Inhibition of Protein Phosphatase 1 Translation by Preventing JNK

Active ERK Contributes to Protein Translation by Preventing JNK-Dependent Inhibition of Protein Phosphatase 1

This information is current as Martha M. Monick, Linda S. Powers, Thomas J. Gross, of September 28, 2021. Dawn M. Flaherty, Christopher W. Barrett and Gary W. Hunninghake J Immunol 2006; 177:1636-1645; ; doi: 10.4049/jimmunol.177.3.1636

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Active ERK Contributes to Protein Translation by Preventing JNK-Dependent Inhibition of Protein Phosphatase 11

Martha M. Monick,2 Linda S. Powers, Thomas J. Gross, Dawn M. Flaherty, Christopher W. Barrett, and Gary W. Hunninghake

Human alveolar macrophages, central to immune responses in the lung, are unique in that they have an extended life span in contrast to precursor monocytes. We have shown previously that the ERK MAPK (ERK) pathway is constitutively active in human alveolar macrophages and contributes to the prolonged survival of these cells. We hypothesized that ERK maintains survival, in part, by positively regulating protein translation. In support of this hypothesis, we have found novel links among ERK, JNK, protein phosphatase 1 (PP1), and the eukaryotic initiation factor (eIF) 2␣. eIF2␣ is active when hypophosphorylated and is essential for initiation of protein translation (delivery of initiator tRNA charged with methionine to the ribosome). Using

[35S]methionine labeling, we found that ERK inhibition signiﬁcantly decreased protein translation rates in alveolar macrophages. Downloaded from Decreased protein translation resulted from phosphorylation (and inactivation) of eIF2␣. We found that ERK inhibition increased JNK activity. JNK in turn inactivated (via phosphorylation) PP1, the phosphatase responsible for maintaining the hypophosphorylated state of eIF2␣. As a composite, our data demonstrate that in human alveolar macrophages, constitutive ERK activity positively regulates protein translation via the following novel pathway: active ERK inhibits JNK, leading to activation of PP1␣, eIF2␣ dephosphorylation, and translation initiation. This new role for ERK in alveolar macrophage homeostasis may help to explain the survival characteristic of these cells within their unique high oxygen and stress microenvironment. The Journal of http://www.jimmunol.org/ Immunology, 2006, 177: 1636–1645.

uman alveolar macrophages survive for long periods in (caspase 9 and BimEL) (14, 15). ERK has also been shown to the lung (1). Survival occurs even in the face of expo- contribute to cell survival via other mechanisms. In one case, ERK H sure to chemical pollutants, reactive oxygen species, in- has been shown to regulate Hdm2 protein expression (Hdm2 in- flammatory mediators, and infectious agents (2). This ability to hibits p53 accumulation) by enhancing association of Hdm2 tran- adapt to stress is crucial to their survival. We have shown that scripts with polyribosomes (16). This effect of ERK on protein human alveolar macrophages are characterized by high constitu- translation led to our present hypothesis that in human alveolar by guest on September 28, 2021 tive activity of two survival pathways, PI3K/protein kinase B (Akt) macrophages constitutive ERK activity positively regulates protein and ERK MAPK (3). The survival effects of Akt are well described translation and survival. and include inhibition of BH3-only proapoptotic proteins, Ongoing protein translation is important for the continued health caspases, and the transcription factor, FoxOa (4, 5). The survival of cells and is regulated at a number of points, including initiation, mechanisms of ERK activity are less well described and appear to elongation, and termination. The primary rate-limiting step is ini- be cell type and system specific. Although ERK has been linked to tiation of translation, and this is regulated at multiple checkpoints apoptosis in a few cases (6), in the majority of studies, ERK ac- (17). Stresses, including DNA damage, misfolded proteins, nutri- tivity is linked to cell survival, and the specific mechanisms of its ent deprivation, and viral infection, induce a temporary shutdown survival activity are unknown (3, 7–11). of the translation machinery (17). In addition to the temporary ERK is a member of the MAPK family of serine/threonine ki- shutdown of translation induced by stress, a prolonged shutdown nases. It is activated by phosphorylation of a tyrosine ϫ threonine of protein translation accompanies apoptosis generated by many motif (TEY) and in turn phosphorylates downstream substrates stimuli (18, 19). The prolonged survival of alveolar macrophages containing a consensus proline-directed serine or threonine site suggests ongoing functional translation machinery. (PX(S/T)P) (12, 13). Among known ERK substrates are a number The two main pathways leading to initiation of translation are of proapoptotic proteins that are inhibited by ERK activity the eukaryotic initiation factor (eIF)3 4E pathway that brings mRNAs with 5Ј caps to the ribosome and the eIF2 pathway that is responsible for bringing the initiator methionyl-tRNA (Met- University of Iowa Carver College of Medicine and Veterans Administration Medical tRNA met) to the start site. ERK activity is already linked in some Center, Iowa City, IA 52242 i systems to translation initiation via a described effect on Mnk1 Received for publication January 25, 2006. Accepted for publication May 11, 2006. phosphorylation of eIF4E (20). In contrast to the described link The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance between ERK and eIF4E, there is no described link between ERK with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a Department of Veterans Affairs Merit Review grant; National Institutes of Health Grants HL-60316, HL-077431, and HL079901-01A1; and Grant RR00059 from the General Clinical Research Centers Program, National 3 Abbreviations used in this paper: eIF, eukaryotic initiation factor; Met-tRNA met, Center for Research Resources, National Institutes of Health. i initiator methionyl-tRNA; Cdk, cyclin-dependent kinase; ER, endoplasmic reticulum; 2 Address correspondence and reprint requests to Dr. Martha M. Monick, Division of GCN2, general control nonderepressing 2 kinase; HRI, heme-regulated inhibitor ki- Pulmonary, Critical Care, and Occupational Medicine, Room 100, Eckstein Medical nase; MKP, MAPK phosphatase; PARP, poly(ADP-ribose) polymerase; PERK, pro- Research Building, Carver College of Medicine, University of Iowa, Iowa City, IA tein kinase R-like ER kinase; PKR, protein kinase R; PP1, protein phasphatase 1; 52242. E-mail address: [email protected] PVDF, polyvinylidene difluoride.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 1637 signaling and the eIF2 pathway (17, 21–23). In preliminary stud- (9101) were obtained from Cell Signaling Technology, and JNK (559309) ies, we found no effect of ERK on the eIF4E pathway in human from Calbiochem. Cleaved caspase 7 (9491) and 9 (9509) and cleaved poly(ADP-ribose) polymerase (PARP) (9541) and eIF2-␣ (nonphospho) alveolar macrophages, but we did ﬁnd that ERK activity modu- ␣ ␣ (9722) Abs were also obtained from Cell Signaling Technology. PP1 lated the phosphorylation state of eIF2 (dephosphorylation of (sc-6105), JNK2 (7345), PP1␣ agarose conjugate (sc-6105-AC), HRP-con- serine 51 is required for activity). jugated Abs anti-rabbit (sc-2004), anti-mouse (sc-2005), and anti-goat (sc- Four kinases have been described that can phosphorylate serine 2020) were all obtained from Santa Cruz Biotechnology. We tried a num- 51 on eIF2␣ and decrease protein translation after stress (24). Pro- ber of MAPK phosphatase-7 (MKP-7) Abs, including ones from Abcam (ab15698), Imgenix (IMX-3042), and an Ab provided by M. Collins (Royal tein kinase R (PKR) is activated by dimerization following exposure to dsRNA (25). Heme-regulated inhibitor kinase (HRI) is activated by low heme levels (26). PKR-like endoplasmic reticulum kinase (PERK) senses endoplasmic reticulum (ER) stress (27) and general control nonderepressing 2 kinase (GCN2) responds to amino acid deprivation (28). Activation of any of these kinases can lead to eIF2␣ phosphorylation on serine 51 and decreased protein translation. The exceptions to this block in translation are a few stress-related proteins whose translation actually goes up in the setting of eIF2␣ phosphorylation (17). As well as the described kinases, the phosphorylation and activity of eIF2␣ are also regulated by the activity of protein phasphatase 1␣ (PP1␣) phosphatase Downloaded from (29). The balance between upstream kinase activity and PP1␣ phosphatase activity determines the translation outcome (21). This study was performed in human alveolar macrophages obtained from normal volunteers. Alveolar macrophages are a distinct cell population with a unique environment. They are both

similar to and distinct from other macrophage populations. We http://www.jimmunol.org/ have recently shown differences in survival pathways between alveolar macrophages and their precursors, blood monocytes (3, 30). In both studies, we found that alveolar macrophages have constitutive activity of ERK and the PI3K/Akt pathways that is not present in monocytes. The role of the baseline ERK activity is the focus of this study. There are other studies that define important differences between alveolar macrophages and other tissue macrophages, especially the studies by Curtis and colleagues (31) and Peters-Golden et al. (32–37). This study focuses on human alve- by guest on September 28, 2021 olar macrophages and does not attempt to define which other macrophages exhibit the described phenotype. We consider this to be an important component of further studies. In this study, we investigated the novel hypothesis that in un- stimulated human alveolar macrophages, long-term viability is maintained via ERK-dependent effects on the phosphatase, PP1␣, and protein translation. We demonstrate that inhibition of ERK FIGURE 1. A, Inhibition of ERK decreases protein translation rates. results in decreased protein translation, JNK-dependent inactiva- Human alveolar macrophages were put in culture with and without U0126 tion of PP1␣, and phosphorylation of eIF2␣. The end result of (20 ␮M) for 2 h. During the last 30 min of this culture time, the cells were these events (downstream of ERK inhibition) is shortened survival moved to methionine-free medium (U0126 replaced where needed). For of alveolar macrophages, due, at least in part, to decreased protein the labeling period, the following was added: actinomycin D (10 ␮M), translation. [35S]methionine (0.2 mCi/ml), and in some cases U0126 (20 ␮M). Cells were incubated for a further 2 h, and then whole cell lysates were obtained and an SDS-PAGE gel was run, dried, and visualized on a phosphor im- Materials and Methods ager. For the TCA data, lysates from three separate experiments were Materials treated with TCA to precipitate proteins, the samples were run through a Millipore filter, and the precipitated protein was counted on a liquid scin- Chemicals were obtained from Sigma-Aldrich. The MEK inhibitor UO126 tillation counter. The graph shows percentage of control based on the con- (662005), p38 inhibitor SB203580 (559389), JNK inhibitor II (SP600125 trol value of experiment 1 control (control/sample ϫ 100) (mean Ϯ SE). B, (420119)), PP1␣ inhibitor tautomycin (580551), ERK activation inhibitor peptide II (328005), actinomycin D (114666), and recombinant rabbit mus- Human alveolar macrophages have constitutive ERK activity. Human al- cle PP1␣ isoform (539493) were obtained from Calbiochem. Recombinant veolar macrophages were placed in culture for 1 h immediately after iso- human active JNK2␣2 peptide (PHO3011) was purchased from BioSource lation with and without added U0126 (20 ␮M). Whole cell lysates were International. Radionuclides [35S]methionine (NEG-709A) and obtained and Western blot analysis was performed for the active (phos- [␥-32P]ATP Easytides (NEG-502Z) were purchased from PerkinElmer. phorylated) form of ERK. On the right is composite densitometry from the Ethidium homodimer (E1169) was obtained from Molecular Probes. West- three samples. The data demonstrate constitutive ERK activity in human ern blotting reagents include Calbiochem’s phosphatase inhibitor mixture alveolar macrophages. C, U0126 primarily inhibits ERK. Human alveolar (524625), complete minitab protease inhibitors (11836170001) from macrophages were placed in culture with and without U0126 (20 ␮M) for Roche, ECL (RPN2106) and ECLϩ (RPN2132) chemiluminescent detec- tion reagents from Amersham Biosciences, and acrylimide (161-0158), 1 h. Specificity was examined by determining the effect of U0126 on the buffers (161-0798 and 161-0799), polyvinylidene difluoride (PVDF) mem- activity of ERK (phosphorylated on threonine 202 and tyrosine 204), p38 branes (162-0174), and Bradford protein assay reagent (500-0006) from (phosphorylated on threonine 180 and tyrosine 182), and Akt (phosphor- Bio-Rad. Abs used in this study were obtained from a variety of sources. ylated on serine 473) by Western blot analysis. (The relationship between Phosphorylation-specific Abs to eIF2-␣ (9721), PP1␣ (2581), and ERK ERK inhibition and JNK activity is addressed later in the manuscript.) 1638 HUMAN ALVEOLAR MACROPHAGES AND ERK MAPK

Free and University College Medical School, London, U.K.). We found all TCA precipitation of labeled proteins

of these Abs problematic in terms of visualizing endogenous proteins. We 35 finally ended up using an Ab from Calbiochem (MKP-7; PC726). ␤-Actin Some of the protein from the S-labeling experiments was used to obtain 35 ␮ 35 (A5316) was obtained from Sigma-Aldrich. Culture medium used in ex- relative S counts of the labeled proteins. A total of 20 lofthe S- ␮ periments was serum-free RPMI 1640 tissue culture medium plus Glu- labeled cell suspension was added to 100 l of BSA (1 mg/ml) and sodium tamax (61870-036) from Invitrogen Life Technologies and methionine/ nitrate (NaN3, 0.02%). One milliliter of ice-cold 10% w/v TCA was added cysteine-free RPMI 1640 medium (R7513) from Sigma-Aldrich. Kits used to each tube, and the samples were vortexed vigorously. The solution was in this study were pNPP Phosphatase Activity Kit (POPN-10K) from Bio- filtered through a 2.5-cm glass microfiber filter (Millipore) and the filters Assay Systems and CellTiter-Glo Luminescent Cell Viability Assay were washed twice with 5 ml of ice-cold 10% TCA. The discs were washed (G7571) from Promega. two more times in ethanol, dried, and placed in vials with 5–10 ml of scintillation mixture, and radioactivity was determined with a scintillation Isolation of human alveolar macrophages counter set on a wide open channel. Starting values (total) were determined by spotting 20 ␮l of the original cell lysate onto a filter and counting. Alveolar macrophages were obtained from normal nonsmoking volunteers, Calculations were as follows: sample counts/total counts ϫ 100 yields as previously described (38). Briefly, normal volunteers with a lifetime percentage of 35S incorporation. Fold increase was determined by dividing nonsmoking history, no acute or chronic illness, and no current medica- experimental sample values by control values. tions underwent bronchoalveolar lavage. The cell pellet was washed twice in HBSS without Ca2ϩ and Mg2ϩ and suspended in complete RPMI 1640 medium with glutamax (Invitrogen Life Technologies) and added genta- Whole cell protein isolation ␮ micin (80 g/ml). Cells were cultured in special nonadherent plates from Whole cell protein was obtained by lysing the cells on ice for 20 min in 200 Costar (3471, 3473) to minimize effect of adherence on signaling. All ␮l of lysis buffer (0.05 M Tris (pH 7.4), 0.15M NaCl, 1% Nonidet P-40, experiments were conducted in serum-free conditions. Differential cell with added protease and phosphatase inhibitors: 1 protease minitab (Roche

counts were determined using a Wright-Giemsa-stained cytocentrifuge Biochemicals)/10 ml and 100 ␮lof100ϫ phosphatase inhibitor mixture Downloaded from preparation. All cell preparations had between 90 and 100% alveolar mac- (Calbiochem)/10 ml). The lysates were sonicated for 20 s, kept at 4°C for rophages. This study was approved by the Committee for Investigations 30 min, and spun at 15,000 ϫ g for 10 min, and the supernatant was saved. Involving Human Subjects at the University of Iowa. Protein determinations were made using the Bradford protein assay from Bio-Rad. Cell lysates were stored at Ϫ70°C until use. Methionine labeling Alveolar macrophages were put into culture in low adherent 100-mm plates 6 Immunoprecipitation of cellular proteins

(10 cells/ml RPMI 1640 with glutamax and gentamicin). Actinomycin D http://www.jimmunol.org/ (Calbiochem) was added to prevent any new transcription of proteins. After For immunoprecipitation, lysates were combined with 25 ␮gofPP1␣ aga- a time period for isolation recovery, the cells were transferred to methi- rose conjugate (Santa Cruz Biotechnology) overnight rotating at 4 degrees. onine-free RPMI 1640 (Sigma-Aldrich) and cultured for 30 min. [35S]Me- Beads were washed three times with lysis buffer. For Western blot analysis, thionine was added (5 mCi/0.5 ml, 0.2 mCi/ml prewarmed methionine-free the beads were suspended in 50 ␮lof2ϫ sample buffer and heated for 5 RPMI 1640), and the cells were cultured for various times before harvest- min at 95 degrees. The beads were spun down and proteins were analyzed ing. Cells were harvested using standard protein isolation procedures, as by SDS-PAGE. Western blot protocol was followed, as described below. outlined below. Protein levels were measured, and a SDS-PAGE gel was Coimmunoprecipitations were determined by staining the resulting blots run. The gels were dried and autoradiographs were obtained showing 35S- for proteins other than the one pulled down. Equal loading was determined labeled proteins. by stripping the blots and reprobing for the immunoprecipitated protein. by guest on September 28, 2021

FIGURE 2. Inhibition of ERK increases phosphorylation of eIF2␣ on serine 51. A, Three sets of human alveolar macrophages were cultured with U0126 (20 ␮M) for various lengths of time. Whole cell lysates were obtained and Western blot analysis was performed using an Ab specific for eIF2␣ phosphorylated on serine 51. Composite densitometry of the three experiments is shown on the right. B, Hu- man alveolar macrophages were exposed to varying doses of U0126 (0.2, 2, and 20 ␮M) for 1 h. Whole cell lysates were obtained and Western blot analysis was performed for phosphorylated eIF2a (serine 51) and active ERK. Equal loading of the proteins is demonstrated by staining of the blots for ␤-actin. C, Human alveolar macrophages were cultured with a cell-permeable ERK inhibitory peptide (50 ␮M) for various lengths of time. Whole cell lysates were obtained and Western blot analysis was performed using an Ab specific for eIF2␣ phosphorylated on serine 51. D, Human alveolar macrophages were cultured with an ERK inhibitor (U0126, 20 ␮M) for prolonged time periods. Whole cell lysates were obtained and Western blot analysis was performed using an Ab specific for eIF2␣ phosphorylated on serine 51. The Journal of Immunology 1639

Western blot analysis Quantification was by direct cell count. Two hundred cells were counted from a minimum of four different fields. Average viability was determined Western blot analysis for the presence of particular proteins or for phos- and statistics were performed using GraphPad software. phorylated forms of proteins was performed, as previously described (39). Briefly, 30 ␮g of protein was mixed 1:1 with 2ϫ sample buffer (20% ATP assay glycerol, 4% SDS, 10% 2-ME, 0.05% bromphenol blue, and 1.25 M Tris (pH 6.8)), loaded onto a 10% SDS-PAGE gel, and run at 110 V for 2 h. Cell survival was also monitored by using CellTiter-Glo Luminescent Cell Cell proteins were transferred to PVDF membranes with a Bio-Rad semi- Viability Assay from Promega. Alveolar macrophages were cultured (1 dry transfer system, according to the manufacturer’s instructions. Equal million alveolar macrophages treated with either U0126 or ERK Activation loading of the protein groups on the blots was evaluated using Ponceaus S Inhibitor Peptide II for 6 or 24 h) in 24-well plates. ATP, signaling the (Sigma-Aldrich), a staining solution designed for staining total proteins on presence of metabolically active cells, was measured by bringing the plate PVDF membranes. The PVDF was then blocked with 5% milk in TTBS to room temperature and adding CellTiter-Glo directly to each well. The (TBS with 0.1% Tween 20) for 1 h, washed, and then incubated with the plate was mixed on an orbital shaker to induce cell lysis and then the primary Ab at dilutions of 1/500 to 1/2,000 overnight. The blots were sample was read in a luminometer (integration time of 1 s). Data are pre- washed four times with TTBS and incubated for 1 h with HRP-conjugated sented as mean Ϯ SE of luminescent readings from three separate anti-IgG Ab (1/5,000 to 1/20,000). Immunoreactive bands were developed experiments. using a chemiluminescent substrate, ECL Plus or ECL (Amersham Bio- sciences). An autoradiograph was obtained, with exposure times of 10 s to In vitro kinase assay 2 min. Protein levels were quantified using a FluorS scanner and Quantity To evaluate PP1␣ phosphorylation, 250 ng of recombinant human active One software for analysis (Bio-Rad). The data were analyzed, and statistics JNK2␣2 peptide and 10 ␮g of recombinant rabbit muscle PP1␣ isoform were performed using Graphpad software. Densitometry is expressed as were combined with 5 ␮Ci of ATP (32) to evaluate the ability of JNK2 to fold increase (experimental value/control value). phosphorylate PP1␣. The PP1␣ inhibitor tautomycin (1 ␮M) was included

in the reaction buffer (20 mM MOPS, 25 mM glycerol phosphate, 1 mM Downloaded from Phosphatase activity assay NaVO4, 5 mM EGTA, and 1 mM DTT). The experiment was conducted ␣ ␣ ␣ Phosphatase activity was measured using a pNPP phosphatase assay kit with JNK2 2 peptide alone; PP1 isoform alone; JNK2 2 peptide and ␣ ␣ ␣ from BioAssay Systems. To measure PP1␣ phosphatase activity, PP1␣ was PP1 isoform; and JNK2 2 peptide, PP1 isoform, and the JNK inhibitor ␮ immunoprecipitated from whole cell lysates (human alveolar macro- SP600125 at 1 M. Samples were incubated at 30 degrees for 20 min. ϫ phages) using microcystin-Sepharose beads or PP1␣ Ab/protein A-Sepa- Equal volume of 2 sample buffer was added at the end of the incubation, rose beads. Serial 2-fold dilutions of the washed beads were made in 100 and samples were run on a 10% acrylimide gel, transferred to PVDF mem- ␮ brane, and visualized on a phosphor imager (Bio-Rad; Quantity One mM Tris-HCl (pH 7.5) and 10 mM MgCl2. A total of 50 l of each dilution http://www.jimmunol.org/ was placed into a 96-well plate. pNPP substrate solution (50 ␮l) was then software). added to each well. After 5–30 min, the absorbance was read at 405 nM. Dilutions that fit into a linear range of purified standard (PP1␣) were used Results to acquire data. Data are presented as OD units. Inhibition of ERK decreases protein translation Cell survival analysis Human alveolar macrophages were placed in culture with and without the MEK inhibitor (U0126) for 2 h. During the last 30 min For analysis of cell survival, alveolar macrophages were cultured in six- well tissue culture plates with or without pathway inhibitors (ERK, U0126 of culture, the cells were moved to methionine-deficient medium at 20 ␮M or ERK activation inhibitor peptide II (Calbiochem) at 50 ␮M) for 30 min (U0126 was replaced where appropriate). Cells were 35 for the described times. Triplicate cultures were performed on all experi- then treated with [ S]methionine and actinomycin D (to block by guest on September 28, 2021 ments. After the incubation period, the cells were stained with ethidium transcription) and cultured for additional 2 h. Whole cell lysates homodimer (Molecular Probes) at 8 ␮M, and images were obtained of both bright field and fluorescence using a Leica DMRB microscope equipped were obtained and an aliquot was run out on a 10% SDS-PAGE with a Qimaging RETICA 1300 digital camera and imaging system. After gel. The gel was dried and an autoradiogram was obtained. Fig. 1 obtaining images, the percentage of EthD-1-positive cells was determined. demonstrates that ERK inhibition results in decreased translation

FIGURE 3. A, Inhibition of ERK decreases PP1␣ activity. Human alveolar macrophages were cultured for 1 h with either U0126 (20 ␮M) or the PP1 inhibitor, tautomycin (1 ␮M). Whole cell lysates were obtained and PP1␣ was immunoprecipitated. The immunoprecipitated PP1␣ was used in a phosphatase activity assay (pNPP substrate solution). Data are presented as total OD units (340 nM) after a 30-min incubation of immunoprecipitated phosphatase and substrate. B, Blocking PP1␣ activity is sufﬁcient to induce eIF2␣ phosphorylation. Human alveolar macrophages were cultured with PP1␣ inhibitor (tautomycin, 0.2–2.0 ␮M) for 1 h. Whole cell lysates were obtained and Western blot analysis was performed using an Ab speciﬁc for eIF2␣ phosphorylated on serine 51. Densitometry shown as arbitrary units is on the right. 1640 HUMAN ALVEOLAR MACROPHAGES AND ERK MAPK

FIGURE 4. A, Inhibition of ERK induces phosphorylation of PP1␣ on threonine 320. Human alveolar macrophages were cultured with an ERK inhibitor (U0126, 20 ␮M) for 1 or 3 h. Whole cell lysates were obtained and Western blot analysis was performed using an Ab specific for PP1␣ phosphorylated on threonine 320. B, JNK inhibition (and not p38 inhibition) reverses the effect of U0126 on eIF2␣. Human alveolar macrophages were cultured with an ERK inhibitor (U0126, 20 ␮M)for1h.In some groups, either p38 (SB20358, 20 ␮M) or JNK (SP600125, 1 ␮M) was also inhibited. Whole cell lysates were obtained and Western blot analysis was performed using an Ab specific for eIF2␣ phosphorylated on serine 51. Downloaded from http://www.jimmunol.org/ of multiple proteins. Also shown are data from three separate ex- inhibition was more restricted. One possible cause of the discrep- periments in which TCA-precipitable proteins were isolated from ancies may be varied induction of GADD34, a PP1 regulator (and the lysate and 35S counts were analyzed. Data are presented as fold negative feedback mechanism (21, 29)) induced following eIF2␣ difference (U0126/control). The graph demonstrates that in condi- phosphorylation. We found various levels of GADD34 induction tions in which transcription is frozen (actinomycin treatment), in the different donors (data not shown). This would contribute to U0126 decreases the amount of new protein production. To con- altered duration of the eIF2␣ phosphorylation. In all cases, U0126 firm the presence of baseline ERK activity, three sets of macro- caused a prolonged phosphorylation of eIF2␣ (see Fig. 2D). phages were cultured with U0126 for 1 h. Whole cell lysates were We treated normal alveolar macrophages with varying amounts by guest on September 28, 2021 obtained and ERK activity was monitored by Western blot analysis of U0126. Fig. 2B demonstrates that U0126 causes significant for the phosphorylated form of the protein. Fig. 1B demonstrates phosphorylation of eIF2␣ over multiple concentrations. The inhib- significant baseline ERK activity in human alveolar macrophages. itory effect of U0126 on ERK phosphorylation is shown as a con- As a composite, these data suggest that in alveolar macrophages trol. To determine that the U0126 observation was not due to an there is constitutive ERK activity that plays an important role in artifact of the chemical inhibitor, we performed the same experi- maintaining ongoing protein translation. To examine the specific- ment using a small peptide ERK inhibitor. This inhibitor is a 13-aa ity of U0126, we treated alveolar macrophages with U0126 for 1 h peptide from the ERK upstream kinase, MEK, fused to the HIV- and examined activity of the MAPK, p38, and the PI3K pathway TAT membrane translocation peptide via a glycine linker (H- component, Akt. Fig. 1C demonstrates that1hofU0126 com- GYGRKKRRQRRR-G-MPKKKPTPIQ LNP-NH2). The peptide pletely blocks baseline ERK activity, has some negative effect on is membrane permeable and interferes with ERK/MEK interac- p38 activity, and has no effect on the activity of Akt. The effect of tions. Fig. 2C demonstrates that, as with U0126, the ERK inhibi- U0126 on JNK activity is not shown in Fig. 1C, as the activation tory peptide also increased eIF2␣ phosphorylation on serine 51. of JNK by U0126 is a part of this study, and is shown in Figs. 5 Finally, we asked whether the increased eIF2␣ phosphorylation and 6. Inhibition of ERK increases phosphorylation of eIF2␣. Pro- generated by ERK inhibition was a transient or prolonged event. tein translation is primarily regulated at initiation. One major reg- Fig. 2D shows that the increase in eIF2␣ phosphorylation on serine ulatory pathway is the eIF4F pathway, which recruits mRNAs with 51 after ERK inhibition is a prolonged event (extending out to 5Ј caps to the ribosome. The other major pathway is the eIF2 24 h). Consistent with the discussion in the paragraph above, there met ␣ pathway, which brings the start methionine (Met-tRNAi )tothe was some variability in the duration of the eIF2 . However, in ribosome. When we examined the eIF4F pathway (eIF4E, 4EBP-1, most cases, U0126 caused prolonged phosphorylation of eIF2␣. eIF4A, eIF4B, and eIF4G) for alterations in activating and inhib- The data in this study demonstrate, consistent with the effects of iting phosphorylations after ERK inhibition, we found little change ERK inhibition on translation, that eIF2␣ is phosphorylated on the (data not shown). This is despite the described role of ERK as an inhibitory serine 51 site for a prolonged period following ERK upstream activator of Mnk1, which phosphorylates eIF4E. How- inhibition. ever, when we examined phosphorylation of eIF2␣ on serine 51, an inhibiting phosphorylation, we found significant and prolonged phosphorylation of eIF2␣ after U0126 (Fig. 2A). Densitometry Inhibition of ERK decreases PP1 activity from the three separate experiments is also shown. It is important EIF2␣ phosphorylation is regulated by the combined activity of to note that there is individual variation among the donors. In some upstream kinases (PKR, PERK, GCN2, and HRI). It is also regu- cases, the eIF2␣ phosphorylation began as early as 15 min after lated by the dephosphorylating activity of PP1␣. We initially ex- U0126 and continued out to 24 h. In other cases, the time of eIF2␣ amined the effect of ERK inhibition on the upstream kinases and The Journal of Immunology 1641 did not find any significant effect (data not shown). We then ex- The threonine phosphorylation site is shown in bold. Because ERK amined the effect of ERK inhibition on PP1␣ activity. Immuno- was inhibited in these studies, it is not a potential upstream kinase. precipitated PP1␣ was evaluated for in vitro phosphatase activity The only described upstream kinases for this site are the cell cycle using pNPP as a substrate. ERK inhibition significantly decreased regulatory kinases, cyclin-dependent kinase 1 (Cdk1) and Cdk2 PP1␣ activity (Fig. 3A). To further examine whether phosphatase (40, 41). We could find no sign in any change in Cdk1 or Cdk2 inhibition was sufficient to increase phosphorylation of eIF2␣,we activity with ERK inhibition (data not shown). This is consistent treated human alveolar macrophages with the PP1-specific inhib- with a recent study by Romerio and Zella (42), demonstrating that itor, tautomycin. Fig. 3B shows that inhibition of PP1 (tautomycin) IFN-␣ decreases ERK activity leading to decreased Cdk2 activity. results in phosphorylation of eIF2␣. This suggests that inhibiting To investigate other possible upstream kinases of the PP1␣ site, we PP1 is sufficient to increase eIF2␣ phosphorylation in human al- evaluated the effect of inhibiting other MAPKs together with the veolar macrophages and that there is enough baseline activity in ERK inhibition. Fig. 4B demonstrates that inhibiting p38 the upstream kinases to assure phosphorylation of eIF2␣ in con- (SB203580) had no effect on the U0126-induced eIF2␣ phosphor- ditions in which PP1 is inhibited. As a composite, these data sug- ylation. In contrast, blocking JNK activity at the same time as gest that ERK inhibition in alveolar macrophages leads to a de- U0126 resulted in a loss of the U0126-induced phosphorylation of crease in PP1␣ activity, which is sufficient to increase the eIF2␣. As a composite, these experiments demonstrate that inhi- phosphorylation of eIF2␣ at serine 51. bition of baseline ERK activity in alveolar macrophages results in phosphorylation of PP1␣ on threonine 320 and that JNK activity is ␣ ERK inhibition increases PP1 phosphorylation required for the ERK inhibition-dependent phosphorylation of

To determine a possible mechanism by which ERK regulates PP1 eIF2␣. Downloaded from activity, we evaluated the effect of ERK inhibition on an inhibiting phosphorylation of the ␣ catalytic subunit of PP1 (threonine 320). ERK inhibition results in activation of JNK and a decrease in Fig. 4A demonstrates that in alveolar macrophages, ERK inhibition the JNK phosphatase, MKP-7 increases phosphorylation of PP1␣, a phosphorylation event that We next asked whether ERK inhibition increased JNK activity. In inhibits activity of PP1␣. Fig. 5A, we demonstrate that ERK inhibition by either U0126 or an

The phosphorylation site on PP1␣ (threonine 320) is a consen- ERK inhibitory peptide signiﬁcantly increased JNK activity. In http://www.jimmunol.org/ sus MAPK substrate site (PX(S/T)P)(PP1␣: 315 GGRPITPPRNS Fig. 5B, we examine the effect of ERK inhibition on amounts of 325). The MAPK consensus sequence is underlined and in italics. MKP-7. ERK inhibition decreased total MKP-7 protein amounts. by guest on September 28, 2021

FIGURE 5. Inhibition of ERK activates JNK and decreases amounts of the JNK phosphatase, MKP-7. A, Human alveolar macrophages were cultured with either U0126 (20 ␮M) or a cell-permeable ERK inhibitory peptide (50 ␮M) for 1 or 3 h. Whole cell lysates were obtained and Western blot analysis was performed using an Ab speciﬁc for JNK phosphorylated on threonine 183 and tyrosine 185. B, Human alveolar macrophages were cultured with an ERK inhibitor, U0126 (20 ␮M), for 1 or 3 h. Whole cell lysates were obtained and Western blot analysis was performed using an Ab speciﬁc for total MKP-7. 1642 HUMAN ALVEOLAR MACROPHAGES AND ERK MAPK

These data suggest that at baseline in alveolar macrophages, active sults in cleaved caspase 7, cleaved caspase 9, and cleaved PARP, ERK decreases JNK activity by phosphorylating and protecting peaking at 24 h of U0126 exposure. Fig. 7B demonstrates that from degradation the JNK phosphatase, MKP-7. U0126 increases the membrane permeability of alveolar macrophages, as demonstrated by ethidium homodimer uptake (shown ␣ ␣ JNK phosphorylates PP1 in vitro and complexes with eIF2 both by a photomicrograph and by cell counts from three separate ␣ and PP1 experiments). In Fig. 7C, we evaluated ATP depletion as a marker To test the hypothesis that JNK could be a PP1␣ threonine 320 of viability. Exposure to both U0126 and the ERK inhibitory pep- kinase, we performed an in vitro assay using purified active JNK tide caused a significant drop in ATP levels, from 40 to 70% of and purified catalytic domain of PP1␣. Isolated proteins were control at 6 h and from 2 to 5% of control at 24 h. The ATP data mixed with a kinase buffer and 32ATP. The samples were then run are consistent with the enhanced caspase and PARP cleavage we on an SDS-PAGE gel, the gel was transferred to PVDF membrane, saw at 24 h of U0126 exposure. As a composite, the data demon- and radioactivity was determined using a phosphor imager. Fig. 6A strate that baseline ERK activity is crucial for the survival of hu- demonstrates that in vitro, JNK can phosphorylate PP1␣ on thre- man alveolar macrophages. onine 320. We next asked whether ERK inhibition would bring PP1␣ and JNK together in a complex. Alveolar macrophages were treated with an ERK inhibitor and then PP1␣ immunoprecipitated. Discussion The protein complex was then analyzed for JNK binding. Fig. 6B The alveolar macrophage maintains baseline activity of two path- demonstrates that JNK is recruited to PP1␣ under conditions of ways that contribute to prolonged survival. We have shown pre- ERK inhibition. It is also clear that it is the active form of JNK2 viously that both the PI3K/Akt and ERK pathways are activated at Downloaded from that is associating with PP1␣ after ERK inhibition. baseline in alveolar macrophages (3). The prosurvival effects of Akt are well described and were not the subject of these studies. In ERK inhibition shortens survival of human alveolar contrast, little is known about the survival effects of ERK (10, macrophages 43–45). In this study, we examined the role of ERK activity in Our initial hypothesis was that baseline ERK activity in alveolar maintaining adequate levels of protein translation, a necessary con- macrophages is an important survival pathway. Having demon- dition for cell survival. We found that inhibition of ERK signifi- http://www.jimmunol.org/ strated that ERK inhibition leads to a decrease in global protein cantly decreased protein translation in alveolar macrophages. Due translation, we then evaluated the effect of ERK inhibition on al- to the rich oxidant and pathogen exposure in the lung, it is our veolar macrophage survival. Fig. 7 demonstrates that inhibition of hypothesis that this is a lung-specific effect. However, we have not ERK increases markers of apoptosis and death in alveolar macro- yet performed the macrophage comparative experiments that will phages. Fig. 7A demonstrates that ERK inhibition with U0126 re- allow for that conclusion. We have shown previously that survival pathway activity is different between human alveolar macrophages and their precursor cells, blood monocytes (3, 30). We have not yet compared alveolar macrophages and other tissue macrophages. These experiments are part of ongoing projects. by guest on September 28, 2021 Regulation of translation primarily occurs at the initiation step (17, 46). One important pathway is recruitment of ribosomes to mRNA by the eIF4F complex. Despite described links between ERK and eIF4E (20, 47), we found no obvious effect of ERK inhibition on this complex in alveolar macrophages. Another important pathway in the initiation process is binding the initiator met Met-tRNAi to the P site of the small ribosomal subunit by the eIF2 complex (17). When we examined the effect of ERK inhibition on the eIF2 complex, we found a significant change in the phosphorylation status of eIF2␣ (active eIF2␣ is hypophosphory- FIGURE 6. A, Active JNK phosphorylates PP1␣ in vitro. A total of 250 lated on serine 51). Phosphorylation of eIF2␣ normally occurs as ng of active JNK2 (JNK2a2 aa 2–424 produced in Escherichia coli with an part of a stress response (nutrient deprivation, viral infection, ER N-terminal polyhistidine tag and purified via sequential chromatography) stress, and low heme levels). In the case of ERK inhibition, we ␮ ␣ ␣ was mixed in a 1.5-ml microtube with 10 gofPP1 (PP1 isoform from observed no changes in the activity of the stress-related upstream rabbit muscle (sequence around the threonine 320 site is identical with ͗ ͘ kinases (data not shown). We did, however, observe marked human sequence ( www.ncbi.nlm.nih.gov/BLAST ) in buffer A (20 mM ␣ MOPS, 25 mM ␤ glycerol phosphate, 1 mM NaVO , 5 mM EGTA, 1 mM changes in activity of the phosphatase, PP1 , which is responsible 4 ␣ DTT, and 1 ␮M tautomycin) with added 5 ␮Ci of ATP in 0.5 mM cold for maintaining eIF2 in a hypophosphorylated state (29)). ATP buffer/buffer A (total volume ϭ 40 ␮l). Individual samples had vari- ERK inhibition resulted in decreased PP1␣ activity that was due ations of possible proteins (JNK alone, PP1␣ alone, both, and both with to an increase in an inhibitory phosphorylation at a site on the added JNK inhibitor, SP600125, 10 ␮M). The microtubes were vortexed catalytic unit of PP1␣ (threonine 320). Threonine 320 is a MAPK gently and incubated at room temperature for 30 min. A total of 40 ␮lof substrate consensus sequence. Therefore, we looked at other Western sample buffer was added, and the samples were run out on a 10% MAPKs as potential upstream kinases responsible for PP1␣ phos- SDS-PAGE gel. The finished gel was transferred to a PVDF membrane, phorylation. We found that ERK inhibition increased activity of and radioactive bands were identified by phosphor imager. Equal loading the MAPK, JNK. In addition, we found that active JNK can phos- of the PP1␣ protein was confirmed by Western blot analysis using an Ab ␣ ␣ phorylate PP1 in vitro and that JNK inhibition reverses the in- specific for PP1 . B, Inhibition of ERK results in binding of active JNK to ␣ PP1␣. Human alveolar macrophages were cultured with U0126 (20 ␮M) crease in eIF2 phosphorylation seen with ERK inhibition. Fi- for 1 h. Whole cell lysates were obtained and PP1␣ was immunoprecipi- nally, using a number of methods, we show that ERK inhibition tated. Western blot analysis was then performed for the following proteins: significantly shortens survival of alveolar macrophages. (Fig. 8 JNK (whole protein), active JNK (phosphorylated on threonine 183 and contains a diagram demonstrating our hypothesis on the relation- tyrosine 185), and PP1␣ (loading control). ship among ERK, eIF2␣, JNK, and PP1␣.) The Journal of Immunology 1643

FIGURE 7. Inhibition of ERK shortens survival of alveolar macrophages. A, Human alveolar macrophages were cultured with U0126 (20 ␮M) for 1, 3, 6, and 24 h. Whole cell lysates were obtained and Western blot analysis was performed using Abs specific for cleaved caspase 7, cleaved caspase 9, and cleaved PARP. Equal loading of proteins is demonstrated with a ␤-actin blot. B, Human alveolar macrophages were cultured with U0126 (20 ␮M) for 6 or 24 h. Cell survival was analyzed by EthD-1 cell entry. The figure shows the cell monolayer with the fluorescent photomicrographs of the EthD-1-positive cells superimposed on the brightfield images. Downloaded from The images are representative of three separate experiments. The graph depicts the percentage of dead cells found at both 6 and 24 h post-U0126 exposure. The data are from three separate experiments. C, Human alveolar macrophages were cultured with either U0126 (20 ␮M) or a cell-permeable ERK in- http://www.jimmunol.org/ hibitory peptide (50 ␮M) for 6 or 24 h. At the end of the incubation period, total ATP was measured using an ATP luminescence assay. Data are presented as arbitrary light units. At both time periods and with both inhibitors, control values were significantly different from experimental values (p Ͻ 0.01). by guest on September 28, 2021

Translation inhibition (speciﬁcally phosphorylation of eIF2␣) PP1␣). In our case, eIF2␣ phosphorylation has no endogenous and apoptosis have been linked in other systems. One recent paper shutoff mechanism as long as ERK is inactive and the extended links apoptosis induced by proteosome inhibition to the phosphor- shutdown of the translation apparatus leads to cell death. ylation of eIF2␣ by upstream kinases (48). These authors found a Our data demonstrate a decrease in PP1␣ activity with ERK correlation between an increase in eIF2␣ phosphorylation and inhibition and the novel observation that this is linked to ERK caspase activation and apoptosis. This is consistent with our inhibition-dependent activation of JNK. PP1␣ activity is regulated observations. by its regulatory subunits. PP1␣ activity is also negatively regu- In contrast to the proapoptotic effect of eIF2␣ phosphorylation, lated by phosphorylation at threonine 320 (40). There is only one there are also data linking eIF2␣ phosphorylation to activation of previous study linking ERK to PP1. In a paper by Quevedo et al. the prosurvival transcription factor, NF-␬B (23, 48, 49). One study (51), using neuronal cell cultures, insulin-like growth factor 1 was by Jiang et al. (50) examined the hypothesis that NF-␬B activity shown to induce PP1 activity via MEK/ERK activity. In this case, during ER stress requires temporary translational arrest to prevent the endpoint is activation of eIF2B, not eIF2␣. The Quevedo study the rapid resynthesis of the inhibitory subunit I␬B␣. The authors does demonstrate that in another system, ERK can be upstream of suggest that eIF2␣ phosphorylation is necessary for NF-␬B acti- PP1. Our data link ERK to PPI␣ via an effect on phosphorylation vation. Our system differs in a variety of ways. For one, we have of PP1␣ on threonine 320 by JNK. no signiﬁcant induction of the upstream kinases (PERK, PKR, Our conclusion that PP1␣ phosphorylation is via JNK activation GCN2, and HRI). For another, the duration of phosphorylation of is supported by the following data: ERK inhibition by both chem- eIF2␣ with ERK inhibition is extremely long, and this is different ical (U0126) inhibitor and cell-permeable inhibitory peptides re- from the normal stress response. Although ER stress temporarily sults in JNK activation in alveolar macrophages. The threonine inhibits most translation, it induces translation of a subset of stress- 320 site on PP1␣ is part of a consensus MAPK substrate sequence related proteins. One of these stress-related proteins is instrumental (PX(S/T)P). The kinases that preferentially phosphorylate this con- in reactivating PP1␣ and ending the eIF2␣ phosphorylation. This sensus sequence include: MAPKs (ERK, p38, and JNK isoforms), feedback mechanism is not operative in our system. We found no glycogen synthase-3, and Cdks (52). The upstream kinase is ob- induction of GADD34 (the stress-related protein that activates viously not ERK as it is inhibited, and when we examined p38 we 1644 HUMAN ALVEOLAR MACROPHAGES AND ERK MAPK

stress-induced apoptosis requires JNK activity. Interestingly, they link JNK activity to inhibition of tyrosine phosphatases. Our study links JNK activation to inactivation of another class of phosphatases, the serine/threonine phosphatases (PP1␣). In contrast, a study by Himes et al. (59) shows that activation of JNK by CSF-1 is important in macrophage differentiation and survival. Although both our study and the Himes et al. (59) study are about macrophages and JNK, there are important differences between the two studies. The Himes et al. (59) study uses murine bone marrow-derived macrophages instead of human primary tissue macrophages (alveolar). They also are studying the effect of a growth factor, CSF-1, instead of homeostatic conditions. Further- more, their inhibitor level is up to 10 times the amount used in our study (10–20 ␮M compared with 1 ␮M). We believe that the differences in species, model, and inhibitor use can explain the different survival outcomes in the two studies. As a composite, our data demonstrate that in alveolar macrophages, baseline ERK activity maintains protein translation via the

following novel pathway: ERK decreases JNK activity by stabi- Downloaded from ␣ FIGURE 8. Diagrammatic representation of the link among ERK, JNK, lizing MPK-7. This, in turn, leads to activation of PP1 and main- ␣ PP1␣, eIF2␣, and protein translation. In human alveolar macrophages, tenance of hypophosphorylated (active) eIF2 and translation. ERK decreases JNK activity. JNK activity can decrease PP1␣ phosphatase This positive effect of ERK on protein translation is consistent with activity. With low JNK, the higher activity of PP1␣ maintains hypophos- the prolonged survival of human alveolar macrophages. Thus, this phorylated (active) eIF2␣. Inhibition of ERK or PP1␣ leads to eIF2␣ phos- newly described role for ERK in alveolar macrophage homeostasis

phorylation and decreased protein translation. Ongoing protein translation explains, in part, the survival characteristics of these cells. http://www.jimmunol.org/ (driven by ERK activity) contributes to the prolonged survival of these cells, despite the frequently stressful conditions found in the lung. Disclosures The authors have no financial conflict of interest. found that ERK inhibition in alveolar macrophages actually de- References creased p38 activity slightly. We also found no change in the ac- 1. Thomas, E. D., R. E. Ramberg, G. E. Sale, R. S. Sparkes, and D. W. Golde. 1976. tivity of Cdk1, a previously described PP1␣ kinase (40). However, Direct evidence for a bone marrow origin of the alveolar macrophage in man. Science 192: 1016–1018. when we examined JNK activity, we found a significant increase 2. Komuro, I., N. Keicho, A. Iwamoto, and K. S. Akagawa. 2001. 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