Interleukins 2 and 15 Regulate Ets1 Expression via ERK1/2 and MNK1 in Human Natural Killer Cells
2004; Elsevier BV; Volume: 280; Issue: 6 Linguagem: Inglês
10.1074/jbc.m408356200
ISSN1083-351X
AutoresEric M. Grund, Demetri D. Spyropoulos, Dennis K. Watson, Robin C. Muise‐Helmericks,
Tópico(s)IL-33, ST2, and ILC Pathways
ResumoInterleukins (IL)-2 and IL-15 regulate natural killer (NK) cell proliferation, survival, and cytolytic activity. Ets1 is a transcription factor expressed early in NK cell differentiation. Because IL-2Rβ, IL-2Rγ, IL-15, and Ets1 knock-out mice similarly lack NK cells, we explored a molecular connection between IL-2R signaling and Ets1. Here we report the post-transcriptional regulation of Ets1 by IL-2R signaling in human NK cells. IL-2 and IL-15 stimulation leads to increased Ets1 protein levels with no significant change in mRNA levels. Pulse and pulse-chase experiments show that IL-2 stimulation results in both a marked increase in the nascent translation of Ets1 and an increased protein half-life. Pharmacological inhibition of MEK specifically blocks IL-2- and IL-15-induced translation, whereas p38, phosphatidylinositol 3-kinase, and mTOR inhibitors had no effect on Ets1 levels. Fli1, an Ets family member, exhibited a different mechanism of regulation, illustrating the specificity of IL-2R β and γ subunit signaling on the regulation of Ets1 expression. Expression of a dominant negative form of MNK1, a regulator of the translation initiation factor eIF4E, blocks the expression of Ets1 as do the dominant negative forms of the common IL-2R β and γ chains. Expression of Ets1 is regulated similarly in normal peripheral human NK cells. Taken together, our findings provide a direct link between IL-2R subunit signaling and Ets1 expression and helps to explain the interdependence of the IL-2R subunits and Ets1 for NK cell development and function. Interleukins (IL)-2 and IL-15 regulate natural killer (NK) cell proliferation, survival, and cytolytic activity. Ets1 is a transcription factor expressed early in NK cell differentiation. Because IL-2Rβ, IL-2Rγ, IL-15, and Ets1 knock-out mice similarly lack NK cells, we explored a molecular connection between IL-2R signaling and Ets1. Here we report the post-transcriptional regulation of Ets1 by IL-2R signaling in human NK cells. IL-2 and IL-15 stimulation leads to increased Ets1 protein levels with no significant change in mRNA levels. Pulse and pulse-chase experiments show that IL-2 stimulation results in both a marked increase in the nascent translation of Ets1 and an increased protein half-life. Pharmacological inhibition of MEK specifically blocks IL-2- and IL-15-induced translation, whereas p38, phosphatidylinositol 3-kinase, and mTOR inhibitors had no effect on Ets1 levels. Fli1, an Ets family member, exhibited a different mechanism of regulation, illustrating the specificity of IL-2R β and γ subunit signaling on the regulation of Ets1 expression. Expression of a dominant negative form of MNK1, a regulator of the translation initiation factor eIF4E, blocks the expression of Ets1 as do the dominant negative forms of the common IL-2R β and γ chains. Expression of Ets1 is regulated similarly in normal peripheral human NK cells. Taken together, our findings provide a direct link between IL-2R subunit signaling and Ets1 expression and helps to explain the interdependence of the IL-2R subunits and Ets1 for NK cell development and function. Natural killer (NK) 1The abbreviations used are: NK, natural killer; IL, interleukin; MAP, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; IL-2R, IL-2 receptor; MEK, MAP kinase/ERK kinase; GST, glutathione S-transferase; UTR, untranslated region.1The abbreviations used are: NK, natural killer; IL, interleukin; MAP, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; IL-2R, IL-2 receptor; MEK, MAP kinase/ERK kinase; GST, glutathione S-transferase; UTR, untranslated region. cells have the critical innate immune function of independently recognizing and lysing tumor and virally and bacterially infected cells (1Smyth M.J. Hayakawa Y. Takeda K. Yagita H. Nat. Rev. Cancer. 2002; 2: 850-861Crossref PubMed Scopus (573) Google Scholar). In addition to NK cell direct cytotoxicity, they also act to stimulate other cells of the immune system by secretion of immunoregulatory cytokines such as tumor necrosis factor, chemokines, and interleukins that stimulate T cell and B cell responses (2Lauwerys B.R. Garot N. Renauld J.C. Houssiau F.A. J. Immunol. 2000; 165: 1847-1853Crossref PubMed Scopus (183) Google Scholar). Interestingly, NK cells participate in antigen-specific immune responses by responding to IL-2 secreted by T cells and dendritic cells (3Fehniger T.A. Cooper M.A. Nuovo G.J. Cella M. Facchetti F. Colonna M. Caligiuri M.A. Blood. 2003; 101: 3052-3057Crossref PubMed Scopus (652) Google Scholar). Therefore, NK cells are central to both the innate and acquired immune responses. Indeed, there is an impressive correlation between low NK cell function and susceptibility to viral and other microbial infections (4Biron C.A. Byron K.S. Sullivan J.L. N. Engl. J. 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Cytokines act en masse to control NK cellular responses through the activation of signaling pathways, including the MAP kinase pathways (ERK1/2, p38, and c-Jun N-terminal kinase), as well as the PI3K pathway (14Colucci F. Caligiuri M.A. Di Santo J.P. Nat. Rev. Immunol. 2003; 3: 413-425Crossref PubMed Scopus (382) Google Scholar). These signaling pathways transmit external stimuli to the nucleus and activate numerous transcription factors, resulting in both the temporal and spatial changes in gene expression required for cellular proliferation, cytokine secretion, or cytotoxicity (15Gaffen S.L. Cytokine. 2001; 14: 63-77Crossref PubMed Scopus (151) Google Scholar). Thus, a variety of cytokines act in concert to coordinately regulate NK cell processes. Interestingly, both IL-2 and IL-15 selectively induce survival and proliferation of NK cells and other CD56+ cells such as NKT cells but not conventional T cells or B cells (16Dunne J. Lynch S. O'Farrelly C. Todryk S. Hegarty J.E. Feighery C. Doherty D.G. J. Immunol. 2001; 167: 3129-3138Crossref PubMed Scopus (146) Google Scholar). Both the IL-2 and IL-15 receptors (IL-2R and IL-15R) are composed of three different subunits (α, β, and γ) that are involved in differential signaling and ligand binding specificities. The IL-2Rα and IL-15Rα subunit specifically binds IL-2 or IL-15, respectively (17Giri J.G. Kumaki S. Ahdieh M. Friend D.J. Loomis A. Shanebeck K. DuBose R. Cosman D. Park L.S. Anderson D.M. EMBO J. 1995; 14: 3654-3663Crossref PubMed Scopus (560) Google Scholar). Importantly, only the β subunit participates in binding IL-2 and IL-15, whereas the γ subunit participates in binding IL-2, -4, -7, -9, and -15 (15Gaffen S.L. Cytokine. 2001; 14: 63-77Crossref PubMed Scopus (151) Google Scholar). In addition to IL-2 and IL-15, NK cells respond to other cytokines such as IL-18 and IL-12 that do not share receptor subunits with IL-2 or IL-15 (14Colucci F. Caligiuri M.A. Di Santo J.P. Nat. Rev. Immunol. 2003; 3: 413-425Crossref PubMed Scopus (382) Google Scholar). The common signaling components and functions of IL-2 and IL-15 in NK cells suggest common regulation of a shared set of downstream target genes. The founding member of the Ets family of transcription factors, Ets1, plays a pivotal role in the regulation of NK cell function (18Barton K. Muthusamy N. Fischer C. Ting C.N. Walunas T.L. Lanier L.L. Leiden J.M. Immunity. 1998; 9: 555-563Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). The earliest NK cell precursor in bone marrow is characterized by Ets1 transcript expression (19Rosmaraki E.E. Douagi I. Roth C. Colucci F. Cumano A. Di Santo J.P. Eur. J. Immunol. 2001; 31: 1900-1909Crossref PubMed Scopus (286) Google Scholar). Ets1-deficient mice have severe defects in the NK cell lineage showing marked reductions in NK cell number and NK cell cytolytic activity, have reduced interferon γ secretion, and develop tumors upon challenge with NK cell susceptible tumor cells (18Barton K. Muthusamy N. Fischer C. Ting C.N. Walunas T.L. Lanier L.L. Leiden J.M. Immunity. 1998; 9: 555-563Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). In addition to their defects in NK cells, the thymocytes of Ets1-/- mice do not produce normal levels of IL-4 after anti-CD3 stimulation (20Walunas T.L. Wang B. Wang C.R. Leiden J.M. J. Immunol. 2000; 164: 2857-2860Crossref PubMed Scopus (87) Google Scholar). Moreover, their peripheral T cells display a severe proliferative defect in response to multiple activation signals, resulting in spontaneous apoptosis in vitro (21Muthusamy N. Barton K. Leiden J.M. Nature. 1995; 377: 639-642Crossref PubMed Scopus (286) Google Scholar). These data suggest that Ets1 is an essential mediator of NK cell development and function. A NK cell phenotype strikingly similar to that described for Ets1 is displayed by the mice deficient in IL-15R/IL-2R β and γ subunits (30Lian R.H. Kumar V. Semin. Immunol. 2002; 14: 453-460Crossref PubMed Scopus (51) Google Scholar, 31Kennedy M.K. Glaccum M. Brown S.N. Butz E.A. Viney J.L. Embers M. Matsuki N. Charrier K. Sedger L. Willis C.R. Brasel K. Morrissey P.J. Stocking K. Schuh J.C. Joyce S. Peschon J.J. J. Exp. Med. 2000; 191: 771-780Crossref PubMed Scopus (1310) Google Scholar, 32DiSanto J.P. Muller W. Guy-Grand D. Fischer A. Rajewsky K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 377-381Crossref PubMed Scopus (747) Google Scholar, 33Gilmour K.C. Fujii H. Cranston T. Davies E.G. Kinnon C. Gaspar H.B. Blood. 2001; 98: 877-879Crossref PubMed Scopus (82) Google Scholar, 34Suzuki H. Duncan G.S. Takimoto H. Mak T.W. J. Exp. Med. 1997; 185: 499-505Crossref PubMed Scopus (302) Google Scholar). 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Chem. 2002; 277: 29643-29653Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), we report here that IL-2 signaling positively regulates Ets1 expression on the post-transcriptional level via the activation of MEK > ERK1/2. Induction of Ets1 by IL-2R activation correlates with the activation of the translation initiation factor, eIF4E through `1 phosphorylation but not through a PI3K/AKT-dependent mTOR pathway. Expression of a dominant negative form of MNK1 results in a marked decrease in Ets1 expression, suggesting a signaling pathway whereby Ets1 expression is regulated by an Erk1 > MNK1 > eIF4E-dependent pathway that leads to an increased Ets1 translation initiation. We also show that both IL-2 and IL-15 similarly regulate Ets1 and that blockade of either the common β or γ chain of the IL-2R results in a decreased expression of Ets1. These results demonstrate a molecular link between IL-2R signaling and Ets1 and a possible explanation for the commonality of knock-out phenotypes. Cell Culture, Cytokines, and Inhibitors—The nontransformed immortalized human NK cell line, NK92, (35Maki G. Klingemann H.G. Martinson J.A. Tam Y.K. J. Hematother. Stem Cell Res. 2001; 10: 369-383Crossref PubMed Scopus (167) Google Scholar) was obtained from the American Type Tissue Collection and maintained in α-minimum essential medium (Invitrogen) supplemented with 12.5% fetal calf serum (Invitrogen), 12.5% horse serum (Invitrogen), 150 units/ml of IL-2, 0.1 mm 2-mercaptoethanol, and penicillin-streptomycin; maintained at 37 °C and 5% CO2; and passaged every 48 h. Recombinant human IL-2 was made available by the Biological Resources Branch, National Cancer Institute Preclinical Repository. Recombinant human IL-15 was obtained from R &D Systems Inc. Recombinant human IL-18 was purchased from Medical & Biological Laboratories. For IL-2 starvation, NK92 cells were plated in the absence of IL-2 for 48–60 h prior to IL-2 stimulation (20 ng/ml). The concentrations of the other cytokines were IL-15, 10 ng/ml; IL-12, 5 ng/ml; and IL-18, 5 ng/ml. The MEK inhibitor PD98059, the p38 inhibitor SB203580, the PI3K inhibitor wortmannin, and the FK506-binding protein inhibitor rapamycin were purchased from BIOMOL Research Labs, Inc. All of the inhibitors were added 30 min prior to cytokine addition. The concentrations of each are as follows: PD98059, 100 μm; SB203580, 50 μm; wortmannin, 100 nm; and rapamycin, 1 μg/ml. Isolation of Normal Peripheral Human NK Cells—Peripheral blood lymphocytes were isolated from the blood of healthy adult donors by density gradient centrifugation (Histopaque; Sigma). Isolated peripheral blood lymphocytes were removed from the gradient and cultured in complete media containing either 4 ng/ml IL-2 or 2 ng/ml IL-15. After 2 days in culture, peripheral NK cells were isolated using a NK cell negative isolation kit using procedures supplied by the manufacturer (Dynal). NK cells were cultured for 24 h as above in the presence or absence of 100 μm PD98059. Antibodies—Rabbit polyclonal antibodies directed against Ets1 (C-20), Ets2 (C-20), Fli1–1 (C-19), ERK2 (K-23), and cyclin D3 (C-16) were purchased from Santa Cruz Biotechnology. Ets1 monoclonal antibody was obtained from Transduction Laboratories. Antibodies directed against actin were obtained from Oncogene Research Products. Active MAP kinase antibody was purchased from Promega. AKT, phospho-AKT (Ser473), p38, phospho-p38 (Thr180/Tyr182), phospho-MNK1 (Thr197/202), eIF4E, and phospho-eIF4E (Ser209) antibodies were purchased from Cell Signaling. Antibodies directed against GST and PI3K p85 were purchased from Upstate Biotechnology, Inc. Secondary horseradish peroxidase-conjugated antibodies were goat anti-mouse IgM (Sigma), goat F(ab′)2 anti-mouse IgG (H&L), and goat anti-rabbit IgG (H&L) (Caltag). Western Blot Analysis—Total protein was prepared using radioimmune precipitation assay buffer (0.15 m NaCl, 50 mm Tris-HCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) containing complete protease inhibitors (Roche Applied Science), 40 mm NaFl, and 2 mm Na3VO4. Protein concentration was determined by BCA method (Pierce), and equal amounts of total protein were resolved by 10% SDS-PAGE, transferred to polyvinylidene difluoride (Millipore) membrane, and probed with the appropriate antibodies. Primary antibodies were detected with horseradish peroxidase-linked secondary antibodies and visualized using Luminol Reagent (Santa Cruz Biotechnology) according to the manufacturer's protocols. Northern Blot Analysis—Total RNA was isolated using RNA STAT-60 (TEL-TEST, INC.) according to the manufacturer's protocol. Total RNA (10 μg) was separated on a 1% agarose formaldehyde gel, transferred to Duralon-UV membrane (Stratagene), and subjected to UV cross-linking. The Ets1 probe was derived from a full-length cDNA clone and the β-actin probe from I. Maroulakou (Tufts University). Hybridizations were performed using probes generated using a random priming protocol and QuikHyb in procedures provided by the manufacturer (Stratagene). The blots were quantitated using a Bio-Rad Molecular Imaging System. Northern blotting was repeated at least three independent times for each experiment. [35S]Methionine/Cysteine Pulse and Pulse-Chase—NK92 cells were incubated in RPMI medium without methionine and cysteine (Sigma) for 15 min. [35S]Methionine/cysteine (Promix; Amersham Biosciences) was added at 100 μCi/ml of medium and incubated for the times indicated in the text. The cells were lysed using Nonidet P-40 lysis buffer consisting of 150 mm NaCl, 50 mm Tris, pH 7.4, 40 mm NaF, 5% Nonidet P-40, 10 mm Na3VO4. Equal amounts of total protein were immunoprecipitated using antibodies described in the text, resolved by SDS-PAGE, transferred to polyvinylidene difluoride, dried, and exposed to x-ray film. For Pulse-Chase analysis, NK92 cells were resuspended in RPMI medium without methionine and cysteine for 15 min and incubated for 1 h with [35S]methionine/cysteine at 100 μCi/ml. The cells were washed and resuspended in complete α-minimum essential medium with excess methionine (10 mm) and cysteine (3 mm) (Sigma) and allowed to incubate for the times indicated in the text. Equal amounts of total protein were immunoprecipitated with the antibodies described in the text, resolved by SDS-PAGE, transferred to polyvinylidene difluoride, and exposed to x-ray film. Band density was quantitated using a Bio-Rad Molecular Imaging System and expressed as relative units of density. Immunoprecipitation—NK92 cells were lysed in Nonidet P-40 lysis buffer, and equal amounts of total protein were immunoprecipitated using either antibody-bound agarose beads or antibody plus protein A-Sepharose beads (Amersham Biosciences). Protein was immunoprecipitated by overnight incubation at 4 °C with agitation in Nonidet P-40 lysis buffer. Immunoprecipitates were then centrifuged at 2000 cpm, washed extensively in Nonidet P-40 lysis buffer, and resolved on 10% SDS-polyacrylamide gels followed by Western blot analysis. Transfection—pEYFP-N1 was purchased from Clontech, and the dominant negative forms of IL-2R β and γ chains were kindly provided by Warren J. Leonard (National Institutes of Health). Transient transfections were performed by electroporation using a newly defined protocol, 2Grund, E. M., and Muise-Helmericks, R. C. (2005) J. Immunol. Methods, in press. which allows for the efficient transfection of this cell type. Briefly, electroporations were performed using a Bio-Rad Gene Pulser II in freshly prepared 20 mm Hepes buffer containing 137 mm NaCl, 5 mm KCl, 0.7 mm Na2HPO4, 6 mm dextrose, 1.25% Me2SO, and 50 mm trehalose in the presence of 100 μg of plasmid. Flow Cytometry—Sixty hours post-electroporation, the cells were harvested by centrifugation, washed in ice-cold phosphate-buffered saline supplemented with 2% fetal bovine serum, filtered through a 40-μm nylon cell strainer (BD Falcon), and resuspended in the same medium at 106 cells/ml. Sorting was performed on a fluorescence-activated Vantage cell sorter (BD Biosciences). EYFP was excited by Innova 70 laser at 488-nm excitation wavelength. EYFP fluorescence emission was measured using a 530/30-nm band pass filter. IL-2 Regulates Ets1 Post-transcriptionally in NK Cells—The results generated from knock-out animal studies have suggested a molecular link between IL-2R-mediated signaling and the Ets1 transcription factor (18Barton K. Muthusamy N. Fischer C. Ting C.N. Walunas T.L. Lanier L.L. Leiden J.M. Immunity. 1998; 9: 555-563Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 32DiSanto J.P. Muller W. Guy-Grand D. Fischer A. Rajewsky K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 377-381Crossref PubMed Scopus (747) Google Scholar, 34Suzuki H. Duncan G.S. Takimoto H. Mak T.W. J. Exp. Med. 1997; 185: 499-505Crossref PubMed Scopus (302) Google Scholar). The human IL-2-dependent natural killer cell line, NK92, was used to dissect the interdependence of the IL-2R and Ets1. This cell line requires IL-2 for proliferation; removal of IL-2 causes a block in the G1 phase of the cell cycle without an increase in apoptosis (data not shown and Ref. 36Hodge D.L. Schill W.B. Wang J.M. Blanca I. Reynolds D.A. Ortaldo J.R. Young H.A. J. Immunol. 2002; 168: 6090-6098Crossref PubMed Scopus (53) Google Scholar). To determine whether IL-2 withdrawal affects Ets1 expression, NK92 cells were exposed to reduced levels of IL-2 for 48 h and then stimulated with IL-2. As shown in Fig. 1A, Ets1 protein levels drop significantly in low levels of IL-2. In contrast, Ets1 levels are induced 5-fold by 3 h after the addition of IL-2 and accumulate to 10-fold at 24 h post-IL-2 induction. Because IL-2, IL-15, and IL-18 modulate NK cell activity (37Fehniger T.A. Shah M.H. Turner M.J. VanDeusen J.B. Whitman S.P. Cooper M.A. Suzuki K. Wechser M. Goodsaid F. Caligiuri M.A. J. Immunol. 1999; 162: 4511-4520PubMed Google Scholar, 38Lauwerys B.R. Renauld J.C. Houssiau F.A. Cytokine. 1999; 11: 822-830Crossref PubMed Scopus (123) Google Scholar), we tested their effect on Ets1 expression in IL-2-starved NK92 cells. As shown in Fig. 1A both IL-2 and IL-15 induce Ets1 protein levels, whereas IL-18 does not. These data suggest that IL-2 and IL-15 induce Ets1 protein levels via the common IL-2R β and γ subunits. To determine the mechanism by which IL-2 induces the expression of Ets1, we tested the effect of IL-2 on Ets1 mRNA levels. NK92 cells starved of IL-2 for 48 h were incubated with IL-2 for increasing amounts of time and analyzed for Ets1 mRNA expression by Northern blot analysis. Ets1 mRNA levels are not significantly induced by the addition of IL-2 after starvation (Fig. 1B). Furthermore, the addition of different amounts of IL-2 after starvation does not affect Ets1 mRNA steady state levels (Fig. 1C). These results suggest that treatment of NK92 cells with IL-2 primarily regulates Ets1 expression via a post-transcriptional mechanism. IL-2 Regulates Ets1 Protein Synthesis and Stability—To determine whether IL-2 regulates Ets1 via increased translation initiation and/or protein stability, pulse and pulse-chase analyses were performed. IL-2-starved NK92 cells were pulse-labeled with [35S]methionine/cysteine in the presence or absence of IL-2. As shown by the immunoprecipitations and the quantitation in Fig. 2 (A and B), there is a marked increase in the rate of Ets1 synthesis in the presence of IL-2. To determine whether IL-2 also affected the stability of Ets1 protein, pulse-chase analyses were performed. As shown by the immunoprecipitations in Fig. 2C, IL-2 causes a slight increase in the stability of Ets1; however, the most pronounced effect of IL-2 is the induction of Ets1 protein synthesis in NK cells. IL-2 Regulates Ets1 Protein Levels through Erk1/2—Several pharmacological inhibitors were used to investigate the pathway(s) by which IL-2 induces the expression of Ets1 protein. NK92 cells growing in IL-2 were treated with PD98059, a MEK inhibitor, the p38 inhibitor SB203580, or the PI3K inhibitor wortmannin. The MEK inhibitor PD98059 had the largest effect on Ets1 steady state levels (Fig. 3A). The steady state expression of another Ets family member expressed in NK cells, Ets2, was unaffected by these pharmacological agents. To determine whether the MEK inhibitor specifically blocks Ets1 induction by IL-2, IL-2-starved cells were pretreated with PD98059 or SB203580 prior to IL-2 induction (Fig. 3B). Similarly, the most marked reduction of induced Ets1 levels was in the presence of the MEK inhibitor PD98059. To assess the specificity of ERK inhibition on Ets1, we evaluated the affect of these inhibitors on a different Ets family member whose expression is also induced by IL-2, Fli1. The induction of Fli1 is unaffected by ERK1/2 inhibition (Fig. 3B). Interestingly, Fli1 induction is blocked almost exclusively by p38 inhibition, suggesting that IL-2 regulates Ets family member expression through different signaling pathways in NK cells. Loss of Fli1, however, does not affect NK cell function. Because the state of phosphorylation of ERK1/2, p38, and AKT closely mirror their activities, we used phosphospecific antibodies in Western blot analyses to verify the effectiveness of these upstream inhibitors with NK92 cells. As expected, the p38 inhibitor SB203580 caused a reduction in the phosphorylation of p38 (Fig. 3C). Moreover, the phosphorylation of ERK1/2 was reduced by treatment with the MEK inhibitor PD98059 (Fig. 3C). Wortmannin, a PI3K inhibitor, was effective in reducing the phosphorylation status of AKT (Fig. 3C), a downstream effector of PI3K (41Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1510) Google Scholar). Interestingly, inhibition of the MEK > ERK1/2 pathway or the PI3K > AKT pathway resulted in a slight but reproducible increase in the phosphorylation of p38 (Fig. 3C). These data indicate that PD98059, SB203580, and wortmannin block their expected targets in NK92 cells. Taken together our results suggest that Ets1 protein levels are significantly regulated by the MEK > ERK1/2 pathway. Because Ets1 protein levels are not completely blocked by the MEK inhibitor, it is possible that secondary mechanisms of Ets1 regulation exist. To confirm the requirement of ERK1/2 for the IL-2-dependent increased rate of Ets1 translation, IL-2-starved cells were treated with the MEK inhibitor prior to IL-2 stimulation, and the translation rate of Ets1 was measured by pulse label analyses. As shown in Fig. 4A, inhibition of MEK by PD98059 results in a significant decrease in the rate of Ets1 synthesis. The quantitation of these data is shown in Fig. 4B. Taken together, these findings indicate that IL-2 causes an ERK1/2-dependent increase in Ets1 protein expression primarily on the level of translation initiation. Phosphorylation of MNK1 and eIF4E Is Regulated by IL-2 in NK Cells and Is Blocked by MEK Inhibition—Our results suggest that IL-2 signaling through MEK may regulate the rate of Ets1 translation. Multiple signaling pathways including PI3K/AKT and MEK/ERK have roles in the regulation of translation (42Muise-Helmericks R.C. Grimes H.L. Bellacosa A. Malstrom S.E. Tsichlis P.N. Rosen N. J. Biol. Chem. 1998; 273: 29864-29872Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 43Kelleher R.J. II I Govindarajan A. Jung H.Y. Kang H. Tonegawa S. Cell. 2004; 116: 467-479Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar). ERK1/2 has been shown to directly phosphorylate MNK1/2 (44Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (776) Google Scholar). MNK1/2 are serine/threonine kinases that directly phosphorylates eIF4E (45Wang X. Flynn A. Waskiewicz A.J. Webb B.L. Vries R.G. Baines I.A. Cooper J.A. Proud C.G. J. Biol. Chem. 1998; 273: 9373-9377Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), a cap-binding protein that regulates translation initiation by recruiting mRNAs to the ribosome (46Koromilas A.E. Lazaris-Karatzas A. Sonenberg N. EMBO J. 1992; 11: 4153-4158Crossref PubMed Scopus (335) Google Scholar). Phosphorylation by MNK1 serves to activate eIF4E independent of the 4E-binding proteins that bind and inhibit eIF4E. To determine whether IL-2 leads to the increased activity of MNK1 and its downstream substrate eIF4E in NK92 cells, their phosphorylation status was tested by Western blot analyses. As shown in Fig. 5A, IL-2 starvation caused a decrease in MNK1 phosphorylation. Treatment with the MEK inhibitor, PD98059 and not p38 inhibitor, SB203580 blocks the generation of phospho-MNK1. Likewise, eIF4E phosphorylation is induced by IL-2 but blocked by PD98059. Therefore, induction of MNK1 and eIF4E activity correlates with IL-2-induced MEK activation in NK92 cells. Consequently, IL-2-induced MNK1 and eIF4e activity may be linked to Ets1 induction in NK cells. Our pharmacological data suggest that Ets1 translation is independent of PI3K/AKT. AKT activation signals through mTOR to increase translation initiation (42Muise-Helmericks R.C. Grimes H.L. Bellacosa A. Malstrom S.E. Tsichlis P.N. Rosen N. J. Biol. Chem. 1998; 273: 29864-29872Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). To further investigate the role of this pathway on Ets1 levels, IL-2-stimulated NK92 cells were treated with rapamycin. Rapamycin suppresses mRNA translation through direct inhibition of mTOR (47Jefferies H.B. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693
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