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The Phosphorylation of Eukaryotic Initiation Factor eIF4E in Response to Phorbol Esters, Cell Stresses, and Cytokines Is Mediated by Distinct MAP Kinase Pathways

The Phosphorylation of Eukaryotic Initiation Factor eIF4E in Response to Phorbol Esters, Cell... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 16, Issue of April 17, pp. 9373–9377, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Phosphorylation of Eukaryotic Initiation Factor eIF4E in Response to Phorbol Esters, Cell Stresses, and Cytokines Is Mediated by Distinct MAP Kinase Pathways* (Received for publication, December 1, 1997, and in revised form, January 13, 1998) Xuemin Wang‡, Andrea Flynn‡§¶, Andrew J. Waskiewicz§i, Benjamin L. J. Webb‡§, Robert G. Vries‡**, Ian A. Baines‡ ‡‡, Jonathan A. Cooperi, and Christopher G. Proud‡ §§ From the ‡Department of Biosciences, University of Kent at Canterbury, Canterbury, CT2 7NJ, United Kingdom and the iFred Hutchinson Cancer Research Centre, Seattle, Washington 98109 Initiation factor eIF4E binds to the 5*-cap of eukary- tion but can be unwound by eIF4A (1, 2). Binding of eIF4E to eIF4G can be blocked by regulator proteins termed eIF4E- otic mRNAs and plays a key role in the mechanism and regulation of translation. It may be regulated through binding proteins (4E-BPs), which interact with the same re- its own phosphorylation and through inhibitory binding gion of eIF4E that binds eIF4G (4). The best characterized of proteins (4E-BPs), which modulate its availability for them is 4E-BP1 (also called PHAS-I, (4)). It is regulated by initiation complex assembly. eIF4E phosphorylation is phosphorylation at multiple sites, in response, e.g. to insulin, enhanced by phorbol esters. We show, using specific which causes its dissociation from eIF4E (4). inhibitors, that this involves both the p38 mitogen-acti- eIF4E is a phosphoprotein, and its phosphorylation is gen- vated protein (MAP) kinase and Erk signaling pathways. erally enhanced by agents that activate translation (reviewed Cell stresses such as arsenite and anisomycin and the in Refs. 1 and 2). Phosphorylation of eIF4E increases its affin- a and interleukin-1b also cytokines tumor necrosis factor- ity for the cap and for mRNA and may also favor its entry into cause increased phosphorylation of eIF4E, which is abol- initiation complexes (1, 2). Both effects may be important in the ished by the specific p38 MAP kinase inhibitor, SB203580. activation of translation under conditions that increase eIF4E These changes in eIF4E phosphorylation parallel the ac- phosphorylation. The phosphorylation site in eIF4E is Ser tivity of the eIF4E kinase, Mnk1. However other stresses (5, 6), although the identity of the protein kinase responsible O , which also stimu- such as heat shock, sorbitol, and H 2 2 for its phosphorylation in vivo is less clear. We have recently late p38 MAP kinase and increase Mnk1 activity, do not shown that insulin-induced eIF4E phosphorylation requires increase phosphorylation of eIF4E. The latter stresses in- the MAP kinase signaling pathway (also termed the Erk, ex- crease the binding of eIF4E to 4E-BP1, and we show that tracellular signal-regulated kinase pathway, the term used this blocks the phosphorylation of eIF4E by Mnk1 in vitro, here) (7). However, eIF4E is not a substrate for the Erks, and which may explain the absence of an increase in eIF4E we have shown that it is phosphorylated instead at Ser by a phosphorylation under these conditions. novel Erk-activated protein kinase, MAP kinase signal-inte- grating kinase-1 (Mnk1) (8, 9). Mnk1 is also phosphorylated and activated by an additional enzyme related to Erk, p38 MAP Initiation factor eIF4E plays a key role in mRNA translation and its regulation (1, 2). eIF4E binds to the 7-methylguanosine kinase, which lies on a distinct signaling pathway activated by cell stresses and cytokines (8 –10). triphosphate (“cap”) structure found at the 59-end of eukaryotic cytoplasmic mRNAs. eIF4E also interacts with eIF4G, a large Here we show that the increased phosphorylation of eIF4E brought about by the phorbol ester tetradecanoylphorbol 13- scaffolding protein, which itself binds to other translation fac- tors including eIF4A, an RNA helicase, and eIF3, a multimeric acetate (TPA), which activates members of the protein kinase C (PKC) family, requires the Erk and p38 MAP kinase pathways. protein that binds to the 40 S ribosomal subunit (3). The complex of eIF4E, eIF4G, and eIF4A is often termed eIF4F and Furthermore, we show that eIF4E phosphorylation is enhanced by agents, such as certain stresses and cytokines, that activate is believed to be especially important for the translation of mRNAs whose 59-untranslated regions are rich in secondary p38 MAP kinase and that this is blocked by a specific inhibitor of this enzyme. Our data support the identity of Mnk1 as a structure, because such structures in general inhibit transla- physiologically important eIF4E kinase. Certain stresses that activate p38 MAP kinase do not increase eIF4E phosphoryla- * This work was supported by Grant G9411756 from the Medical tion. This is likely to be due to the increased association of Research Council (to C. G. P.), Grant 046110 from the Wellcome Trust eIF4E with 4E-BP1 that these conditions bring about, because (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 4E-BP1 inhibits phosphorylation of eIF4E by Mnk1. by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 MATERIALS AND METHODS solely to indicate this fact. Chemicals and Biochemicals—Unless otherwise stated, chemicals § These authors contributed equally to the work. were obtained as described previously (7, 11). Anti-(P)Erk was from Present address: Laboratoire de Biophysique, Muse ´ e National New England BioLabs. Anti-human eIF4E was raised against a syn- d’Histoire Naturelle, 43 rue Cuviere, 75231 Paris Cedex 05, France. ** Present address: Faculty of Medicine, Dept. of Medical Biochem- istry, Section on Molecular Carcinogenesis, P.O. Box 9503, 2300 RA Leiden, The Netherlands. The abbreviations used are: 4E-BP, eIF4E-binding protein; MAP, ‡‡ Recipient of a Studentship from Pfizer Central Research. mitogen-activated protein kinase; TPA, tetradecanoylphorbol 13-ace- §§ To whom correspondence should be addressed. Present address: tate; PKC, protein kinase C; CHO, Chinese hamster ovary; HUVEC, Dept. of Anatomy & Physiology, Medical Sciences Inst., University of human umbilical vein endothelial cell; MAPKAP-K, MAP kinase-acti- Dundee, Dundee, DD1 4HN, UK. Tel.: 44-1382-344919; Fax: 44-1382- vated protein kinase; GST, glutathione S-transferase; TNFa, tumor 322424; E-mail:[email protected]. necrosis factor-a; IL-1b, interleukin-1b; JNK, c-Jun N-terminal kinase. This paper is available on line at http://www.jbc.org 9373 This is an Open Access article under the CC BY license. 9374 Regulation of Initiation Factor 4E Phosphorylation thetic 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 de- scribed previously (8, 13). Human umbilical vein endothelial cells (HUVECs) were grown in modified MCDB131 medium (Clonetics), sup- plemented with 2% (v/v) fetal calf serum, 10 ng/ml epidermal growth factor, 1 mg/ml hydrocortisone, 50 mg/ml gentamicin, 50 ng/ml ampho- tericin B, and 12 mg/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 down- stream kinase MAP kinase-activated kinase-2 (MAPKAP-K2 (10)) us- ing recombinant hsp25 as substrate (14). Isolation and Analysis of eIF4E—eIF4E was isolated from cell ex- tracts by affinity chromatography on m GTP-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). RESULTS AND DISCUSSION TPA-induced Phosphorylation of eIF4E Requires the Erk and p38 MAP Kinase Pathways—In several cell types, phorbol es- ters 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 re- FIG.1. A, 293 cells were pretreated with or without SB203580 (25 duced the ability of TPA to enhance eIF4E phosphorylation mM) and/or PD098059 (50 nM) for 60 min and then with or without TPA (Fig. 1A) and also completely inhibited the activation of Erk by (150 nM) for a further 15 min, as indicated, prior to extraction. Extracts TPA in 293 cells (Fig. 1B, lanes 1– 4). However, the inhibition of were processed for analysis of eIF4E phosphorylation: the positions of eIF4E phosphorylation was incomplete indicating that other its phosphorylated (4E(P)) and nonphosphorylated (4E) forms are indi- cated. The figure shows a Western blot developed using ECL. Numbers signaling pathways were involved. We therefore tested the below each lane show the percentage of eIF4E in the phosphorylated effect of a specific inhibitor of p38 MAP kinase SB203580 (21) form (% 4E(P)), as determined by densitometric analysis of the ECL on eIF4E phosphorylation. It has been shown not to interfere images. B, 293 cells were treated as for A. Extracts were analyzed either with stress-, cytokine-, or growth factor-induced activation of by SDS-polyacrylamide gel electrophoresis and Western blotting using an antiphospho-Erk antibody (figure is a blot developed using ECL) other signaling pathways such as Erk, JNK, or p70 S6 kinase (lanes 1– 4) or for MAPKAP-K2 activity (figure is an autoradiograph) (21–24). SB203580 partially blocked TPA-induced eIF4E phos- (lanes 5– 8). The positions of Erk and hsp25 are indicated. C, activity of phorylation, and its use together with PD098059 completely Mnk1-GST in 293 cells treated with or without TPA and the inhibitors abolished TPA-induced eIF4E phosphorylation (Fig. 1A). As (as indicated) as described for A. The resulting dried gel was analyzed using a PhosphorImager and the data thus obtained are presented in shown in Fig. 1B (lanes 5– 8), TPA activates MAPKAP-K2, arbitrary PhosphorImager units (control is set at 1.0 and corresponds to which is activated by p38 MAP kinase (10), in 293 cells. These unstimulated (unstim) cells). D, TPA increases eIF4E phosphorylation data suggest that TPA acts through both Erk and p38 MAP in CHO.K1 cells, and this increase is blocked by PD098059/SB203580. kinase to increase eIF4E phosphorylation in 293 cells. The experiments were performed and the data are presented as in A. 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 due to the high basal level of eIF4E phosphorylation (Fig. 1D). transfected 293 cells with a vector encoding wild-type Mnk1 This was partially decreased either by PD098059 or SB203580, fused to GST (8) and subjected the transfected cells to treat- although both were required to completely suppress it (Fig. ment with TPA in the absence or the presence of kinase inhib- 1D). Each compound alone also decreased the level of eIF4E itors. Cells were extracted, and Mnk1-GST was isolated and phosphorylation in TPA-treated CHO.K1 cells, but both were assayed. SB203580 or PD098059 only partially prevented the again required to abolish eIF4E phosphorylation completely activation of Mnk1 by TPA, but use of both completely abol- (Fig. 1D). Taken together, the data reinforce the conclusion ished it (Fig. 1C). The changes in Mnk1 activity parallel the that both the Erk and p38 MAP kinase cascades are involved in alterations in eIF4E phosphorylation observed under these mediating changes in eIF4E phosphorylation. Our data imply conditions, entirely consistent with a key role for Mnk1 in that the phorbol ester-induced phosphorylation of eIF4E is not, mediating eIF4E phosphorylation (8). as has previously been suggested (1), directly mediated by TPA also increases eIF4E phosphorylation in CHO.K1 cells, PKC. A more likely route by which TPA increases eIF4E phos- which involves the so-called conventional isoforms of PKC (11). phorylation is through activation of the Erk/p38 MAP kinase Analysis of the roles of signaling pathways in the phosphoryl- cascades, via PKC, leading to the activation of Mnk1, which ation of eIF4E in these cells is more complex than for 293 cells itself directly phosphorylates eIF4E (8). This is consistent with Regulation of Initiation Factor 4E Phosphorylation 9375 nase pathway (8). Changes in Mnk1 activity again parallel those in eIF4E phosphorylation (Fig. 2C). The ability of arsen- ite 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 arsen- ite-induced eIF4E phosphorylation and p38 MAP kinase acti- vation (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. Ani- somycin also increases eIF4E phosphorylation in NIH 3T3 cells, and this increase is blocked by SB203580 (25). The finding that arsenite stimulates eIF4E phosphorylation is surprising given that arsenite potently inhibits protein syn- thesis (26), whereas eIF4E phosphorylation is normally asso- ciated with its activation. It is likely that arsenite inhibits other steps in translation, and, indeed, we have shown that it increases phosphorylation of the a-subunit of eIF2, which is well known to lead to inhibition of peptide chain initiation (27). Effects of Other Stresses on eIF4E Phosphorylation—Other stresses such as hyperosmolarity (sorbitol) and oxidative stress (hydrogen peroxide) also activate p38 MAP kinase, MAP- KAP-K2 (Fig. 2A) and Mnk1 (Fig. 2C). However, unlike arsen- ite, 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 acti- vate 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 acti- FIG.2. A, measurement of MAPKAP-K2 activity in 293 cells. Cells vate Mnk1? To try to explain this apparent paradox, we ana- were preincubated with or without SB203580 (25 mm) for 60 min prior lyzed the association of eIF4E with its regulator 4E-BP1; we to treatment with arsenite (0.1 mM, 20 min), H O (3 mM, 25 min), 2 2 sorbitol (0.6 mM, 25 min), and heat shock (HS, 44 °C, 30 min and 120 have previously shown that in CHO.K1 cells, most stresses min) as indicated. The figure shows an autoradiograph of the SDS- increase binding of 4E-BP1 to eIF4E (16). This effect is also polyacrylamide gel. The position of hsp25 is indicated. Numbers below seen in 293 cells (Fig. 3A). (The exception here (as in CHO cells) each lane show the relative activity of MAPKAP-K2 (percentage of is arsenite, which does not cause increased binding of 4E-BP1 control), determined by PhosphorImager analysis of the dried gel. B, assessment of eIF4E phosphorylation. Lanes 1–5, 293 cells. Cells were to eIF4E. This is probably because it can activate the rapamy- pretreated with or without SB203580 and/or PD098059 for 60 min and cin-sensitive signaling pathway (28), which leads to the phos- then exposed, where indicated, to sodium arsenite (0.1 mM) for 20 min, phorylation of 4E-BP1 and its dissociation from eIF4E (4).) as indicated. Lanes 6 –9, CHO.K1 cells. Cells were treated as for lanes 1–5 These findings raised the possibility that the association of (except that PD098059 was not used here). Data are presented as Fig. 1A. 4E-BP1 with eIF4E might impair phosphorylation of the latter C, activity of Mnk1-GST in 293 cells. Stimuli and inhibitors were used under the conditions described above. The data were obtained and are by Mnk1. To test this, we examined the effect of 4E-BP1 on the presented as in Fig. 1C. unstim, unstimulated; SB, SB203580; HS, heat ability of Mnk1 to phosphorylate eIF4E in vitro. The data (Fig. shock; SORB, sorbitol; ARS, arsenite; ANISO, anisomycin. D, assessment 3B) clearly show that 4E-BP1 substantially inhibits the phos- of the level of phosphorylation of eIF4E. Lanes 1–5, 293 cells; lanes 6 –10, phorylation of eIF4E by Mnk1. The highest amount of 4E-BP1 CHO.K1 cells. All stress conditions are as same as those in A: con, control; ars, arsenite; HO, hydrogen peroxide; HS, heat shock; sor, used represents saturation of the eIF4E with 4E-BP1 as indi- sorbitol. cated by the fact that addition of further 4E-BP1 resulted in (i) it not being retained on m GTP-Sepharose, i.e. not being asso- all the published data on phorbol ester-induced eIF4E phos- ciated with eIF4E, and (ii) phosphorylation of the excess 4E- phorylation (11, 17–19). BP1 by the Erk present in the activated Mnk1, with only free Arsenite Induces the Phosphorylation of eIF4E, Which Is 4E-BP1 (and not the 4E-BP1/eIF4E complex) being a substrate Blocked by SB203580 —The above data prompted us to ask for Erk (12) (data not shown). SB203580 had no effect on the whether other treatments that activate p38 MAP kinase affect association of eIF4E with 4E-BP1, either under stress or con- eIF4E phosphorylation. Arsenite potently activates the p38 trol conditions (16). 4E-BP1 did not affect the phosphorylation MAP kinase pathway in 293 cells (Fig. 2A) and also markedly of another substrate, the cAMP-response element binding pro- increased eIF4E phosphorylation (Fig. 2B, lanes 1–5). The p38 tein, by Mnk1 (data not shown). This suggests that inhibition of MAP kinase inhibitor SB203580 (21) blocked both this and the eIF4E phosphorylation by 4E-BP1 reflects the inability of arsenite-induced activation of MAPKAP-K2 (Fig. 2, A and B). Mnk1 to phosphorylate eIF4E in the eIF4E/4E-BP1 complex 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 X. Wang, A. Flynn, A. J. Waskiewicz, B. L. J. Webb, R. G. Vries, I. A. phosphorylation appears to be mediated by the p38 MAP ki- Baines, J. A. Cooper, and C. G. Proud, unpublished data. 9376 Regulation of Initiation Factor 4E Phosphorylation FIG.3. A, effects of stress on binding of 4E-BP1 to eIF4E in 293 cells. Cells were subjected to stress conditions as in Fig. 2A and then ex- tracted and analyzed for 4E-BP1 and eIF 4E. The figure shows a Western blot developed using ECL. con, control; ars, arsenite; HO, hydrogen peroxide; sor, sorbitol; HS, heat shock. B, effect of 4E-BP1 on the phosphorylation of eIF4E by Mnk1. Lanes 1– 4, eIF4E was incu- bated with Mnk1-GST that had previously been activated by incubation with Erk1 and ATP/Mg, followed by washing of the Mnk1-GST bound to glutathione-Sepharose with LiCl (0.5 M) to remove as much Erk1 as possible (8). Reactions for the phosphorylation of eIF4E by Mnk1-GST were performed in the absence (lane 1) or the presence (lanes 2– 4)of 4E-BP1 and radiolabeled ATP. Lanes 2– 4 contain increasing amounts of 4E-BP1 (in the ratio 1:3:7). Numbers below each lane indicate the relative labeling of eIF4E as determined by PhosphorImager analysis. FIG.4. Cytokines increase eIF4E phosphorylation. A, HUVECs C, dissociation of eIF4F complexes in stressed cells (see Fig. 2A), as- were pretreated with or without SB203580 (25 mM) for 60 min before sessed by analyzing samples isolated as described under “Materials and exposure to TNFa (5 ng/ml) for 10 min, as indicated, and extracts were Methods” (m GTP-Sepharose-bound material) on an 8% polyacrylamide analyzed for eIF4E phosphorylation. Data are presented as in Fig. 1A. gel followed by blotting with anti-eIF4G. B, CHO.K1 cells were treated for 20 min with or without IL-1b (indi- cated concentration). Cells were then extracted, and extracts were analyzed for eIF4E phosphorylation. Data are presented as Fig. 1A. and is likely to be specific for this substrate. The inhibition of Mnk1-catalyzed eIF4E phosphorylation by 4E-BP1 provides an explanation for the differing effects of phosphorylation of eIF4E through the p38 MAP kinase path- stresses on eIF4E phosphorylation, and, in particular, for the way. In CHO.K1 cells, another cytokine, interleukin-1b (IL-1b) ability of heat shock to reduce eIF4E phosphorylation (re- activates p38 MAP kinase, although less markedly than TNFa viewed in Ref. 29). Some studies have shown that rapamycin does in HUVECs (data not shown). IL-1b (5 ng/ml) increased reduces the level of eIF4E phosphorylation (25, 30). Our data the phosphorylation of eIF4E in CHO.K1 cells (Fig. 4B). This suggest that this may be due to increased association of eIF4E effect, like that of TNFa in HUVECs, was prevented by with 4E-BP1 caused by rapamycin (due to dephosphorylation of SB203580 (data not shown). 4E-BP1 (4)) and resulting inhibition of Mnk1-catalyzed eIF4E Conclusions—The ability of activators of p38 MAP kinase to phosphorylation. 4E-BP1 also blocks the phosphorylation of increase eIF4E phosphorylation was seen in three different cell eIF4E by PKC (31). The binding site for 4E-BP1 in eIF4E has types, human embryonic kidney (293) cells, CHO cells, and recently been identified (32). Because it is some distance from HUVECs and in response to stresses and cytokines. In all Ser in the three-dimensional structure of the protein, it cases, SB203580 blocked the phosphorylation of eIF4E. The seems unlikely that 4E-BP1 actually occludes the phosphoryl- data for TNFa in HUVECs are of particular note given that ation site. The effect of 4E-BP1 may instead reflect interference the TNFa-stimulated induction of the cell adhesion molecule with the interaction between these kinases and other regions of V-CAM is mediated by the p38 MAP kinase pathway (34) and the eIF4E protein required for kinase/substrate binding. involves post-transcriptional effects that might be related to The increased binding of 4E-BP1 to eIF4E was accompanied by changes in eIF4E phosphorylation. Both the stress stimuli a decrease in the binding of eIF4G to eIF4E (Fig. 3C), as expected (arsenite and anisomycin) and TNFa also activate the JNK from the mutually competitive nature of their interactions (33). pathway. However, the ability of SB203580 (which does not Consistent with its lack of effect on 4E-BP1 binding, arsenite also affect JNK activity in the cells used here) to block eIF4E had no effect on the association of eIF4E with eIF4G. phosphorylation indicates that the JNK pathway is not in- Regulation of eIF4E Phosphorylation by Cytokines That Ac- volved in modulating eIF4E phosphorylation. tivate p38 MAP Kinase—It was important to ascertain whether We have previously shown that insulin-induced phosphoryl- treatment of cells with physiological activators of p38 MAP ation of eIF4E requires the Erk pathway (7). Taken together, kinase, such as cytokines, also altered the phosphorylation of our findings show that eIF4E phosphorylation can be mediated eIF4E. Tumor necrosis factor-a (TNFa) is a physiological reg- by two distinct signaling pathways, the Erk and p38 MAP ulator of endothelial cell function (34), and in HUVECs it kinase pathways, depending on the stimulus, consistent with markedly activates the p38 MAP kinase pathway without any the established regulatory properties of the eIF4E kinase apparent effect on Erk activity (data not shown and Ref. 34). Mnk1, which is a target for activation by both (8, 9). Changes in We therefore studied its effect on the phosphorylation of eIF4E. eIF4E phosphorylation largely mirror alterations in Mnk1 activ- TNFa increased the phosphorylation of eIF4E (Fig. 4A), and ity, consistent with a physiological role for this kinase in eIF4E this increase was prevented by SB203580, which blocked acti- phosphorylation. The only exceptions are stress conditions that vation of the p38 MAP kinase pathway and hence of MAPKAP- increase binding of 4E-BP1 to eIF4E. In almost all cases, such K2. 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The Phosphorylation of Eukaryotic Initiation Factor eIF4E in Response to Phorbol Esters, Cell Stresses, and Cytokines Is Mediated by Distinct MAP Kinase Pathways

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 16, Issue of April 17, pp. 9373–9377, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Phosphorylation of Eukaryotic Initiation Factor eIF4E in Response to Phorbol Esters, Cell Stresses, and Cytokines Is Mediated by Distinct MAP Kinase Pathways* (Received for publication, December 1, 1997, and in revised form, January 13, 1998) Xuemin Wang‡, Andrea Flynn‡§¶, Andrew J. Waskiewicz§i, Benjamin L. J. Webb‡§, Robert G. Vries‡**, Ian A. Baines‡ ‡‡, Jonathan A. Cooperi, and Christopher G. Proud‡ §§ From the ‡Department of Biosciences, University of Kent at Canterbury, Canterbury, CT2 7NJ, United Kingdom and the iFred Hutchinson Cancer Research Centre, Seattle, Washington 98109 Initiation factor eIF4E binds to the 5*-cap of eukary- tion but can be unwound by eIF4A (1, 2). Binding of eIF4E to eIF4G can be blocked by regulator proteins termed eIF4E- otic mRNAs and plays a key role in the mechanism and regulation of translation. It may be regulated through binding proteins (4E-BPs), which interact with the same re- its own phosphorylation and through inhibitory binding gion of eIF4E that binds eIF4G (4). The best characterized of proteins (4E-BPs), which modulate its availability for them is 4E-BP1 (also called PHAS-I, (4)). It is regulated by initiation complex assembly. eIF4E phosphorylation is phosphorylation at multiple sites, in response, e.g. to insulin, enhanced by phorbol esters. We show, using specific which causes its dissociation from eIF4E (4). inhibitors, that this involves both the p38 mitogen-acti- eIF4E is a phosphoprotein, and its phosphorylation is gen- vated protein (MAP) kinase and Erk signaling pathways. erally enhanced by agents that activate translation (reviewed Cell stresses such as arsenite and anisomycin and the in Refs. 1 and 2). Phosphorylation of eIF4E increases its affin- a and interleukin-1b also cytokines tumor necrosis factor- ity for the cap and for mRNA and may also favor its entry into cause increased phosphorylation of eIF4E, which is abol- initiation complexes (1, 2). Both effects may be important in the ished by the specific p38 MAP kinase inhibitor, SB203580. activation of translation under conditions that increase eIF4E These changes in eIF4E phosphorylation parallel the ac- phosphorylation. The phosphorylation site in eIF4E is Ser tivity of the eIF4E kinase, Mnk1. However other stresses (5, 6), although the identity of the protein kinase responsible O , which also stimu- such as heat shock, sorbitol, and H 2 2 for its phosphorylation in vivo is less clear. We have recently late p38 MAP kinase and increase Mnk1 activity, do not shown that insulin-induced eIF4E phosphorylation requires increase phosphorylation of eIF4E. The latter stresses in- the MAP kinase signaling pathway (also termed the Erk, ex- crease the binding of eIF4E to 4E-BP1, and we show that tracellular signal-regulated kinase pathway, the term used this blocks the phosphorylation of eIF4E by Mnk1 in vitro, here) (7). However, eIF4E is not a substrate for the Erks, and which may explain the absence of an increase in eIF4E we have shown that it is phosphorylated instead at Ser by a phosphorylation under these conditions. novel Erk-activated protein kinase, MAP kinase signal-inte- grating kinase-1 (Mnk1) (8, 9). Mnk1 is also phosphorylated and activated by an additional enzyme related to Erk, p38 MAP Initiation factor eIF4E plays a key role in mRNA translation and its regulation (1, 2). eIF4E binds to the 7-methylguanosine kinase, which lies on a distinct signaling pathway activated by cell stresses and cytokines (8 –10). triphosphate (“cap”) structure found at the 59-end of eukaryotic cytoplasmic mRNAs. eIF4E also interacts with eIF4G, a large Here we show that the increased phosphorylation of eIF4E brought about by the phorbol ester tetradecanoylphorbol 13- scaffolding protein, which itself binds to other translation fac- tors including eIF4A, an RNA helicase, and eIF3, a multimeric acetate (TPA), which activates members of the protein kinase C (PKC) family, requires the Erk and p38 MAP kinase pathways. protein that binds to the 40 S ribosomal subunit (3). The complex of eIF4E, eIF4G, and eIF4A is often termed eIF4F and Furthermore, we show that eIF4E phosphorylation is enhanced by agents, such as certain stresses and cytokines, that activate is believed to be especially important for the translation of mRNAs whose 59-untranslated regions are rich in secondary p38 MAP kinase and that this is blocked by a specific inhibitor of this enzyme. Our data support the identity of Mnk1 as a structure, because such structures in general inhibit transla- physiologically important eIF4E kinase. Certain stresses that activate p38 MAP kinase do not increase eIF4E phosphoryla- * This work was supported by Grant G9411756 from the Medical tion. This is likely to be due to the increased association of Research Council (to C. G. P.), Grant 046110 from the Wellcome Trust eIF4E with 4E-BP1 that these conditions bring about, because (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 4E-BP1 inhibits phosphorylation of eIF4E by Mnk1. by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 MATERIALS AND METHODS solely to indicate this fact. Chemicals and Biochemicals—Unless otherwise stated, chemicals § These authors contributed equally to the work. were obtained as described previously (7, 11). Anti-(P)Erk was from Present address: Laboratoire de Biophysique, Muse ´ e National New England BioLabs. Anti-human eIF4E was raised against a syn- d’Histoire Naturelle, 43 rue Cuviere, 75231 Paris Cedex 05, France. ** Present address: Faculty of Medicine, Dept. of Medical Biochem- istry, Section on Molecular Carcinogenesis, P.O. Box 9503, 2300 RA Leiden, The Netherlands. The abbreviations used are: 4E-BP, eIF4E-binding protein; MAP, ‡‡ Recipient of a Studentship from Pfizer Central Research. mitogen-activated protein kinase; TPA, tetradecanoylphorbol 13-ace- §§ To whom correspondence should be addressed. Present address: tate; PKC, protein kinase C; CHO, Chinese hamster ovary; HUVEC, Dept. of Anatomy & Physiology, Medical Sciences Inst., University of human umbilical vein endothelial cell; MAPKAP-K, MAP kinase-acti- Dundee, Dundee, DD1 4HN, UK. Tel.: 44-1382-344919; Fax: 44-1382- vated protein kinase; GST, glutathione S-transferase; TNFa, tumor 322424; E-mail:[email protected]. necrosis factor-a; IL-1b, interleukin-1b; JNK, c-Jun N-terminal kinase. This paper is available on line at http://www.jbc.org 9373 This is an Open Access article under the CC BY license. 9374 Regulation of Initiation Factor 4E Phosphorylation thetic 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 de- scribed previously (8, 13). Human umbilical vein endothelial cells (HUVECs) were grown in modified MCDB131 medium (Clonetics), sup- plemented with 2% (v/v) fetal calf serum, 10 ng/ml epidermal growth factor, 1 mg/ml hydrocortisone, 50 mg/ml gentamicin, 50 ng/ml ampho- tericin B, and 12 mg/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 down- stream kinase MAP kinase-activated kinase-2 (MAPKAP-K2 (10)) us- ing recombinant hsp25 as substrate (14). Isolation and Analysis of eIF4E—eIF4E was isolated from cell ex- tracts by affinity chromatography on m GTP-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). RESULTS AND DISCUSSION TPA-induced Phosphorylation of eIF4E Requires the Erk and p38 MAP Kinase Pathways—In several cell types, phorbol es- ters 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 re- FIG.1. A, 293 cells were pretreated with or without SB203580 (25 duced the ability of TPA to enhance eIF4E phosphorylation mM) and/or PD098059 (50 nM) for 60 min and then with or without TPA (Fig. 1A) and also completely inhibited the activation of Erk by (150 nM) for a further 15 min, as indicated, prior to extraction. Extracts TPA in 293 cells (Fig. 1B, lanes 1– 4). However, the inhibition of were processed for analysis of eIF4E phosphorylation: the positions of eIF4E phosphorylation was incomplete indicating that other its phosphorylated (4E(P)) and nonphosphorylated (4E) forms are indi- cated. The figure shows a Western blot developed using ECL. Numbers signaling pathways were involved. We therefore tested the below each lane show the percentage of eIF4E in the phosphorylated effect of a specific inhibitor of p38 MAP kinase SB203580 (21) form (% 4E(P)), as determined by densitometric analysis of the ECL on eIF4E phosphorylation. It has been shown not to interfere images. B, 293 cells were treated as for A. Extracts were analyzed either with stress-, cytokine-, or growth factor-induced activation of by SDS-polyacrylamide gel electrophoresis and Western blotting using an antiphospho-Erk antibody (figure is a blot developed using ECL) other signaling pathways such as Erk, JNK, or p70 S6 kinase (lanes 1– 4) or for MAPKAP-K2 activity (figure is an autoradiograph) (21–24). SB203580 partially blocked TPA-induced eIF4E phos- (lanes 5– 8). The positions of Erk and hsp25 are indicated. C, activity of phorylation, and its use together with PD098059 completely Mnk1-GST in 293 cells treated with or without TPA and the inhibitors abolished TPA-induced eIF4E phosphorylation (Fig. 1A). As (as indicated) as described for A. The resulting dried gel was analyzed using a PhosphorImager and the data thus obtained are presented in shown in Fig. 1B (lanes 5– 8), TPA activates MAPKAP-K2, arbitrary PhosphorImager units (control is set at 1.0 and corresponds to which is activated by p38 MAP kinase (10), in 293 cells. These unstimulated (unstim) cells). D, TPA increases eIF4E phosphorylation data suggest that TPA acts through both Erk and p38 MAP in CHO.K1 cells, and this increase is blocked by PD098059/SB203580. kinase to increase eIF4E phosphorylation in 293 cells. The experiments were performed and the data are presented as in A. 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 due to the high basal level of eIF4E phosphorylation (Fig. 1D). transfected 293 cells with a vector encoding wild-type Mnk1 This was partially decreased either by PD098059 or SB203580, fused to GST (8) and subjected the transfected cells to treat- although both were required to completely suppress it (Fig. ment with TPA in the absence or the presence of kinase inhib- 1D). Each compound alone also decreased the level of eIF4E itors. Cells were extracted, and Mnk1-GST was isolated and phosphorylation in TPA-treated CHO.K1 cells, but both were assayed. SB203580 or PD098059 only partially prevented the again required to abolish eIF4E phosphorylation completely activation of Mnk1 by TPA, but use of both completely abol- (Fig. 1D). Taken together, the data reinforce the conclusion ished it (Fig. 1C). The changes in Mnk1 activity parallel the that both the Erk and p38 MAP kinase cascades are involved in alterations in eIF4E phosphorylation observed under these mediating changes in eIF4E phosphorylation. Our data imply conditions, entirely consistent with a key role for Mnk1 in that the phorbol ester-induced phosphorylation of eIF4E is not, mediating eIF4E phosphorylation (8). as has previously been suggested (1), directly mediated by TPA also increases eIF4E phosphorylation in CHO.K1 cells, PKC. A more likely route by which TPA increases eIF4E phos- which involves the so-called conventional isoforms of PKC (11). phorylation is through activation of the Erk/p38 MAP kinase Analysis of the roles of signaling pathways in the phosphoryl- cascades, via PKC, leading to the activation of Mnk1, which ation of eIF4E in these cells is more complex than for 293 cells itself directly phosphorylates eIF4E (8). This is consistent with Regulation of Initiation Factor 4E Phosphorylation 9375 nase pathway (8). Changes in Mnk1 activity again parallel those in eIF4E phosphorylation (Fig. 2C). The ability of arsen- ite 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 arsen- ite-induced eIF4E phosphorylation and p38 MAP kinase acti- vation (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. Ani- somycin also increases eIF4E phosphorylation in NIH 3T3 cells, and this increase is blocked by SB203580 (25). The finding that arsenite stimulates eIF4E phosphorylation is surprising given that arsenite potently inhibits protein syn- thesis (26), whereas eIF4E phosphorylation is normally asso- ciated with its activation. It is likely that arsenite inhibits other steps in translation, and, indeed, we have shown that it increases phosphorylation of the a-subunit of eIF2, which is well known to lead to inhibition of peptide chain initiation (27). Effects of Other Stresses on eIF4E Phosphorylation—Other stresses such as hyperosmolarity (sorbitol) and oxidative stress (hydrogen peroxide) also activate p38 MAP kinase, MAP- KAP-K2 (Fig. 2A) and Mnk1 (Fig. 2C). However, unlike arsen- ite, 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 acti- vate 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 acti- FIG.2. A, measurement of MAPKAP-K2 activity in 293 cells. Cells vate Mnk1? To try to explain this apparent paradox, we ana- were preincubated with or without SB203580 (25 mm) for 60 min prior lyzed the association of eIF4E with its regulator 4E-BP1; we to treatment with arsenite (0.1 mM, 20 min), H O (3 mM, 25 min), 2 2 sorbitol (0.6 mM, 25 min), and heat shock (HS, 44 °C, 30 min and 120 have previously shown that in CHO.K1 cells, most stresses min) as indicated. The figure shows an autoradiograph of the SDS- increase binding of 4E-BP1 to eIF4E (16). This effect is also polyacrylamide gel. The position of hsp25 is indicated. Numbers below seen in 293 cells (Fig. 3A). (The exception here (as in CHO cells) each lane show the relative activity of MAPKAP-K2 (percentage of is arsenite, which does not cause increased binding of 4E-BP1 control), determined by PhosphorImager analysis of the dried gel. B, assessment of eIF4E phosphorylation. Lanes 1–5, 293 cells. Cells were to eIF4E. This is probably because it can activate the rapamy- pretreated with or without SB203580 and/or PD098059 for 60 min and cin-sensitive signaling pathway (28), which leads to the phos- then exposed, where indicated, to sodium arsenite (0.1 mM) for 20 min, phorylation of 4E-BP1 and its dissociation from eIF4E (4).) as indicated. Lanes 6 –9, CHO.K1 cells. Cells were treated as for lanes 1–5 These findings raised the possibility that the association of (except that PD098059 was not used here). Data are presented as Fig. 1A. 4E-BP1 with eIF4E might impair phosphorylation of the latter C, activity of Mnk1-GST in 293 cells. Stimuli and inhibitors were used under the conditions described above. The data were obtained and are by Mnk1. To test this, we examined the effect of 4E-BP1 on the presented as in Fig. 1C. unstim, unstimulated; SB, SB203580; HS, heat ability of Mnk1 to phosphorylate eIF4E in vitro. The data (Fig. shock; SORB, sorbitol; ARS, arsenite; ANISO, anisomycin. D, assessment 3B) clearly show that 4E-BP1 substantially inhibits the phos- of the level of phosphorylation of eIF4E. Lanes 1–5, 293 cells; lanes 6 –10, phorylation of eIF4E by Mnk1. The highest amount of 4E-BP1 CHO.K1 cells. All stress conditions are as same as those in A: con, control; ars, arsenite; HO, hydrogen peroxide; HS, heat shock; sor, used represents saturation of the eIF4E with 4E-BP1 as indi- sorbitol. cated by the fact that addition of further 4E-BP1 resulted in (i) it not being retained on m GTP-Sepharose, i.e. not being asso- all the published data on phorbol ester-induced eIF4E phos- ciated with eIF4E, and (ii) phosphorylation of the excess 4E- phorylation (11, 17–19). BP1 by the Erk present in the activated Mnk1, with only free Arsenite Induces the Phosphorylation of eIF4E, Which Is 4E-BP1 (and not the 4E-BP1/eIF4E complex) being a substrate Blocked by SB203580 —The above data prompted us to ask for Erk (12) (data not shown). SB203580 had no effect on the whether other treatments that activate p38 MAP kinase affect association of eIF4E with 4E-BP1, either under stress or con- eIF4E phosphorylation. Arsenite potently activates the p38 trol conditions (16). 4E-BP1 did not affect the phosphorylation MAP kinase pathway in 293 cells (Fig. 2A) and also markedly of another substrate, the cAMP-response element binding pro- increased eIF4E phosphorylation (Fig. 2B, lanes 1–5). The p38 tein, by Mnk1 (data not shown). This suggests that inhibition of MAP kinase inhibitor SB203580 (21) blocked both this and the eIF4E phosphorylation by 4E-BP1 reflects the inability of arsenite-induced activation of MAPKAP-K2 (Fig. 2, A and B). Mnk1 to phosphorylate eIF4E in the eIF4E/4E-BP1 complex 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 X. Wang, A. Flynn, A. J. Waskiewicz, B. L. J. Webb, R. G. Vries, I. A. phosphorylation appears to be mediated by the p38 MAP ki- Baines, J. A. Cooper, and C. G. Proud, unpublished data. 9376 Regulation of Initiation Factor 4E Phosphorylation FIG.3. A, effects of stress on binding of 4E-BP1 to eIF4E in 293 cells. Cells were subjected to stress conditions as in Fig. 2A and then ex- tracted and analyzed for 4E-BP1 and eIF 4E. The figure shows a Western blot developed using ECL. con, control; ars, arsenite; HO, hydrogen peroxide; sor, sorbitol; HS, heat shock. B, effect of 4E-BP1 on the phosphorylation of eIF4E by Mnk1. Lanes 1– 4, eIF4E was incu- bated with Mnk1-GST that had previously been activated by incubation with Erk1 and ATP/Mg, followed by washing of the Mnk1-GST bound to glutathione-Sepharose with LiCl (0.5 M) to remove as much Erk1 as possible (8). Reactions for the phosphorylation of eIF4E by Mnk1-GST were performed in the absence (lane 1) or the presence (lanes 2– 4)of 4E-BP1 and radiolabeled ATP. Lanes 2– 4 contain increasing amounts of 4E-BP1 (in the ratio 1:3:7). Numbers below each lane indicate the relative labeling of eIF4E as determined by PhosphorImager analysis. FIG.4. Cytokines increase eIF4E phosphorylation. A, HUVECs C, dissociation of eIF4F complexes in stressed cells (see Fig. 2A), as- were pretreated with or without SB203580 (25 mM) for 60 min before sessed by analyzing samples isolated as described under “Materials and exposure to TNFa (5 ng/ml) for 10 min, as indicated, and extracts were Methods” (m GTP-Sepharose-bound material) on an 8% polyacrylamide analyzed for eIF4E phosphorylation. Data are presented as in Fig. 1A. gel followed by blotting with anti-eIF4G. B, CHO.K1 cells were treated for 20 min with or without IL-1b (indi- cated concentration). Cells were then extracted, and extracts were analyzed for eIF4E phosphorylation. Data are presented as Fig. 1A. and is likely to be specific for this substrate. The inhibition of Mnk1-catalyzed eIF4E phosphorylation by 4E-BP1 provides an explanation for the differing effects of phosphorylation of eIF4E through the p38 MAP kinase path- stresses on eIF4E phosphorylation, and, in particular, for the way. In CHO.K1 cells, another cytokine, interleukin-1b (IL-1b) ability of heat shock to reduce eIF4E phosphorylation (re- activates p38 MAP kinase, although less markedly than TNFa viewed in Ref. 29). Some studies have shown that rapamycin does in HUVECs (data not shown). IL-1b (5 ng/ml) increased reduces the level of eIF4E phosphorylation (25, 30). Our data the phosphorylation of eIF4E in CHO.K1 cells (Fig. 4B). This suggest that this may be due to increased association of eIF4E effect, like that of TNFa in HUVECs, was prevented by with 4E-BP1 caused by rapamycin (due to dephosphorylation of SB203580 (data not shown). 4E-BP1 (4)) and resulting inhibition of Mnk1-catalyzed eIF4E Conclusions—The ability of activators of p38 MAP kinase to phosphorylation. 4E-BP1 also blocks the phosphorylation of increase eIF4E phosphorylation was seen in three different cell eIF4E by PKC (31). The binding site for 4E-BP1 in eIF4E has types, human embryonic kidney (293) cells, CHO cells, and recently been identified (32). Because it is some distance from HUVECs and in response to stresses and cytokines. In all Ser in the three-dimensional structure of the protein, it cases, SB203580 blocked the phosphorylation of eIF4E. The seems unlikely that 4E-BP1 actually occludes the phosphoryl- data for TNFa in HUVECs are of particular note given that ation site. The effect of 4E-BP1 may instead reflect interference the TNFa-stimulated induction of the cell adhesion molecule with the interaction between these kinases and other regions of V-CAM is mediated by the p38 MAP kinase pathway (34) and the eIF4E protein required for kinase/substrate binding. involves post-transcriptional effects that might be related to The increased binding of 4E-BP1 to eIF4E was accompanied by changes in eIF4E phosphorylation. Both the stress stimuli a decrease in the binding of eIF4G to eIF4E (Fig. 3C), as expected (arsenite and anisomycin) and TNFa also activate the JNK from the mutually competitive nature of their interactions (33). pathway. However, the ability of SB203580 (which does not Consistent with its lack of effect on 4E-BP1 binding, arsenite also affect JNK activity in the cells used here) to block eIF4E had no effect on the association of eIF4E with eIF4G. phosphorylation indicates that the JNK pathway is not in- Regulation of eIF4E Phosphorylation by Cytokines That Ac- volved in modulating eIF4E phosphorylation. tivate p38 MAP Kinase—It was important to ascertain whether We have previously shown that insulin-induced phosphoryl- treatment of cells with physiological activators of p38 MAP ation of eIF4E requires the Erk pathway (7). Taken together, kinase, such as cytokines, also altered the phosphorylation of our findings show that eIF4E phosphorylation can be mediated eIF4E. Tumor necrosis factor-a (TNFa) is a physiological reg- by two distinct signaling pathways, the Erk and p38 MAP ulator of endothelial cell function (34), and in HUVECs it kinase pathways, depending on the stimulus, consistent with markedly activates the p38 MAP kinase pathway without any the established regulatory properties of the eIF4E kinase apparent effect on Erk activity (data not shown and Ref. 34). Mnk1, which is a target for activation by both (8, 9). Changes in We therefore studied its effect on the phosphorylation of eIF4E. eIF4E phosphorylation largely mirror alterations in Mnk1 activ- TNFa increased the phosphorylation of eIF4E (Fig. 4A), and ity, consistent with a physiological role for this kinase in eIF4E this increase was prevented by SB203580, which blocked acti- phosphorylation. The only exceptions are stress conditions that vation of the p38 MAP kinase pathway and hence of MAPKAP- increase binding of 4E-BP1 to eIF4E. In almost all cases, such K2. 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Published: Apr 1, 1998

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