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The Role of TTP Phosphorylation in the Regulation of Inflammatory Cytokine Production by MK2/3

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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! Every submission reviewed by practicing scientists by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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.
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  • The Combination of TPL2 Knockdown and Tnfα Causes Synthetic Lethality Via Caspase-8 Activation in Human Carcinoma Cell Lines

    The Combination of TPL2 Knockdown and Tnfα Causes Synthetic Lethality Via Caspase-8 Activation in Human Carcinoma Cell Lines

    The combination of TPL2 knockdown and TNFα causes synthetic lethality via caspase-8 activation in human carcinoma cell lines Oksana B. Serebrennikovaa, Maria D. Paraskevopouloua, Elia Aguado-Frailea,1, Vasiliki Tarasliaa, Wenying Rena,2, Geeta Thapaa, Jatin Ropera,3, Keyong Dua,2, Carlo M. Croceb,c,4, and Philip N. Tsichlisa,b,c,4 aMolecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111; bDepartment of Cancer Biology and Genetics, The Ohio State University, Columbus, OH 43210; and cThe Ohio State University Comprehensive Cancer Center, Columbus, OH 43210 Contributed by Carlo M. Croce, May 21, 2019 (sent for review January 29, 2019; reviewed by Emad S. Alnemri and Wafik El-Deiry) Most normal and tumor cells are protected from tumor necrosis subset of tumor cells and we delineate the relevant TPL2-regulated factor α (TNFα)-induced apoptosis. Here, we identify the MAP3 pathway(s). kinase tumor progression locus-2 (TPL2) as a player contributing TPL2 is an oncoprotein that is activated by provirus insertion to the protection of a subset of tumor cell lines. The combination in Moloney murine leukemia virus-induced rodent lymphomas of TPL2 knockdown and TNFα gives rise to a synthetic lethality and mammary tumor virus-induced mammary adenocarcinomas phenotype via receptor-interacting serine/threonine-protein ki- (13, 14). Expression of constitutively active TPL2 from a thymus- nase 1 (RIPK1)-dependent and -independent mechanisms. Whereas targeted transgene confirmed its oncogenic potential (15). Sub- wild-type TPL2 rescues
  • A Post-Transcriptional Mechanism Pacing Expression of Neural Genes with Precursor Cell Differentiation Status

    A Post-Transcriptional Mechanism Pacing Expression of Neural Genes with Precursor Cell Differentiation Status

    ARTICLE Received 24 Sep 2014 | Accepted 21 May 2015 | Published 6 Jul 2015 DOI: 10.1038/ncomms8576 OPEN A post-transcriptional mechanism pacing expression of neural genes with precursor cell differentiation status Weijun Dai1, Wencheng Li2, Mainul Hoque2, Zhuyun Li1, Bin Tian2 & Eugene V. Makeyev1,3 Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 30-untranslated regions (30 UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36), an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly, TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand, inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons. 1 School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore. 2 Department of Microbiology, Biochemistry, and Molecular Genetics, Rutgers New Jersey Medical School, Newark, New Jersey 07103, USA. 3 MRC Centre for Developmental Neurobiology, King’s College London, London SE1 1UL, UK. Correspondence and requests for materials should be addressed to E.V.M.
  • RIPK1, Active Recombinant Mouse Protein Expressed in Sf9 Cells

    RIPK1, Active Recombinant Mouse Protein Expressed in Sf9 Cells

    Catalog # Aliquot Size R07M-11G -05 5 µg R07M-11G -10 10 µg RIPK1, Active Recombinant mouse protein expressed in Sf9 cells Catalog # R07M-11G Lot # V2253-5 Product Description Specific Activity Recombinant mouse RIPK1 (1-327) was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag. 20,000 The RIPK1 gene accession number is NM_009068. 15,000 Gene Aliases 10,000 D330015H01Rik; Rinp; RIP; Rip1 Activity (cpm) 5,000 0 Formulation 0 120 240 360 480 Recombinant protein stored in 50mM Tris-HCl, pH 7.5, Protein (ng) 150mM NaCl, 10mM glutathione, 0.1mM EDTA, 0.25mM The specific activity of RIPK1 was determined to be 2.6 nmol DTT, 0.1mM PMSF, 25% glycerol. /min/mg as per activity assay protocol. Storage and Stability Purity Store product at –70oC. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple The purity of RIPK1 was determined freeze/thaw cycles. to be >70% by densitometry, approx. MW 62 kDa. Scientific Background RIPK1 or Receptor Interacting Protein Kinase 1 is a serine/threonine kinase that was originally identified as interacting with the cytoplasmic domain of FAS. RIPK1 has been deemed as an important element in the signal transduction machinery that mediates programmed cell death. RIPK1 has been shown to interact with TRADD, TRAF1, TRAF2 and TRAF3 and TRADD can act as an RIPK1, Active adaptor protein to recruit RIPK1 to the TNFR1 complex in a Recombinant mouse protein expressed in Sf9 cells TNF-dependent process (1).