The Role of TTP Phosphorylation in the Regulation of Inflammatory Cytokine Production by MK2/3 This information is current as Natalia Ronkina, Nelli Shushakova, Christopher Tiedje, of September 28, 2021. Tatiana Yakovleva, Maxim A. X. Tollenaere, Aaron Scott, Tanveer Singh Batth, Jesper Velgaard Olsen, Alexandra Helmke, Simon Holst Bekker-Jensen, Andrew R. Clark, Alexey Kotlyarov and Matthias Gaestel J Immunol published online 16 September 2019 Downloaded from http://www.jimmunol.org/content/early/2019/09/13/jimmun ol.1801221 Supplementary http://www.jimmunol.org/content/suppl/2019/09/13/jimmunol.180122 http://www.jimmunol.org/ Material 1.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published September 16, 2019, doi:10.4049/jimmunol.1801221 The Journal of Immunology The Role of TTP Phosphorylation in the Regulation of Inflammatory Cytokine Production by MK2/3 Natalia Ronkina,*,1 Nelli Shushakova,†,‡,1 Christopher Tiedje,x Tatiana Yakovleva,* Maxim A. X. Tollenaere,x,2 Aaron Scott,{ Tanveer Singh Batth,‖ Jesper Velgaard Olsen,‖ Alexandra Helmke,† Simon Holst Bekker-Jensen,‖ Andrew R. Clark,{ Alexey Kotlyarov,* and Matthias Gaestel* Tristetraprolin (TTP) is an RNA-binding protein and an essential factor of posttranscriptional repression of cytokine biosynthesis in macrophages. Its activity is temporally inhibited by LPS-induced p38MAPK/MAPKAPK2/3–mediated phosphorylation, leading to a rapid increase in cytokine expression. We compared TTP expression and cytokine production in mouse bone marrow–derived macrophages of different genotypes: wild type, MAPKAP kinase 2 (MK2) deletion (MK2 knockout [KO]), MK2/3 double deletion Downloaded from (MK2/3 double KO [DKO]), TTP-S52A-S178A (TTPaa) knock-in, as well as combined MK2 KO/TTPaa and MK2/3 DKO/TTPaa. The comparisons reveal that MK2/3 are the only LPS-induced kinases for S52 and S178 of TTP and the role of MK2 and MK3 in the regulation of TNF biosynthesis is not restricted to phosphorylation of TTP at S52/S178 but includes independent processes, which could involve other TTP phosphorylations (such as S316) or other substrates of MK2/3 or p38MAPK. Furthermore, we found differences in the dependence of various cytokines on the cooperation between MK2/3 deletion and TTP mutation ex vivo. In the cecal ligation and puncture model of systemic inflammation, a dramatic decrease of cytokine production in MK2/3 DKO, TTPaa, http://www.jimmunol.org/ and DKO/TTPaa mice compared with wild-type animals is observed, thus confirming the role of the MK2/3/TTP signaling axis in cytokine production also in vivo. These findings improve our understanding of this signaling axis and could be of future relevance in the treatment of inflammation. The Journal of Immunology, 2019, 203: 000–000. any cytokine mRNAs display adenylate-uridinylate–rich Under steady-state conditions, TTP is ubiquitously expressed at a elements (AREs) in their 39 untranslated region, which basal level. Inflammatory stimuli like LPS and various cytokines M allows specific binding of regulatory proteins and a mediate transcriptional and posttranscriptional induction of TTP resulting posttranscriptional regulation of cytokine biosynthesis expression in hematopoietic cell lineages (2, 3). Rapid upregula- at the level of mRNA stability and translation (1). Tristetraprolin tion of TTP expression in the early inflammatory response is by guest on September 28, 2021 (TTP) is one of such ARE-binding proteins, which destabilizes accompanied by its transient phosphorylation at multiple sites. mRNAs, inhibits their translation, and thereby suppresses cytokine At least two phosphorylation sites of TTP, S52 and S178, were production. Its constitutive deletion in mice leads to increased shown to be phosphorylated directly by the p38MAPK-activated basal levels of cytokines, such as TNF, and symptoms of cachexia (2). kinases MK2 and MK3 (3, 4). Inactivation of p38MAPK/MK2/3 signaling by p38 or MK2/3 inhibitors or by MK2/3 deletion in mice resulted in a dramatic decrease of TTP-targeted inflammatory *Institute of Cell Biochemistry, Center of Biochemistry, Hannover Medical School, D-30625 Hannover, Germany; †Division of Nephrology and Hypertension, Hannover cytokines and TTP expression, indicating a transient inhibitory Medical School, D-30625 Hannover, Germany; ‡Phenos GmbH, D-30625 Hannover, role of p38/MK2/3 signaling on TTP’s suppressive activity (4–6). Germany; xCenter for Healthy Aging, Department of Cellular and Molecular { Loss of the inhibitory effect of MK2/3-mediated phosphorylation Medicine, University of Copenhagen, DK-2200 Copenhagen, Denmark; Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University on TTP activity was also observed in knock-in mice, in which the ‖ of Birmingham, B15 2TT Birmingham, United Kingdom; and Proteomics Program, TTP gene was altered to code for a nonphosphorylatable mutant, Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark TTP-S52A-S178A (TTPaa) (2). Similar to MK2/3-deficient (MK2/3 aa/aa 1 double knockout [DKO]) mice, the TTP knock-in mice (TTPaa N.R. and N.S. contributed equally to this work as first authors. mice) demonstrate significant reduction of TTP-targeted inflamma- 2Current address: LEO Pharma A/S, Ballerup, Denmark. tory cytokines and TTP expression, reflecting the inability of TTPaa ORCIDs: 0000-0003-3772-4136 (N.S.); 0000-0002-3013-6967 (C.T.); 0000-0002-2653- 0777 (M.A.X.T.); 0000-0001-9325-5026 (A.S.); 0000-0003-4996-8322 (A.R.C.); 0000- to be inactivated by p38 MAPK/MK2/3 signaling. These observa- 0002-4944-4652 (M.G.). tions indicate that transient phosphorylation of TTP inhibits its Received for publication September 10, 2018. Accepted for publication August 20, suppressive function and enables its target cytokines to be 2019. expressed and elucidate the inflammatory response before TTP Address correspondence and reprint requests to Prof. Matthias Gaestel, Institute of Cell dephosphorylation reactivates its suppressive function, leading Biochemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, to decreased cytokine production resolving inflammation. This Germany. E-mail address: [email protected] is also in agreement with the finding that phospho-TTP can be The online version of this article contains supplemental material. replaced by the mRNA-stabilizing factor HuR at the AREs (3). Abbreviations used in this article: ARE, AU-rich element; BMDM, bone marrow– derived macrophage; CLP, cecal ligation and puncture; DKO, double knockout; Interestingly, TTPaa protein is induced to lower levels than KO, knockout; PL, peritoneal lavage; PMN, polymorphonuclear neutrophil; SILAC, wild-type (WT) TTP in LPS-stimulated bone marrow–derived stable isotope labeling by amino acids in cell culture; TTP, tristetraprolin; TTPaa, macrophages (BMDMs) (2). This can be explained by two distinct TTP-S52A-S178A; WT, wild-type. phenomena. First, an ARE in TTP’s own mRNA allows negative Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 feedback control of TTP expression (4). Constitutively active TTPaa www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801221 2 TTP PHOSPHORYLATION IN THE REGULATION OF CYTOKINES BY MK2/3 2 13 15 13 15 is therefore able to suppress its own expression more effectively H4 (medium), or L-arginine C6- N4 and L-lysine C6- N2 (heavy; by this feedback mechanism, leading to lower TTPaa levels than Cambridge Isotope Laboratories). To induce TTP expression, cells were m WT TTP (2). Second, the phosphorylation of TTP at serines 52 induced with 1 g/ml doxycycline overnight and pretreated with the MK2 inhibitor PF3644022 (catalog no. PZ0188, 10 mM; Sigma-Aldrich) for and 178 not only impairs the mRNA-destabilizing activity of TTP, 60 min before stimulation with anisomycin (1 mg/ml; Sigma-Aldrich) for it also protects TTP against proteasome-dependent degradation. 60 min. GFP immunoprecipitations were carried out using a magnetic Hence, the nonphosphorylatable mutant TTPaa is both highly active GFP-Trap binding resin (Chromotek). Beads were eluted in 23 Laemmli and highly unstable (2, 5, 6). buffer, and the resulting supernatants were mixed and loaded onto a 4–12% NuPAGE Bis-Tris Gel (Novex; Thermo Fisher Scientific). Further processing Analyses of TTP phosphorylation revealed many in vitro phos- of samples and mass spectrometry was performed as described before (21). phorylation sites for various protein kinases (7) and several sites phosphorylated in transfected cells (2, 8, 9). However, the above- Cecal ligation and puncture–induced sepsis model described signaling pathway targets only two sites of the MK2/3- Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) substrate TTP downstream of the p38MAPK/MK2/3 axis. So far, it under light isoflurane anesthesia in 10- to 12-wk-old mice. A 1-cm ventral is not clear whether other substrates of MK2/3 or downstream to midline abdominal incision was made. The cecum was then exposed, ligated the p38MAPK/MK2/3 axis [such as small Hsps (10), RTN4/NOGO-B with 4-0 silk sutures just distal to the ileocecal valve (comprising 70% of the cecum and sparing the cecal vessels) to avoid intestinal obstruction, and (11), SRF (12), RIPK1 (13–15), or UBE2J1 (16)] contributes to punctured through with a 24-gauge needle.