which activates members of the protein kinase C (PKC) family, requires the Erk and p38 MAP kinase pathways. Furthermore, we show that eIF4E phosphorylation is enhanced by agents, such as certain stresses and cytokines, that activate p38 MAP kinase and that this is blocked by a specific inhibitor of this enzyme. Our data support the identity of Mnk1 as a physiologically important eIF4E kinase. Certain stresses that activate p38 MAP kinase do not increase eIF4E phosphorylation. This is likely to be due to the increased association of eIF4E with 4E-BP1 that these conditions bring about, because 4E-BP1 inhibits phosphorylation of eIF4E by Mnk1.
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MATERIALS AND METHODS |
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Chemicals and Biochemicals-- Unless otherwise stated, chemicals were obtained as described previously (7, 11). Anti-(P)Erk was from New England BioLabs. Anti-human eIF4E was raised against a synthetic peptide corresponding to residues 5-23 of the protein. Antibodies to eIF4G were a kind gift from S. J. Morley (Sussex). The anti-4E-BP1 antibody was raised against a peptide corresponding to residues 101-113 of human 4E-BP1. Recombinant Mnk1-GST and 4E-BP1 were prepared as described previously (8, 12). The expression vector for 4E-BP1 was a generous gift from R. M. Denton (Bristol).
Cell Culture, Treatment, and Extraction-- Human embryonic kidney 293 and Chinese hamster ovary (CHO.K1) cells were grown as described previously (8, 13). Human umbilical vein endothelial cells (HUVECs) were grown in modified MCDB131 medium (Clonetics), supplemented with 2% (v/v) fetal calf serum, 10 ng/ml epidermal growth factor, 1 µg/ml hydrocortisone, 50 µg/ml gentamicin, 50 ng/ml amphotericin B, and 12 µg/ml bovine brain extract. Where used, inhibitors were added 1 h prior to treatment of the cells. Cells were extracted in our standard buffer, which contains a mixture of protein phosphatase and proteinase inhibitors (13). The transfection protocol for 293 cells was described previously (8).
Assessment of Protein Kinase Activities-- Mnk1 was assayed using eIF4E as substrate as described by Waskiewicz et al. (8). p38 MAP kinase activity was assessed by measuring the activity of the downstream kinase MAP kinase-activated kinase-2 (MAPKAP-K2 (10)) using recombinant hsp25 as substrate (14).
Isolation and Analysis of eIF4E-- eIF4E was isolated from cell extracts by affinity chromatography on m7GTP-Sepharose as described previously (11). Its phosphorylation state was assessed by isoelectric focusing/immunoblotting as described previously (11). Its association with 4E-BP1 and with components of eIF4F was analyzed by Western blotting using antisera to eIF4G and 4E-BP1 (15, 16).
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RESULTS AND DISCUSSION |
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TPA-induced Phosphorylation of eIF4E Requires the Erk and p38 MAP Kinase Pathways-- In several cell types, phorbol esters that activate PKC enhance eIF4E phosphorylation (11, 17-19). Exposure of 293 cells to TPA increased the level of eIF4E phosphorylation from almost zero to about 30% (Fig. 1A). To test whether, as for insulin (7), this effect required the Erk pathway, we used the compound PD098059, a specific inhibitor of MEK activation (20). As expected, PD098059 reduced the ability of TPA to enhance eIF4E phosphorylation (Fig. 1A) and also completely inhibited the activation of Erk by TPA in 293 cells (Fig. 1B, lanes 1-4). However, the inhibition of eIF4E phosphorylation was incomplete indicating that other signaling pathways were involved. We therefore tested the effect of a specific inhibitor of p38 MAP kinase SB203580 (21) on eIF4E phosphorylation. It has been shown not to interfere with stress-, cytokine-, or growth factor-induced activation of other signaling pathways such as Erk, JNK, or p70 S6 kinase (21-24). SB203580 partially blocked TPA-induced eIF4E phosphorylation, and its use together with PD098059 completely abolished TPA-induced eIF4E phosphorylation (Fig. 1A). As shown in Fig. 1B (lanes 5-8), TPA activates MAPKAP-K2, which is activated by p38 MAP kinase (10), in 293 cells. These data suggest that TPA acts through both Erk and p38 MAP kinase to increase eIF4E phosphorylation in 293 cells. Waskiewicz et al. (8) previously showed that Mnk1 could be activated in vitro by either Erk or p38 MAP kinase. To assess Mnk1 activity in 293 cells subjected to these treatments, we transfected 293 cells with a vector encoding wild-type Mnk1 fused to GST (8) and subjected the transfected cells to treatment with TPA in the absence or the presence of kinase inhibitors. Cells were extracted, and Mnk1-GST was isolated and assayed. SB203580 or PD098059 only partially prevented the activation of Mnk1 by TPA, but use of both completely abolished it (Fig. 1C). The changes in Mnk1 activity parallel the alterations in eIF4E phosphorylation observed under these conditions, entirely consistent with a key role for Mnk1 in mediating eIF4E phosphorylation (8).
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Arsenite Induces the Phosphorylation of eIF4E, Which Is Blocked by SB203580-- The above data prompted us to ask whether other treatments that activate p38 MAP kinase affect eIF4E phosphorylation. Arsenite potently activates the p38 MAP kinase pathway in 293 cells (Fig. 2A) and also markedly increased eIF4E phosphorylation (Fig. 2B, lanes 1-5). The p38 MAP kinase inhibitor SB203580 (21) blocked both this and the arsenite-induced activation of MAPKAP-K2 (Fig. 2, A and B). Arsenite did not activate Erk in 293 cells (data not shown), and the MEK inhibitor PD098059 did not affect arsenite-induced eIF4E phosphorylation (Fig. 2B). Thus, arsenite-induced eIF4E phosphorylation appears to be mediated by the p38 MAP kinase pathway (8). Changes in Mnk1 activity again parallel those in eIF4E phosphorylation (Fig. 2C). The ability of arsenite to increase eIF4E phosphorylation is not restricted to 293 cells because the same effect was also observed in CHO.K1 cells (Fig. 2B, lanes 6-9), and again, SB203580 blocked both arsenite-induced eIF4E phosphorylation and p38 MAP kinase activation (Fig. 2B and data not shown). Anisomycin activates Mnk1 in 293 cells, and this activation was blocked by SB203580 (Ref. 8 and Fig. 2C). Anisomycin increased eIF4E phosphorylation in 293 cells (to about 80%, data not shown), and this effect was also completely blocked by SB203580. Anisomycin also increases eIF4E phosphorylation in NIH 3T3 cells, and this increase is blocked by SB203580 (25).
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Effects of Other Stresses on eIF4E Phosphorylation-- Other stresses such as hyperosmolarity (sorbitol) and oxidative stress (hydrogen peroxide) also activate p38 MAP kinase, MAPKAP-K2 (Fig. 2A) and Mnk1 (Fig. 2C). However, unlike arsenite, they did not increase eIF4E phosphorylation (Fig. 2D, lanes 3-5). In 293 cells, where the basal eIF4E phosphorylation is low, no change was seen (Fig. 2D). In CHO.K1 cells, where the basal eIF4E phosphorylation is significant, they led to a fall in eIF4E phosphorylation (Fig. 2D). Heat shock did not appreciably activate p38 MAP kinase in 293 cells but did in CHO.K1 cells (16), and this activation is blocked by SB203580. Despite this, heat shock actually caused a decrease in eIF4E phosphorylation (Fig. 2D).
Why do some stresses increase eIF4E phosphorylation, whereas others cause a decrease, even though they also activate Mnk1? To try to explain this apparent paradox, we analyzed the association of eIF4E with its regulator 4E-BP1; we have previously shown that in CHO.K1 cells, most stresses increase binding of 4E-BP1 to eIF4E (16). This effect is also seen in 293 cells (Fig. 3A). (The exception here (as in CHO cells) is arsenite, which does not cause increased binding of 4E-BP1 to eIF4E. This is probably because it can activate the rapamycin-sensitive signaling pathway (28), which leads to the phosphorylation of 4E-BP1 and its dissociation from eIF4E (4).) These findings raised the possibility that the association of 4E-BP1 with eIF4E might impair phosphorylation of the latter by Mnk1. To test this, we examined the effect of 4E-BP1 on the ability of Mnk1 to phosphorylate eIF4E in vitro. The data (Fig. 3B) clearly show that 4E-BP1 substantially inhibits the phosphorylation of eIF4E by Mnk1. The highest amount of 4E-BP1 used represents saturation of the eIF4E with 4E-BP1 as indicated by the fact that addition of further 4E-BP1 resulted in (i) it not being retained on m7GTP-Sepharose, i.e. not being associated with eIF4E, and (ii) phosphorylation of the excess 4E-BP1 by the Erk present in the activated Mnk1, with only free 4E-BP1 (and not the 4E-BP1/eIF4E complex) being a substrate for Erk (12) (data not shown). SB203580 had no effect on the association of eIF4E with 4E-BP1, either under stress or control conditions (16).2 4E-BP1 did not affect the phosphorylation of another substrate, the cAMP-response element binding protein, by Mnk1 (data not shown). This suggests that inhibition of eIF4E phosphorylation by 4E-BP1 reflects the inability of Mnk1 to phosphorylate eIF4E in the eIF4E/4E-BP1 complex and is likely to be specific for this substrate.
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Regulation of eIF4E Phosphorylation by Cytokines That Activate p38
MAP Kinase--
It was important to ascertain whether treatment of
cells with physiological activators of p38 MAP kinase, such as
cytokines, also altered the phosphorylation of eIF4E. Tumor necrosis
factor- (TNF
) is a physiological regulator of endothelial cell
function (34), and in HUVECs it markedly activates the p38 MAP kinase pathway without any apparent effect on Erk activity (data not shown and
Ref. 34). We therefore studied its effect on the phosphorylation of
eIF4E. TNF
increased the phosphorylation of eIF4E (Fig.
4A), and this increase was
prevented by SB203580, which blocked activation of the p38 MAP kinase
pathway and hence of MAPKAP-K2. These data show for the first time that
cytokines increase phosphorylation of eIF4E through the p38 MAP kinase
pathway. In CHO.K1 cells, another cytokine, interleukin-1
(IL-1
)
activates p38 MAP kinase, although less markedly than TNF
does in
HUVECs (data not shown). IL-1
(5 ng/ml) increased the
phosphorylation of eIF4E in CHO.K1 cells (Fig. 4B). This
effect, like that of TNF
in HUVECs, was prevented by SB203580 (data
not shown).
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Conclusions--
The ability of activators of p38 MAP kinase to
increase eIF4E phosphorylation was seen in three different cell types,
human embryonic kidney (293) cells, CHO cells, and HUVECs and in
response to stresses and cytokines. In all cases, SB203580 blocked the phosphorylation of eIF4E. The data for TNF in HUVECs are of
particular note given that the TNF
-stimulated induction of the cell
adhesion molecule V-CAM is mediated by the p38 MAP kinase pathway
(34) and involves post-transcriptional effects that might be related to
changes in eIF4E phosphorylation. Both the stress stimuli (arsenite and
anisomycin) and TNF
also activate the JNK pathway. However, the
ability of SB203580 (which does not affect JNK activity in the cells
used here)2 to block eIF4E phosphorylation indicates that
the JNK pathway is not involved in modulating eIF4E
phosphorylation.
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ACKNOWLEDGEMENTS |
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We thank Pfizer Central Research for kindly providing the SB203580 used in this study, Michèle Heaton (Kent) for recombinant eIF4E, Drs. Nick Morrice and Robert Mackintosh (Dundee) for Erk, and Jashmin Patel (Kent) for recombinant 4E-BP1.
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FOOTNOTES |
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* This work was supported by Grant G9411756 from the Medical Research Council (to C. G. P.), Grant 046110 from the Wellcome Trust (to C. G. P.), and Grant CA73987 from the U. S. Public Health Service (to J. A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to the work.
¶ Present address: Laboratoire de Biophysique, Musée National d'Histoire Naturelle, 43 rue Cuviere, 75231 Paris Cedex 05, France.
** Present address: Faculty of Medicine, Dept. of Medical Biochemistry, Section on Molecular Carcinogenesis, P.O. Box 9503, 2300 RA Leiden, The Netherlands.
Recipient of a Studentship from Pfizer Central Research.
§§ To whom correspondence should be addressed. Present address: Dept. of Anatomy & Physiology, Medical Sciences Inst., University of Dundee, Dundee, DD1 4HN, UK. Tel.: 44-1382-344919; Fax: 44-1382-322424; E-mail:CGPROUD{at}bad.dundee.ac.uk.
1
The abbreviations used are: 4E-BP, eIF4E-binding
protein; MAP, mitogen-activated protein kinase; TPA,
tetradecanoylphorbol 13-acetate; PKC, protein kinase C; CHO, Chinese
hamster ovary; HUVEC, human umbilical vein endothelial cell; MAPKAP-K,
MAP kinase-activated protein kinase; GST, glutathione
S-transferase; TNF, tumor necrosis factor-
; IL-1
,
interleukin-1
; JNK, c-Jun N-terminal kinase.
2 X. Wang, A. Flynn, A. J. Waskiewicz, B. L. J. Webb, R. G. Vries, I. A. Baines, J. A. Cooper, and C. G. Proud, unpublished data.
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REFERENCES |
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