Repression of eEF2K transcription by NF-κB tunes translation elongation to inflammation and dsDNA-sensing

Christopher Biancoa, Letitia Thompsona, and Ian Mohra,b,1

aDepartment of Microbiology, NYU School of Medicine, New York, NY 10016; and bLaura and Isaac Perlmutter Institute, NYU School of Medicine, New York, NY 10016

Edited by Nahum Sonenberg, McGill University, Montreal, Canada, and approved September 26, 2019 (received for review May 29, 2019) expression is rapidly remodeled by infection and inflamma- Posttranscriptional control of at the level of tion in part via transcription factor NF-κB activation and regulated mRNA translation enables swift changes to the proteome in synthesis. While protein synthesis is largely controlled by response to physiological and environmental challenges and plays mRNA translation initiation, whether cellular translation elonga- a key role in infection and immune responses (18). Integrating tion factors are responsive to inflammation and infection remains cellular translation factors into a variety of cell signaling pathways, poorly understood. Here, we reveal a surprising mechanism whereby especially those responsive to the mechanistic target of rapamycin NF-κB restricts of the critical translation elongation complex 1 (mTORC1), allows protein synthesis to be tuned to factor eEF2, which catalyzes the protein synthesis translocation step. different fundamental parameters like energy, nutrient, oxygen, κ – α Upon exposure to NF- B activating stimuli, including TNF , human and growth factor sufficiency (19). Translation initiation is a highly cytomegalovirus infection, or double-stranded DNA, eEF2 phosphor- regulated, rate-limiting step whereby a multisubunit m7GTP cap- ylation on Thr56, which slows elongation to limit protein synthesis, binding complex assembles on the mRNA 5′-end to recruit the and the overall abundance of eEF2 (eEF2K) are reduced. κ 40S ribosome subunit (20). This process is regulated in part by the Significantly, this reflected a p65 NF- B subunit-dependent reduction 4E-BP family of translational repressor , which are sub- in eEF2K pre-mRNA, indicating that NF-κB activation represses eEF2K strates of mTORC1 (21). Following AUG start codon recognition BIOCHEMISTRY transcription to decrease eEF2K protein levels. Finally, we demon- and 60S subunit joining, translation elongation commences and is strate that reducing eEF2K abundance regulates protein synthesis dependent upon eukaryotic elongation factor 2 (eEF2), which in response to a bacterial toxin that inactivates eEF2. This estab- lishes that NF-κB activation by diverse physiological effectors con- mediates ribosome translocation along the mRNA and is regu- trols eEF2 activity via a transcriptional repression mechanism that lated by eEF2 kinase (eEF2K) (22, 23). In response to physio- reduces eEF2K polypeptide abundance to preclude eEF2 phosphory- logical stress including inhibition of mTORC1, eEF2K is activated lation, thereby stimulating translation elongation and protein syn- and phosphorylates eEF2 T56, thereby reducing the affinity of thesis. Moreover, it illustrates how nuclear transcription regulation eEF2 for the ribosome and slowing translation elongation (24, 25). shapes translation elongation factor activity and exposes how Viral and bacterial pathogens also target eEF2 to regulate protein eEF2 is integrated into innate immune response networks orches- synthesis. Human cytomegalovirus (HCMV) remains a significant tratedbyNF-κB. cause of morbidity and mortality in transplant recipients and a leading cause of congenital infections (26–30). In response to translation elongation | eEF2 and eEF2K | NF-κB activation | HCMV-induced mTORC1 activation, eEF2 mRNA translation is HCMV infection | double-strand DNA-sensing stimulated to raise intracellular levels of the critical elongation

nfection and inflammation profoundly impact ongoing protein Significance Isynthesis and often trigger activation of the nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) transcription Following infection and inflammation, activation of the tran- factor family (1). Diverse effectors, including inflammatory cyto- scription factor NF-κB and stimulation of mRNA translation kines like TNFα and nucleic acid pathogen-associated molecular initiation remodel cellular gene expression. In contrast, how patterns (PAMPs) indicative of infection, stimulate NF-κB, which translation elongation factor 2, which performs the trans- normally is contained in an inhibitory complex within the cyto- location step in protein synthesis, responds is not understood. plasm. Upon subsequent translocation into the nucleus, activated We show that eEF2 Thr56 phosphorylation, which slows elon- NF-κB transcription factors orchestrate a bevy of cellular re- gation, and eEF2 kinase (eEF2K) abundance are reduced by κ – κ sponses by regulating transcription of hundreds of (1). In- multiple NF- B activating stimuli. Significantly, NF- B activa- tion repressed eEF2K transcription to stimulate eEF2 by pre- deed, NF-κB activation figures prominently in the development venting eEF2 phosphorylation. In addition, eEF2K abundance and pathogenesis of autoimmunity and inflammation (2–7), aging – regulates protein synthesis upon eEF2 inactivation by a bac- and cancer (1, 8 12), and the deployment of innate host defenses, terial toxin. This demonstrates how eEF2 is integrated into NF- including type I IFN production and induction of IFN-stimulated κB innate immune response networks and reveals an un- – genes (ISGs) (1, 13 15). Significantly, many ISGs encode func- expected mechanism whereby regulated nuclear transcription tions vital for cell-intrinsic innate defenses that curb intracellular impacts translation elongation. pathogen reproduction by restricting infected cell protein syn- thesis, foiling efforts by infectious agents to co-opt control of the Author contributions: C.B., L.T., and I.M. designed research; C.B. and L.T. performed re- cellular protein synthesis machinery and subvert host defenses (16, search; C.B., L.T., and I.M. analyzed data; and C.B. and I.M. wrote the paper. 17). While NF-κB activation indirectly impacts protein synthesis The authors declare no competing interest. via stimulating IFN production and the subsequent activities of This article is a PNAS Direct Submission. induced ISG-encoded products, whether NF-κB acts through an- Published under the PNAS license. cillary mechanisms to regulate protein synthesis remains largely 1To whom correspondence may be addressed. Email: [email protected]. unknown. First published October 21, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1909143116 PNAS | November 5, 2019 | vol. 116 | no. 45 | 22583–22590 Downloaded by guest on October 3, 2021 factor eEF2 (31). In contrast, toxins produced by Corynebacterium activates eEF2. Overall, this work reveals a surprising mechanism diphtheriae and Pseudomonas aeruginosa ADP ribosylate eEF2 on whereby transcriptional repression by NF-κB might modulate diphthamide, a modified histidine residue unique to eEF2, to translation elongation in response to pathogens or inflammatory block elongation and host protein synthesis (32). cytokines. To investigate how protein synthesis responds to different NF- κB–activating stimuli, including TNFα exposure, HCMV infection, Results and dsDNA-sensing, we utilize a primary human fibroblast model. TNFα Stimulates Fibroblast Protein Synthesis and Translation Factors. Tissue-resident fibroblasts are critical long-lived sentinel cells that To investigate whether exposure to TNFα impacts global protein play a fundamental role coordinating acute resolving vs. chronic synthesis, normal human dermal fibroblasts (NHDFs) treated inflammation, adaptive immunity, and tissue remodeling and re- with TNFα for 24 h were metabolically labeled with 35S-containing pair. This is accomplished in part by conditioning tissue micro- amino acids (35S-aa). Fractionation of total protein by SDS/PAGE environments through changes in gene expression, some of which followed by autoradiography and quantification of acid-insoluble are regulated by cytokines or PAMPs that stimulate NF-κB(33– radioactivity by counting in liquid scintillant revealed that TNFα 35). Here, we show that TNFα treatment stimulates global protein treatment increased overall protein synthesis by ∼50% after 24 h synthesis in fibroblasts. Besides promoting initiation via inhibiting compared to untreated cells (Fig. 1A). Next, we examined how key the 4E-BP1 translation repressor, we establish that TNFα unex- regulators of mRNA translation responded to TNFα treatment. pectedly regulates elongation by preventing phosphorylated eEF2 Phosphorylation of 4E-BP1, a repressor of cap-dependent trans- accumulation and reducing eEF2K abundance. Infection with lation initiation, was evaluated by SDS/PAGE followed by im- HCMV also effectively reduced eEF2K protein levels and eEF2 munoblotting, which distinguishes hypophosphorylated 4E-BP1 Thr56 phosphorylation, as did exposure of uninfected primary fi- active repressor isoforms (faster-migrating species) from hyper- broblasts to immunostimulatory dsDNA. Significantly, the reduc- phosphorylated, inactivated isoforms (slower-migrating species). tion in eEF2K protein was accompanied by a corresponding Fig. 1B shows that TNFα treatment reduced hypophosphorylated decrease in eEF2K mRNA transcription that was dependent upon and increased hyperphosphorylated 4E-BP1 abundance (Fig. 1B, the NF-κB subunit p65. Furthermore, eEF2K abundance regu- compare lanes 1 to 3). The mTORC1-selective inhibitor rapamy- lates protein synthesis upon exposure to a bacterial toxin that in- cin antagonized 4E-BP1 hyperphosphorylation induced by TNFα

A + B C kDa - :TNF 2.0 170 2.0 130 * * * 100 TNF (h): 1.5 60 24 70 1.5 rapamycin: - + - + eEF2K 55 TNF : - - + + -hyper 40 eEF2 p-T56 1.0 4E-BP1 1.0 - hypo 35 eEF2 Protein Synthesis actin 0.5 Protein Synthesis 0.5 actin 25 1 2 3 4

tubulin 0 0 TNF (h): 0624 rapamycin: - - + TNF : - + + D E F G dox: - + - + dox: - - + + rapamycin: - - + + TNF : - - + + DAPI TNF : - + - + - + - + TNF : eEF2K FLAG eEF2K eEF2 p-T56 eEF2K eEF2 GAPDH

FLAG actin actin

1 2 3 4

Fig. 1. Control of fibroblast protein synthesis by TNFα.(A) NHDFs were untreated or TNFα-treated prior to metabolic labeling with 35S-radioactive amino acids for 30 min. (Left) Cultures were metabolically labeled after 24 h. Following fractionation of total protein by SDS/PAGE, the gel was fixed, dried, and exposed to X-ray film (Top) or analyzed by immunoblotting using an antibody specific for tubulin (Bottom). (Right) Cultures were metabolically labeled after 6 and 24 h. Amino acid incorporation was quantified by counting acid-insoluble radioactivity in liquid scintillant (*P < 0.05 by Student’s t test). (B) NHDFs were treated with rapamycin and TNFα as indicated. (Left) At 24 h posttreatment, total protein was isolated, separated by SDS/PAGE, and analyzed by immu- noblotting using antibodies specific for 4E-BP1 and actin. (Right) Cultures were metabolically labeled after 24 h and amino acid incorporation quantified as in A (*P < 0.05; ns, not significant by Student’s t test). (C)AsinA except immunoblotting was performed using antibodies specific for eEF2K, eEF2 phospho-T56, eEF2, and actin. (D)AsinB except immunoblotting was performed using antibodies specific for eEF2K and GAPDH. (E) NHDFs transduced with a doxycycline (dox)-inducible FLAG-eEF2K lentivirus were stained with DAPI to visualize nuclei, and indirect immunofluorescence staining was performed using a FLAG- specific antibody. (F) NHDFs transduced as in E were treated with dox and TNFα as indicated. At 24 h post-TNFα treatment, total protein was collected and immunoblotting was performed as in C.(G)AsinF except immunoblotting was performed using antibodies specific for FLAG, eEF2K, and actin.

22584 | www.pnas.org/cgi/doi/10.1073/pnas.1909143116 Bianco et al. Downloaded by guest on October 3, 2021 (Fig. 1B, compare lanes 3 and 4), consistent with reported TNFα- Regulation of eEF2K mRNA Abundance by TNFα Is Dependent upon dependent activation of mTORC1 (36–38), and inhibited the Canonical NF-κB Signaling and the p65 NF-κB Subunit. NHDFs TNFα-induced stimulation of protein synthesis (Fig. 1B). Rapa- expressing doxycycline (dox)-inducible, FLAG-tagged eEF2K were α mycin also reduced 4E-BP1 phosphorylation in control untreated used to investigate whether eEF2K abundance regulated TNF - NHDFs (Fig. 1B, compare lanes 1 and 2). Despite being reduced induced eEF2 phosphorylation. Following dox addition, 100% of by rapamycin, 4E-BP1 phosphorylation in TNFα-treated com- the cells exhibited uniform cytoplasmic FLAG-antigen staining with nuclear sparing by indirect immunofluorescence (Fig. 1E). pared to untreated cells was potentially less sensitive to rapamycin. α Overall eEF2K and eEF2 p-T56 levels increased in dox-treated The exact reasons remain unclear, but could reflect TNF -induced cultures while total eEF2 levels remained similar (Fig. 1F,com- modifications to mTORC1 that might influence the efficacy with pare lanes 1 and 2). Significantly, whereas the abundance of which rapamycin inhibits mTORC1 action on select substrates like eEF2K and eEF2 p-T56 was reduced in response to TNFα (Fig. 4E-BP1 (39, 40). 1F, compare lanes 1 to 3), dox-induced eEF2K expression re- Protein synthesis is also regulated by phosphorylation of the versed this effect (Fig. 1F, compare lanes 3 and 4) and largely critical translation elongation factor eEF2 on T56 by eEF2K, restored eEF2 p-T56 levels to those observed in cells not exposed which slows translation and allows it to be tuned in response to to TNFα (Fig. 1F, compare lanes 4 and 1). Taken together, these physiological and environmental changes (24, 25). To establish findings suggest that eEF2 phosphorylation in response to TNFα whether TNFα regulates eEF2 phosphorylation, total protein is regulated by eEF2K abundance. As FLAG-eEF2K levels in dox- isolated from untreated or TNFα-treated NHDFs was fraction- induced cells were also reduced by TNFα (Fig. 1G), post- ated by SDS/PAGE and analyzed by immunoblotting. Compared transcriptional or combinatorial mechanisms involving transcrip- to untreated cultures, overall levels of T56-phosphorylated eEF2 tional regulation could be involved. were reduced by TNFα while the abundance of total eEF2 was Although the ubiquitin system reportedly regu- lates eEF2K abundance in response to DNA damage checkpoint not detectably lowered (Fig. 1C). While eEF2K is inhibited upon α phosphorylation by p70S6K, which is activated by mTORC1 or activation (41), TNF treatment is known to substantially sculpt the cellular transcriptome by impacting transcriptional activa- p90 RSK1, the reduction in eEF2 phosphorylation was un- tion, repression, and mRNA decay (42, 43). Following exposure expectedly accompanied by a substantial decline in eEF2K pro- to TNFα, analysis of total RNA isolated from NHDFs by RT- tein levels (Fig. 1C) that was not detectably influenced by qPCR revealed a ∼75% reduction in eEF2K mRNA after 6 h

inhibiting mTORC1 using rapamycin (Fig. 1D). This raised the and a 65% reduction after 24 h compared to untreated cells (Fig. BIOCHEMISTRY possibility that eEF2 phosphorylation status in response to TNFα 2 A and B). Overall eEF2K protein abundance also declined over was controlled in part by a different mechanism involving eEF2K this same time period, as did levels of T-56 phosphorylated eEF2 abundance instead of solely via the canonical posttranslational (Fig. 1C). Next, the possibility that the TNFα-induced decrease modification mechanism involving p70S6K. in eEF2K mRNA abundance was dependent upon stimulation of

A 1.5 C D E F

DMSO BAY-11 -+-+ Control p65 :siRNA Control NEMO :siRNA :TNF 1.0 ** NEMO I B p65 actin tubulin Akt 0.5 eEF2K mRNA 2.0 1.5 1.5

0 - + :TNF 1.5 B 1.5 1.0 1.0 ** * ** ** 1.0 1.0 eEF2K mRNA

0.5 eEF2K mRNA 0.5 eEF2K mRNA 0.5

0.5 eEF2K mRNA Control Control 0 0 p65 Control Control 0 NEMO :siRNA --++:TNF :siRNA 0 - + :TNF TNF TNF

Fig. 2. Regulation of eEF2K mRNA abundance by the canonical NF-κB–activating pathway and p65. (A) NHDFs were untreated or TNFα-treated. At 6 h posttreatment, total RNA was isolated and RT-qPCR analysis was performed for eEF2K mRNA. (B)AsinA except cells were treated with TNFα for 24 h. (C) Diagram representing the canonical pathway by which TNFα activates NF-κB transcription factors. (D) NHDFs were transfected with nonsilencing (control) or NEMO-targeting siRNAs. (Upper) At 3 d posttransfection, total protein was collected, separated by SDS/PAGE, and analyzed by immunoblotting using an- tibodies specific for NEMO and tubulin. (Lower) Cultures were untreated or TNFα-treated. At 6 h posttreatment, total RNA was isolated and RT-qPCR was performed for eEF2K mRNA. (E) NHDF cells were treated with DMSO or BAY 11–7082 (10 μM) with or without TNFα.(Upper) At 1 h posttreatment, total protein was fractionated by SDS/PAGE and analyzed by immunoblotting using antibodies specific for IκBα and Akt. (Lower) Total RNA was isolated from cells 6 h posttreatment, and RT-qPCR analysis was performed for eEF2K mRNA. (F) NHDFs were transfected with ns siRNA or p65/RELA siRNA. (Upper)At3d posttransfection, total protein was collected, separated by SDS/PAGE, and analyzed by immunoblotting using antibodies specific for p65 and actin. (Lower) NHDFs were untreated or TNFα-treated. At 6 h posttreatment, total RNA was isolated, and RT-qPCR was performed for eEF2K mRNA. Error bars (A, B,and D–F) indicate SEM (*P ≤ 0.05 and **P ≤ 0.01, Student’s t test).

Bianco et al. PNAS | November 5, 2019 | vol. 116 | no. 45 | 22585 Downloaded by guest on October 3, 2021 the canonical NF-κB pathway by TNFα was investigated (Fig. NF-κB to regulate eEF2 phosphorylation following exposure to 2C). RNA interference was used to deplete IKKγ/NEMO, an stimuli other than TNFα, such as virus infection, was evaluated. IκB kinase (IKK) complex subunit required for degradation of Although NF-κB is rapidly activated in HCMV-infected cells NF-κB inhibitor alpha (IκB) (44). Preventing IκB degradation (45) and eEF2 levels increase via mTORC1-dependent trans- interferes with nuclear translocation of the NF-κB transcription lational control (31), the impact of infection on eEF2 phos- factors p65 and p50 and restricts NF-κB activation. Indeed, NEMO phorylation has not been investigated. Fig. 3A shows that levels abundance was reduced in NHDFs transfected with NEMO of T56 phosphorylated eEF2 decline precipitously as early as siRNA compared to control, nonsilencing (ns) siRNA (Fig. 2D). 6 hpi, well in advance of the substantial virus-induced rise in Furthermore, while eEF2K mRNA abundance was reduced in ns eEF2 abundance that is readily detectable by 24 hpi. By 6 hpi, a siRNA-treated cells exposed to TNFα, eEF2K mRNA levels were modest decline in the abundance of the NF-κB inhibitor, IκB, significantly greater in NEMO-depleted NHDFs following TNFα was observed along with induction of a canonical NF-κB–responsive exposure (Fig. 2D). Similarly, treatment of cells with BAY-11– gene (IL6), consistent with NF-κB being activated under these ex- 7082, a chemical inhibitor that blocks IκB degradation, effectively perimental conditions (Fig. 3 B and C). The decline in phosphory- prevented the TNFα-induced reduction in IκB protein abundance lated eEF2 is accompanied by a reduction in eEF2K levels that is and eEF2K mRNA levels (Fig. 2E). To test directly whether the apparent by 6 hpi and proceeds to its nadir by 24 hpi (Fig. 3A). decrease in eEF2K mRNA abundance in response to TNFα was Phosphorylated eEF2 remains below detection limits through 72 hpi dependent upon NF-κB, the p65 transcription factor NF-κBsubunit despite a limited recovery in eEF2K levels (Fig. 3A). Infection of was depleted using RNAi. Fig. 2F shows that p65 depletion antago- NHDFs with UV-inactivated HCMV, which disables viral gene nized the TNFα-induced reduction in eEF2K mRNA compared to expression, was at least as effective as active virus in reducing overall cultures treated with nonsilencing siRNA. Taken together, this eEF2K levels (Fig. 3D). This suggested that an HCMV virion protein indicates that the reduction of eEF2K mRNA abundance following component or the viral dsDNA genome itself, a potent PAMP ca- TNFα exposure is dependent upon IκB degradation, which regulates pable of inducing a panoply of inflammatory responses, might rep- NF-κB p50/65 subunit nuclear translocation, and the NF-κBtran- resent the underlying trigger for reducing eEF2K abundance. scription factor subunit p65. It further suggests that p65 controls To determine if dsDNA could regulate eEF2 phosphorylation, translation elongation by regulating eEF2K expression. uninfected primary fibroblasts were transfected with and without a synthetic, immunostimulatory dsDNA, and levels of eEF2K, DNA-Sensing Inhibits eEF2 Phosphorylation by Regulating eEF2K eEF2, and T56 phosphorylated eEF2 were measured by immu- mRNA Abundance. As NF-κB activation integrates responses to noblotting. Although eEF2 pT56 and eEF2K were readily de- numerous inflammatory and infectious agents, the capacity of tected in uninfected, transfected NHDFs that were not exposed

A BCD HCMV (hpi)

Uninfected 6244872 100 **

eEF2K Uninfected HCMV HCMV-UV HCMV (hpi) 75 eEF2K 0 3 6 12 24 eEF2 p-T56 IkB 50 IE1/IE2 eEF2 IL6 mRNA Akt 25 UL44 UL44

0 actin actin HCMV: - + EF2.0 G dox: - - + + dsDNA(h): 60 24 1.5 dsDNA: - + - + * IFIT2 eEF2K

eEF2K 1.0 MDA5

eEF2 p-T56 eEF2K mRNA IFIT2 0.5 eEF2 ISG15

0 GAPDH

GAPDH cGAS Control Control p65 : siRNA

+ dsDNA

Fig. 3. Regulation of eEF2 phosphorylation by dsDNA-sensing. (A) NHDFs were infected with HCMV (multiplicity of infection = 3 pfu per cell). At the in- dicated times, total protein was collected, fractionated by SDS/PAGE, and analyzed by immunoblotting using the indicated antibodies. (B)AsinA.(C) NHDFs were mock-infected (uninfected) or infected with HCMV as in A. After 6 h, total RNA was isolated and RT-qPCR was performed using primers specific for IL6 mRNA (**P < 0.01 by Student’s t test). (D)AsinA except cells were infected with HCMV or UV-inactivated HCMV and total protein harvested at 24 hpi. (E) NHDFs were transfected with no DNA or synthetic immunostimulatory dsDNA. At the specified times posttransfection, total protein was collected and im- munoblotting was performed using the indicated antibodies. (F) NHDFs were transfected with nonsilencing siRNA (control), p65/RELA siRNA, or cGAS siRNA and transfected with no DNA or dsDNA. At 24 h posttreatment, total RNA was isolated and RT-qPCR analysis was performed for eEF2K mRNA. Error bars indicate SEM (*P ≤ 0.05; **P ≤ 0.01; and n.s., nonsignificant by Student’s t test). (G) NHDFs transduced with a doxycycline (dox)-inducible FLAG-eEF2K lentivirus were transfected with (+) or without (−) synthetic dsDNA and treated with dox as indicated. After 24 h, total protein was collected and analyzed by immu- noblotting with the indicated antibodies.

22586 | www.pnas.org/cgi/doi/10.1073/pnas.1909143116 Bianco et al. Downloaded by guest on October 3, 2021 to dsDNA, their overall abundance was markedly reduced over A 24 h by dsDNA exposure, while levels of total eEF2 did not detectably decline (Fig. 3E). The dsDNA treatment effectively stimulated type I IFN production, a well-characterized response to dsDNA-sensing as evidenced by the accumulation of the IFN- stimulated gene product IFIT2 (Fig. 3E). The reduction in eEF2K protein levels was accompanied by a decline in eEF2K mRNA abundance and was abrogated by depleting either the NF-κB transcription factor subunit p65 or the dsDNA sensor cGAS (Fig. 3F). Even though eEF2K levels decreased in re- sponse to dsDNA, artificially counteracting the decline in eEF2K abundance using dox-inducible eEF2K-expressing cells to over- express eEF2K did not detectably change the overall abundance BC1.5 1.5 of 3 representative proteins encoded by IFN-stimulated genes (Fig. 3G). This does not preclude the possibility, however, that their rates of synthesis might be impacted. Nevertheless, these *** findings together demonstrate that dsDNA exposure and sensing 1.0 1.0 regulates phosphorylation of the translation elongation factor *** eEF2 and reduces the abundance of its primary regulator eEF2K. Moreover, it establishes that multiple inflammatory triggers in- α 0.5 0.5 eEF2K pre-mRNA cluding TNF and dsDNA modulate eEF2 phosphorylation via eEF2K pre-mRNA an NF-κB–dependent mechanism that regulates eEF2K mRNA abundance. 0

0 Control Control +TNF p65 κ No RT -TNF Repression of eEF2K mRNA Transcription by NF- B p65. Although :siRNA p65 largely functions as a transcriptional activator, NF-κB also reportedly can function as a transcriptional repressor of select

target genes (46, 47). This raised the possibility that the re- TNF BIOCHEMISTRY duction in steady-state eEF2K mRNA levels in response to NF- α κB activation resulted from reduced transcription as opposed to Fig. 4. Repression of eEF2K mRNA transcription by TNF is p65-dependent. (A, Top) eEF2K pre-mRNA exons (1, 2; blue boxes) and intron 1 (solid black posttranscriptional control by mRNA decay. To test whether line). The location of primers specific for the pre-mRNA intron (green arrows) eEF2K mRNA transcription might be repressed by p65, the and those specific for exons 1 and 2 (gold arrows) are shown. (A, Bottom) abundance of mRNA containing the first intron of eEF2K pre- Mature eEF2K mRNA, which is comprised of 18 exons. The 5-terminal mRNA mRNA were measured by RT-qPCR (Fig. 4A). As intron re- cap is represented by m7Gppp, and the polyadenylated 3′-end is shown as moval by mRNA splicing happens cotranscriptionally, this method A(n). (B) NHDFs were untreated or TNFα-treated. At 6 h posttreatment, total provides a suitable proxy for transcription rates (48). In response RNA was isolated and RT-qPCR analysis was performed for eEF2K pre-mRNA. to TNFα exposure, eEF2K pre-mRNA levels declined sub- (C) NHDFs were transfected with nonsilencing siRNA (control) or p65/RELA α stantially in a manner that was largely counteracted by p65 siRNA and treated with TNF . At 6 h posttreatment, total RNA was isolated depletion (Fig. 4 B and C). This demonstrates that eEF2K pre- and RT-qPCR analysis was performed for eEF2K pre-mRNA. (A and B)Acon- trol siRNA-treated culture was not exposed to TNFα as a reference for eEF2K mRNA levels are reduced in a manner dependent upon the p65 ≤ κ mRNA levels in untreated NHDFs. Error bars indicate SEM (***P 0.001, NF- B transcription factor subunit. Furthermore, it is consistent Student’s t test). with eEF2K transcription being repressed by p65 and with eEF2K being a negatively regulated NF-κB–responsive gene. Indeed, data from chromatin immunoprecipitation studies avail- eEF2K abundance by comparing overall protein synthesis in able on the ENCODE database map p65 (RelA) occupancy to the NHDFs where eEF2K was depleted using siRNA to NHDFs eEF2K promoter, supporting the notion that eEF2K is an NF-κB– treated with nonsilencing siRNA (Fig. 5B). Remarkably, eEF2K responsive gene (49, 50). depletion significantly interfered with ETA-induced inhibition of protein synthesis at 2 toxin concentrations (Fig. 5 C and D). This eEF2K Regulates the Responsiveness of Protein Synthesis to Exotoxin κ – demonstrates that the sensitivity of fibroblast protein synthesis to A. To test whether NF- B dependent eEF2K repression could ETA is dependent upon eEF2K abundance. Thus, regulating impact host responses, we sought a model to evaluate whether eEF2K abundance can in fact control protein synthesis and fibroblast protein synthesis would respond to levels of active change the sensitivity of fibroblasts to an ADP ribosylating toxin versus inactive, T-56 phosphorylated eEF2. Determining whether that selectively targets eEF2. the stimulation of TNFα-induced protein synthesis was dependent upon eEF2 elongation activity was difficult because (i) eEF2 acts Discussion catalytically and is among the most abundant of cellular proteins By virtue of the rapid responses engendered, regulating mRNA (51) and (ii)TNFα also stimulates protein synthesis initiation by promoting 4E-BP1 hyperphosphorylation (Fig. 1B and refs. 36– translation is ideally suited to tune gene expression to inflam- 38). To circumvent these confounding parameters, protein syn- matory and infectious insults. While translation initiation is rate- thesis following exposure to a bacterial toxin selective for eEF2 limiting and has been intensively investigated (56), much less is was investigated. P. aeruginosa is a Gram-negative bacteria known known regarding how infection and inflammation impact cellular to activate NF-κB(52–54) that produces an ADP ribosylating translation elongation factors. Here, we reveal an unexpected , exotoxin A (ETA), which irreversibly inactivates eEF2 mechanism whereby the critical translation elongation factor (55). Incubation of NHDFs with ETA concentrations greater than eEF2 is stimulated upon activation of the transcription factor 8 ng/mL profoundly inhibited global protein synthesis as measured NF-κB, a universal coordinator of cell-intrinsic responses to in- by metabolic labeling with 35S amino acids (Fig. 5A). Having de- flammation and infection (1). Phosphorylation of eEF2 on T56 fined concentrations at which ETA efficiently reduced protein by eEF2K regulates translation elongation by reducing the af- synthesis, we next asked whether ETA toxicity was impacted by finity of eEF2 for the 80S ribosome and thereby slowing the

Bianco et al. PNAS | November 5, 2019 | vol. 116 | no. 45 | 22587 Downloaded by guest on October 3, 2021 A focus exclusively on human fibroblasts and show that NF-κB ETA activation effectively stimulates translation by preventing eEF2K kDa 170 from slowing elongation. Preserving eEF2 activity by limiting 130 eEF2K abundance could better enable fibroblasts to coordinate 100 70 B responses that resolve inflammation and repair tissue damage. 55 Besides translation elongation, initiation is also stimulated by 40 Control eEF2K :siRNA TNFα as mTORC1 activation inhibits the cellular repressor of 35 eEF2K cap-dependent translation 4E-BP1, which is consistent with pub- 25 lished reports (36–38). Furthermore, mTORC1 also stimulates actin p70S6K1, which phosphorylates eEF2K S366 (61) and inhibits 15 eEF2K activity. Due to the quick kinetics of TNFα-induced 10 mTORC1 activation (62), this could represent a rapid initial re- sponse to stimulate protein synthesis by inactivating 4E-BP1 and/ C D or eEF2K through posttranscriptional modification. Coupling this Control siRNA eEF2K siRNA κ – kDa 01531 01531: ETA (ng/mL) initial response to an NF- B dependent, transcriptionally medi- 170 0.6 ated decrease in eEF2K protein abundance could maintain high 130 100 * eEF2 activity as time progresses and allow unrestricted elongation 70 even if initiation on mRNAs relying upon a cap-dependent 0.4 55 translation mechanism was tempered by inhibiting mTORC1. 40 Likewise, limiting eEF2K abundance could allow maximal eEF2 35 0.2 activity on transcripts that rely upon noncanonical initiation 25

Protein Synthesis mechanisms when functional eIF4F levels are limited by inhibiting

(ETA treated/untreated) mTORC1. This could explain earlier studies showing that that Control 0 eEF2K 15 :siRNA insulin stimulates muscle protein synthesis in neonates during 10 acute endotoxemia despite suppression of translation initiation, suggesting that an mTOR-independent process regulates muscle Fig. 5. eEF2K depletion renders cells refractory to protein synthesis in- protein synthesis (63). Additionally, as eEF2K inhibits the trans- hibition by exotoxin A. (A) NHDFs were treated with increasing concentra- lation rate of TNFα mRNA (64), TNFα-induced reduction in tions (0 to 2,000 ng/mL) of exotoxin A (ETA). At 24 h posttreatment, cells eEF2K protein abundance could positively regulate TNFα syn- 35 were metabolically radiolabeled with S-containing amino acids for 30 min. thesis to amplify inflammatory signaling. Total protein was isolated and separated by SDS/PAGE, and the fixed, dried Using NF-κB to repress eEF2K likely represents a mechanism gel was exposed to X-ray film. (B) NHDFs were transfected with nonsilencing to support translation of the hundreds of transcripts induced by (control) or eEF2K targeting siRNAs. At 3 d posttransfection, total protein κ was collected, separated by SDS/PAGE, and analyzed by immunoblotting NF- B in response to infection or nucleic acid sensing (1, 65, 66). using antibodies specific for eEF2K and actin. (C)AsinB, except that, 3 d Limiting eEF2K levels prevents eEF2 T56 phosphorylation, after siRNA transfection, cells were treated with the indicated concentra- which would slow elongation and cellular protein synthesis and tions of ETA. (D)AsinB, except that, 3 d after siRNA transfection, cells were potentially impact infection outcome. Our finding that eEF2K treated with 0 ng/μL or 31 ng/μL ETA. At 24 h posttreatment, cells were depletion influences the inhibition of protein synthesis in re- 35 metabolically radiolabeled with S-containing amino acids for 30 minutes, sponse to ETA is consistent with this hypothesis and supports a and the amount of acid-insoluble radioactivity was quantified by counting in role for eEF2K in influencing host responses to pathogens. liquid scintillant. *P < 0.05 by Student’s t test. Translation rates have also been shown to inversely correlate with cotranslational folding efficiency as well as translational translocation step in protein synthesis (24, 25). In response to accuracy (67). While accelerating elongation by repressing eEF2K different NF-κB–activating stimuli including TNFα, HCMV in- is expected to enable rapid protein production, this potentially fection, and synthetic immunostimulatory dsDNA, we show that compromises translational accuracy and fidelity (68) upon NF-κB the overall abundance of T56 phosphorylated eEF2 is reduced activation by inflammatory or infection stress. It also provides a together with a corresponding decrease in eEF2K overall levels. means to reduce accuracy of mRNA decoding through elongation The decrease in eEF2K protein abundance was dependent upon while mTORC1 is inactivated. Significantly, regulating eEF2K the classical NF-κB activation pathway and the p65 NF-κB tran- levels by NF-κB allows infection and/or inflammation to influence scription factor. Surprisingly, the reduction in eEF2K pre-mRNA elongation rates independent of mTORC1/p70S6K. This distin- levels was dependent upon p65, suggesting that eEF2K tran- guishes regulation of elongation in response to infection stress from scription is repressed by NF-κB activation. Finally, we demon- energy insufficiency or nutrient insufficiency (69). Reducing eEF2K strate that manipulating eEF2K abundance regulates protein levels by NF-κB activation could reduce fidelity of viral mRNA synthesis in response to a bacterial toxin that inactivates eEF2. translation as part of a broader defense strategy or impact synthesis This establishes that NF-κB activation by physiological effectors of cellular and or viral proteins from alternate reading frames and/ controls eEF2 activity via a transcriptional repression mechanism or non-AUG initiator codons. Indeed, analysis of cis-acting se- that limits eEF2K abundance. Moreover, it illustrates how eEF2, quences controlling translation of innate immune effectors including which catalyzes the translocation step in translation elongation, MAVS revealed they did not fit classic Kozak consensus sites (70). can be integrated into the network of innate immune responses Instead, there were hundreds of transcripts that are predicted to orchestrated by NF-κB and provides a powerful example of how encode more than one protein product. Alternatively, speeding up nuclear transcription regulation shapes translation elongation in elongation would resolve ribosome queuing, which can form in re- eukaryotes. sponse to protein synthesis inhibitors or occur naturally to regulate Previous reports on how TNFα and NF-κB impact protein polyamine synthesis via translation of an inhibitory upstream ORF synthesis have been conflicting, with some demonstrating an (71). In either case, reducing eEF2K levels in direct response to enhancement in protein synthesis and others inhibition (57–60), NF-κB–activating stimuli provides a powerful mechanism to tune perhaps due to differing cell types and culture conditions. In translation elongation to a wide range of physiological and path- addition, prior genome-wide studies did not identify eEF2K ological processes impacted by NF-κB, including cancer, infection among genes transcriptionally repressed by TNF (43). Here, we biology, development, inflammation, and immunity.

22588 | www.pnas.org/cgi/doi/10.1073/pnas.1909143116 Bianco et al. Downloaded by guest on October 3, 2021 Materials and Methods previously described (77). Briefly, sense (5′-TAAGACACGATGCGATAAAAT- ′ Cell Culture, Viruses, Transfections, Metabolic Labeling with Radioactive Amino CTGTTTGTAAAATTTATTAAGGGTACAAATTGCCCTAGC-3 ) and anti-sense Acids, RT-qPCR, and Immunoblotting. Normal human dermal fibroblasts (Lonza; (5′- GCTAGGGCAATTTGTACCCTTAATAAATTTTACAAACAGATTTTATCGC- CC-2509) were maintained and serum-deprived as previously described (72). ATCGTGTCTTA-3′) strands were annealed by heating at 95 °C for 3 min Human cytomegalovirus (AD169-GFP) (73) propagation and infections, trans- and cooling to room temperature in annealing buffer (10 mM Tris·HCl, fections of dsDNA and siRNA, immunoblotting, metabolic labeling with ra- pH 7.5, 100 mM NaCl, and 1 mM EDTA). dioactive amino acids, and RT-qPCR were previously described (74). GAPDH was used for normalization of all RT-qPCR experiments. UV-inactivated HCMV Antibodies, Chemicals, and Cytokines. Antibodies used in this study: anti-4E- 2 was prepared as described (72) using 6 pulses of 125 mJ/cm . The pINDUCER21- BP1 (Bethyl; A300-501A), anti-tubulin (Sigma; T5168), anti-eEF2K (Cell Signaling; plasmid encoding dox-inducible eEF2K (75) was generously provided by Fabio 3692), anti-eEF2 (Cell Signaling; 2332), anti-eEF2 phospho Thr-56 (Cell Signaling; Martinon (University of Lausanne, Lausanne, Switzerland) and used to gen- 2331), anti-actin (Cell Signaling; 3700), anti-NEMO/IKKγ (Cell Signaling; 2685), erate lentivirus as described previously (76). anti-IκBα (Proteintech; 10268–1-AP), anti-p65/RELA (Proteintech; 10745–1-AP), anti-Akt (Cell Signaling; 9272), and anti-Flag (Cell Signaling; 2368S). Doxycycline Nucleotide Sequences. Primer sequences used in this study were eEF2K for- μ α ward (5′-GCACAGCCAGAGTTACTGGAC-3′), eEF2K reverse (5′-CTCTGCCAA- (Sigma; D3072) was used at 0.125 g/mL (PeproTech; – CTCCTCTGGAC-3′), eEF2K pre-mRNA forward (5′-CGCTTCTCCTCTGGTTGTT- 300 02A) was dissolved in water and used at 50 ng/mL P. aeruginosa 3′), eEF2K pre-mRNA reverse (5′-TTCCATATCCACATCGCCTTTA-3′), GAPDH exotoxin A was obtained from List Biological Labs (no. 160) and dissolved forward (5′-TCTTTTGCGTCGCCAGCCGA-3′), and GAPDH reverse (5′-ACCAGGCG- in water. Rapamycin was dissolved in DMSO and used at 100 ng/mL. CCCAATACGACC-3′). Sequences of siRNAs used in this study were NEMO/IKBKG (5′-GAGAGA- ACKNOWLEDGMENTS. We thank members of the Mohr laboratory, Michael CUCGGCCUGGAGA[dT][dT]-3′), p65/RELA (5′-GGAAUCCAGUGUGUGAAGA[dT] Garabedian, and Angus Wilson for helpful discussions and Dr. Fabio Martinon [dT]-3′), cGAS (5′-GCUACUAUGAGCACGUGAA[dT][dT]-3′), and eEF2K (5′- for the eEF2K expression vector. This work was supported by National CUCAUCACAUCCUAGCCGA [dT][dT]-3′). Institutes of Health Grants GM056927 and AI073898 to I.M. Public Health Synthetic double-strand DNA (dsDNA) was produced by annealing sense Service Institutional Research Training Awards (AI007180, AI07647) in part and anti-sense 60-mer oligonucleotides derived from HSV-1 sequences as supported both C.B. and L.T.

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