Receptor-interacting protein kinase-1 (RIPK1), a master regulator of cell fate decisions, was identified as a direct substrate of MAPKAP kinase-2 (MK2) by phosphoproteomic screens using LPS-treated macrophages and stress-stimulated embryonic fibroblasts. p38MAPK/MK2 interact with RIPK1 in a cytoplasmic complex and MK2 phosphorylates mouse RIPK1 at Ser321/336 in response to pro-inflammatory stimuli, such as TNF and LPS, and infection with the pathogen Yersinia enterocolitica. MK2 phosphorylation inhibits RIPK1 autophosphorylation, curtails RIPK1 integration into cytoplasmic cytotoxic complexes, and suppresses RIPK1-dependent apoptosis and necroptosis. In Yersinia-infected macrophages, RIPK1 phosphorylation by MK2 protects against infection-induced apoptosis, a process targeted by Yersinia outer protein P (YopP). YopP suppresses p38MAPK/MK2 activation to increase Yersinia-driven apoptosis. Hence, MK2 phosphorylation of RIPK1 is a crucial checkpoint for cell fate in inflammation and infection that determines the outcome of bacteria–host cell interaction.
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Christofferson, D. E., Li, Y. & Yuan, J. Control of life-or-death decisions by RIP1 kinase. Annu. Rev. Physiol. 76, 129–150 (2014).
Ofengeim, D. & Yuan, J. Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death. Nat. Rev. Mol. Cell Biol. 14, 727–736 (2013).
Pasparakis, M. & Vandenabeele, P. Necroptosis and its role in inflammation. Nature 517, 311–320 (2015).
Weinlich, R. & Green, D. R. The two faces of receptor interacting protein kinase-1. Mol. Cell 56, 469–480 (2014).
Cusson-Hermance, N., Khurana, S., Lee, T. H., Fitzgerald, K. A. & Kelliher, M. A. Rip1 mediates the Trif-dependent toll-like receptor 3- and 4-induced NF-κB activation but does not contribute to interferon regulatory factor 3 activation. J. Biol. Chem. 280, 36560–36566 (2005).
Meylan, E. et al. RIP1 is an essential mediator of Toll-like receptor 3-induced NF-κB activation. Nat. Immunol. 5, 503–507 (2004).
Dannappel, M. et al. RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis. Nature 513, 90–94 (2014).
Lalaoui, N. et al. Targeting p38 or MK2 enhances the anti-leukemic activity of Smac-mimetics. Cancer Cell 29, 145–158 (2016).
Ea, C.-K., Deng, L., Xia, Z.-P., Pineda, G. & Chen, Z. J. Activation of IKK by TNFα requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol. Cell 22, 245–257 (2006).
Li, H., Kobayashi, M., Blonska, M., You, Y. & Lin, X. Ubiquitination of RIP is required for tumor necrosis factor α-induced NF-κB activation. J. Biol. Chem. 281, 13636–13643 (2006).
Dondelinger, Y. et al. NF-κB-independent role of IKKα/IKKβ in preventing RIPK1 kinase-dependent apoptotic and necroptotic cell death during TNF signaling. Mol. Cell 60, 63–76 (2015).
Cho, Y. S. et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137, 1112–1123 (2009).
Degterev, A. et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat. Chem. Biol. 4, 313–321 (2008).
Gaestel, M. MAPKAP kinases—MKs—two’s company, three’s a crowd. Nat. Rev. Mol. Cell Biol. 7, 120–130 (2006).
Gaestel, M., Kotlyarov, A. & Kracht, M. Targeting innate immunity protein kinase signalling in inflammation. Nat. Rev. Drug Discov. 8, 480–499 (2009).
Ronkina, N. et al. Stress induced gene expression: a direct role for MAPKAP kinases in transcriptional activation of immediate early genes. Nucl. Acids Res. 39, 2503–2518 (2011).
Heidenreich, O. et al. MAPKAP kinase 2 phosphorylates serum response factor in vitro and in vivo. J. Biol. Chem. 274, 14434–14443 (1999).
Tiedje, C. et al. The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation. Nucl. Acids Res. 44, 7418–7440 (2016).
Kotlyarov, A. et al. MAPKAP kinase 2 is essential for LPS-induced TNF-α biosynthesis. Nat. Cell Biol. 1, 94–97 (1999).
Ronkina, N. et al. The mitogen-activated protein kinase (MAPK)-activated protein kinases MK2 and MK3 cooperate in stimulation of tumor necrosis factor biosynthesis and stabilization of p38 MAPK. Mol. Cell Biol. 27, 170–181 (2007).
Lehner, M. D. et al. Mitogen-activated protein kinase-activated protein kinase 2-deficient mice show increased susceptibility to Listeria monocytogenes infection. J. Immunol. 168, 4667–4673 (2002).
Tiedje, C. et al. The p38/MK2-driven exchange between tristetraprolin and HuR regulates AU-rich element-dependent translation. PLoS Genet. 8, e1002977 (2012).
Menon, M. B. et al. Endoplasmic reticulum-associated ubiquitin-conjugating enzyme Ube2j1 is a novel substrate of MK2 (MAPKAP kinase-2) involved in MK2-mediated TNFα production. Biochem. J. 456, 163–172 (2013).
Tiedje, C. et al. p38MAPK/MK2-mediated phosphorylation of RBM7 regulates the human nuclear exosome targeting complex. RNA 21, 262–278 (2015).
Lee, T. H., Shank, J., Cusson, N. & Kelliher, M. A. The kinase activity of Rip1 is not required for tumor necrosis factor-α-induced IκB kinase or p38 MAP kinase activation or for the ubiquitination of Rip1 by Traf2. J. Biol. Chem. 279, 33185–33191 (2004).
Sun, X., Yin, J., Starovasnik, M. A., Fairbrother, W. J. & Dixit, V. M. Identification of a novel homotypic interaction motif required for the phosphorylation of receptor-interacting protein (RIP) by RIP3. J. Biol. Chem. 277, 9505–9511 (2002).
Sanz, L., Sanchez, P., Lallena, M. J., Diaz-Meco, M. T. & Moscat, J. The interaction of p62 with RIP links the atypical PKCs to NF-κB activation. EMBO J. 18, 3044–3053 (1999).
Feoktistova, M. et al. cIAPs block Ripoptosome formation, a RIP1/caspase-8 containing intracellular cell death complex differentially regulated by cFLIP isoforms. Mol. Cell 43, 449–463 (2011).
Tenev, T. et al. The Ripoptosome, a signaling platform that assembles in response to genotoxic stress and loss of IAPs. Mol. Cell 43, 432–448 (2011).
Schwermann, J. et al. MAPKAP kinase MK2 maintains self-renewal capacity of haematopoietic stem cells. EMBO J. 28, 1392–1406 (2009).
Berger, S. B. et al. Cutting Edge: RIP1 kinase activity is dispensable for normal development but is a key regulator of inflammation in SHARPIN-deficient mice. J. Immunol. 192, 5476–5480 (2014).
Newton, K. RIPK1 and RIPK3: critical regulators of inflammation and cell death. Trends Cell Biol. 25, 347–353 (2015).
de Almagro, M. C., Goncharov, T., Newton, K. & Vucic, D. Cellular IAP proteins and LUBAC differentially regulate necrosome-associated RIP1 ubiquitination. Cell Death Dis. 6, e1800 (2015).
Trosky, J. E., Liverman, A. D. B. & Orth, K. Yersinia outer proteins: Yops. Cell. Microbiol. 10, 557–565 (2008).
Mukherjee, S. et al. Yersinia YopJ acetylates and inhibits kinase activation by blocking phosphorylation. Science 312, 1211–1214 (2006).
Mittal, R., Peak-Chew, S.-Y. & McMahon, H. T. Acetylation of MEK2 and I κ B kinase (IKK) activation loop residues by YopJ inhibits signaling. Proc. Natl Acad. Sci. USA 103, 18574–18579 (2006).
Paquette, N. et al. Serine/threonine acetylation of TGFβ-activated kinase (TAK1) by Yersinia pestis YopJ inhibits innate immune signaling. Proc. Natl Acad. Sci. USA 109, 12710–12715 (2012).
He, S., Liang, Y., Shao, F. & Wang, X. Toll-like receptors activate programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway. Proc. Natl Acad. Sci. USA 108, 20054–20059 (2011).
Dondelinger, Y., Vandenabeele, P. & Bertrand, M. J. M. Regulation of RIPK1’s cell death function by phosphorylation. Cell Cycle 15, 5–6 (2016).
Koppe, C. et al. IκB kinaseα/β control biliary homeostasis and hepatocarcinogenesis in mice by phosphorylating the cell-death mediator receptor-interacting protein kinase 1. Hepatology 64, 1217–1231 (2016).
Jaco, I. et al. MK2 phosphorylates RIPK1 to prevent TNF-induced cell death. Mol. Cell 66, 698–710.e5 (2017).
Dillon, C. P. & Balachandran, S. StIKKing it to a death kinase: IKKs prevent TNF-α-induced cell death by phosphorylating RIPK1. Cytokine 78, 47–50 (2016).
Reinhardt, H. C. et al. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 11, 175–189 (2007).
Stoecklin, G. et al. MK2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and ARE-mRNA decay. EMBO J. 23, 1313–1324 (2004).
Blasius, M., Wagner, S. A., Choudhary, C., Bartek, J. & Jackson, S. P. A quantitative 14-3-3 interaction screen connects the nuclear exosome targeting complex to the DNA damage response. Genes Dev. 28, 1977–1982 (2014).
Dondelinger, Y., Darding, M., Bertrand, M. J. M. & Walczak, H. Poly-ubiquitination in TNFR1-mediated necroptosis. Cell. Mol. Life Sci. 73, 2165–2176 (2016).
Vandendriessche, B. et al. MAPK-activated protein kinase 2-deficiency causes hyperacute tumor necrosis factor-induced inflammatory shock. BMC Physiol. 14, 5 (2014).
Dondelinger, Y. et al. MK2 phosphorylation of RIPK1 regulates TNF-mediated cell death. Nat. Cell Biol. http://doi.org/10.1038/ncb3608 (2017).
Weng, D. et al. Caspase-8 and RIP kinases regulate bacteria-induced innate immune responses and cell death. Proc. Natl Acad. Sci. USA 111, 7391–7396 (2014).
Philip, N. H. et al. Caspase-8 mediates caspase-1 processing and innate immune defense in response to bacterial blockade of NF-κB and MAPK signaling. Proc. Natl Acad. Sci. USA 111, 7385–7390 (2014).
Burke, J. R. et al. BMS-345541 is a highly selective inhibitor of I κ B kinase that binds at an allosteric site of the enzyme and blocks NF-κB-dependent transcription in mice. J. Biol. Chem. 278, 1450–1456 (2003).
Mourey, R. J. et al. A benzothiophene inhibitor of mitogen-activated protein kinase-activated protein kinase 2 inhibits tumor necrosis factor α production and has oral anti-inflammatory efficacy in acute and chronic models of inflammation. J. Pharmacol. Exp. Ther. 333, 797–807 (2010).
Kaiser, W. J. et al. Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J. Biol. Chem. 288, 31268–31279 (2013).
Vanlangenakker, N. et al. cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production. Cell Death Differ. 18, 656–665 (2011).
Tan, L. et al. Discovery of type II inhibitors of TGFβ-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2). J. Med. Chem. 58, 183–196 (2015).
Kelliher, M. A. et al. The death domain kinase RIP mediates the TNF-induced NF-κB signal. Immunity 8, 297–303 (1998).
Kang, S. et al. Caspase-8 scaffolding function and MLKL regulate NLRP3 inflammasome activation downstream of TLR3. Nat. Commun. 6, 7515 (2015).
Mack, C., Sickmann, A., Lembo, D. & Brune, W. Inhibition of proinflammatory and innate immune signaling pathways by a cytomegalovirus RIP1-interacting protein. Proc. Natl Acad. Sci. USA 105, 3094–3099 (2008).
Rothe, J. et al. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature 364, 798–802 (1993).
Novikova, L. et al. Cell death triggered by Yersinia enterocolitica identifies processing of the proinflammatory signal adapter MyD88 as a general event in the execution of apoptosis. J. Immunol. 192, 1209–1219 (2014).
Ruckdeschel, K. et al. Yersinia outer protein P of Yersinia enterocolitica simultaneously blocks the nuclear factor-κ B pathway and exploits lipopolysaccharide signaling to trigger apoptosis in macrophages. J. Immunol. 166, 1823–1831 (2001).
Ting, A. T., Pimentel-Muiños, F. X. & Seed, B. RIP mediates tumor necrosis factor receptor 1 activation of NF-κB but not Fas/APO-1-initiated apoptosis. EMBO J. 15, 6189–6196 (1996).
Salomonis, N. et al. Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation. Proc. Natl Acad. Sci. USA 107, 10514–10519 (2010).
Weber, K., Bartsch, U., Stocking, C. & Fehse, B. A multicolor panel of novel lentiviral ‘gene ontology’ (LeGO) vectors for functional gene analysis. Mol. Ther. 16, 698–706 (2008).
Menon, M. B. et al. p38 MAP kinase and MAPKAP kinases MK2/3 cooperatively phosphorylate epithelial keratins. J. Biol. Chem. 285, 33242–33251 (2010).
Micheau, O. & Tschopp, J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114, 181–190 (2003).
Degterev, A., Zhou, W., Maki, J. L. & Yuan, J. Assays for necroptosis and activity of RIP kinases. Meth. Enzymol. 545, 1–33 (2014).
Moquin, D. M., McQuade, T. & Chan, F. K.-M. CYLD deubiquitinates RIP1 in the TNFα-induced necrosome to facilitate kinase activation and programmed necrosis. PLoS ONE 8, e76841 (2013).
Vanden Berghe, T. et al. Determination of apoptotic and necrotic cell death in vitro and in vivo. Methods 61, 117–129 (2013).
We would like to acknowledge K. A. Fitzgerald, M. A. Kelliher, A. T. Ting and W. Brune for the gift of RIPK1-deficient cell lines, G. Tiegs and C. Wegscheid for providing the Tnfr1−/− mice, A. Pich for MS analysis, G. Evan, V. M. Dixit, J. Tschopp, and M. Treier for sharing expression vectors, A. Schambach for providing pLBID lentiviral vector, M. Windheim for discussion of results, K. Laaß for experimental support and T. Yakovleva for technical help. We thank A. Gossler for critical reading of the manuscript, J. C. Silva for help with phosphoproteomics, and I. Braren and the UKE HEXT Vector Facility for establishment of a lentiviral transduction system. This study was supported by grants from the Deutsche Forschungsgemeinschaft DFG to K.R. (Ru788/3-2 and Ru788/6-1) and M.G. (SFB566, TP B12).
The authors declare no competing financial interests.
Integrated supplementary information
a. Classification of phospho-peptides identified in anisomycin-stimulated MEFs and LPS-stimulated macrophages (BMDM), based on the gene-ontology class of the target proteins. b. Peptides detected in Phosphoscan analysis and fold changes dependent on the presence of MK2 (N.D: peptide not detected in KO samples). c and d. Mass-spectrometric analysis of GST-mRIPK1 purified from anisomycin-stimulated HEK293T cells. Total coverage (c) and phospho-sites identified (d), listed with additional available information on the respective sites based on literature and database searches. e. S313 is not required for p38/MK2-mediated phosphorylation of mRIPK1. HEK293T cells were transfected and subjected to GST-pull down and immunoblotting analysis as in Figs 1d and 2d. Neither the mobility shift nor the phospho-PKD substrate motif antibody signals depend on the integrity of the S313 residue. f. The RIPK1 inhibitor Nec-1 does not inhibit the TNF-induced shift of murine RIPK1 even at concentration as high as 100 μM, while the shift is abrogated by two independent p38 MAPK inhibitors (BIRB796, 1 μM and SB202190, 5 μM). g. IKKβ inhibitor BMS345541 does not inhibit the TNF-induced MK2-dependent band-shift of RIPK1. h. Both MAPKAP kinases, MK2 and MK3 can mediate stress induced RIPK1 phosphorylation. MK2/3 DKO MEF cells were reconstituted with MK2, catalytic dead-MK2 (K79R), MK3 expression vector or an empty vector control. The cells were stimulated with anisomycin and subjected to immunoblot analysis to monitor the electrophoretic mobility shift of RIPK1. An antibody against MK2-T222 was used to detect activating phosphorylations of MK2 and MK3, Hsc70 is shown as loading control. The phosphorylation of bonafide substrates of MK2/3, HSPB1 (S86 phosphorylation) and UBE2j1 (band-shift), is shown as positive control. Results shown are representative of 2 (Supplementary Fig. 1e, h) and 3 (Supplementary Fig. 1f, g) independent experiments, respectively.
Supplementary Figure 2 MK2-RIPK1 interaction requires RIPK1-dimerisation and is independent of RIPK3.
a,b,c. Indicated RIPK1 expression vectors (a,b) and MK2 vectors (c) were co-transfected in HEK 293T cells and RIPK1 self-association (a,b) and RIPK1-MK2 interaction were probed by using GST-pull down experiments. d. Schematic presentation of the RIPK1 mutants used in the study and summary of the findings indicating the capability of the different mutants to self-associate as well as to interact with MK2. Self-association or dimerization capacity seems to be a prerequisite for interaction with MK2. e. Recruitment of RIPK1 to MK2 does not depend on RIPK3, but MK2/RIPK1/RIPK3 can form a ternary complex. HEK293T cells were transfected with expression constructs for GST-MK2, Myc-RIPK1, FLAG-RIPK3 and control empty vector controls. 24 h post transfection, cell lysates were subjected to GST-pull down and the pull downs together with input controls were probed by western blotting. While Myc-RIPK1 is strongly and specifically enriched by GST-MK2, FLAG-RIPK3 enrichment is significant only when co-expressed with Myc-RIPK1, suggesting that RIPK1 bridges the MK2-RIPK3 interaction. Results shown in Supplementary Fig. 2a–c, e are representative of 2 independent experiments.
Supplementary Figure 3 MK2 suppresses RIPK1-dependent cell death and RIPK1 activity, but not receptor associated ubiquitination.
a,b. MK2 KO MEFs transduced with MK2 or empty vector control were treated as indicated for 5 h and cell viability was assessed by WST-1-assay. Average values are plotted ± s.d. (∗p = 0.00303; ∗∗p = 8.2 × 10−6, ∗∗∗p = 3.9 × 10−7 n = 3 independent wells). (c). Cells were treated with 15 μg ml−1 cycloheximide (CHX) or 5 μM SM for 1 h, followed by 2 h with 20 ng ml−1 TNF. Consistent with death assay data in panel 3b, immunoblot analysis of the lysates shows strong autophosphorylation of RIPK1 in response to TNF + SM treatment in MK2-deficient cells. The samples in the spliced panel were obtained and processed simultaneously. d. Flow cytometric analysis of RIPK1 expression in WT and RIPK1-KO-MEFs reconstituted with the indicated RIPK1 expression vectors or empty vector controls (top panel). The lower panel shows the median fluorescent intensity of RIPK1 staining in the positive-gated population. RIPK1-WT reconstituted cells show higher RIPK1 expression than WT MEFs and RIPK1-SSAA rescued cells. e. Percentages of necrostatin-sensitive RIPK1-dependent death from the data presented in Fig. 4e were quantified and plotted as mean ± s.e.m. (n = 4, ∗p = 0.0227). f. RIPK1-SSAA mutation was combined with the K45A mutation and death assays were performed as in Fig. 4e. A representative experiment performed in duplicate is shown. g. RIPK1-WT- or RIPK1-SSAA-reconstituted RIPK1-KO MEFs were treated as in Fig. 4f and immunoprecipitated RIPK1 was subjected to autophosphorylation in in vitro-kinase assay. RIPK1-westernblot is shown as IP-control. h. RIPK1 KO MEFs reconstituted with RIPK1-WT or RIPK1-S321D/S336D (RIPK1-SSDD) mutant were treated as indicated for 21 h and cell death was assessed as in Fig. 4e. Quantitative results are expressed as means ± s.e.m. from n = 3 independent experiments (∗p = 0.0417). i. MK2 KO MEFs transduced with MK2 expression vector or empty vector control were stimulated with 1 μg ml−1 FLAG-tagged TNF and the TNF-receptor complex was immunoprecipitated using αFLAG-M2 affinity beads. The immunoprecipitates and input lysates were probed with the indicated antibodies. Statistical analysis was performed using two-tailed unpaired t-test. Viability and cell death data of Supplementary Fig. 3a, b, f are representative of two (Supplementary Fig. 3b) or three (Supplementary Fig. 3a, f) independent experiments. Statistical source data and results of independent repeat experiments are provided in Supplementary Table 1. Shown immunoblot results are representative of 2 (Supplementary Fig. 3c, d, g, j) and 3 (Supplementary Fig. 3i) independent experiments, respectively.
Supplementary Figure 4 Yersinia-induced apoptosis in macrophages: dissecting involvement of IKK, TAK1, p38/MK2 inhibition by YopP in RIPK1-dependent cytotoxicity.
a. J774A.1 macrophages treated with lentiviral RIPK1 shRNA or scrambled control shRNA were infected with wild type Y. enterocolitica Ye, or YopP-negative Ye-ΔyopP. Dead cells were quantified after 5 h. Results are expressed as means ± s.e.m. from n = 3 independent experiments (∗∗∗p = 0.0003). RIPK1 protein levels were analyzed by immunoblotting and equal loading of the gels was controlled by determining the actin levels. b,c. RIPK1 phosphorylation occurs independent of RIPK1 activity. J774A.1 macrophages were infected with yersiniae in presence of the RIPK1 inhibitor Nec-1s where indicated (b). In c, J774A.1 macrophages were transfected with Myc-tagged wild type RIPK1 or kinase-inactive K45A-RIPK1 mutant (KM) before infection. Empty pcDNA3 vector was used as control (vector). RIPK1 electrophoretic mobility was analyzed by immunoblotting in cell lysates prepared after 90 (b), or 35 min of stimulation (c) using anti-RIPK1 (b) or anti-myc antibodies (c). d,e. Primary bone-marrow derived macrophages (BMDM) from WT (+/+) or TNFR1-KO (−/−) mice were infected with WT (Ye) or YopP-negative Yersinia (Ye-ΔyopP) and the mobility shift of RIPK1 was monitored by immunoblotting (d). Cell death was quantified after 6.5 h and results are expressed as means from two independent experiments (e). f. Inhibitors of TAK1, p38, and MK2 suppress RIPK1 phosphorylation induced by Yersinia. Immortalized RIPK1+/+ fetal liver mouse macrophages were infected with YopP-negative Ye-ΔyopP in presence of inhibitors for TAK1 (NP, NG25), IKKβ (BMS), IKK (TPCA), p38 (SB203580), MK2 (Inh III, PF3644022), JNK (SP600125), or MEK1/ERK (PD098059). The phosphorylation of RIPK1 was analyzed by immunoblotting in cell lysates prepared after 75 min of infection. g,h. Infected macrophages treated with specific inhibitors of TAK1 or IKK undergo RIPK1-mediated cell death. RIPK1+/+ and −/− immortalized fetal liver macrophages (g) or immortalized BMDM (h) were stimulated with Ye-ΔyopP or LPS in presence of inhibitors for TAK1 (NP), IKK (TPCA), IKKβ (BMS), and Nec-1s as indicated. Cell death was quantified after 5 h (g) or 4 h (h). i,j. NP and BMS trigger RIPK1-dependent apoptosis. Caspase-3 activation was monitored by analyzing DEVD-AMC cleavage in cell lysates prepared after 3 h of infection (i). The generation of the active caspase-8 p18 (CASP8 p18) was assessed after 3.5 h of infection (j). Results in g,h,i are expressed as means ± s.e.m. from independent experiments (g: ∗p = 0.0002, ∗∗p = 0.0007, ∗∗∗p = 0.0063, n = 3; h: ∗p < 0.0001, ∗∗p = 0.0002, ∗∗∗p = 0.0108, ∗∗∗∗p = 0.0006, ∗∗∗∗∗p = 0.0028, ∗∗∗∗∗∗p = 0.0341, n = 3; i: ∗p = 0.005, n = 3; ∗∗p = 0.0036, n = 4). k. J774A.1 macrophages were treated with LPS in presence of inhibitors for p38 (SB203580, BIRB796, VX-745) or MK2 (Inh III). RIPK1 phosphorylation was analyzed in cell lysates prepared after 90 min. The samples in the spliced panel of Fig. 4k were obtained and processed simultaneously. Statistical analysis was performed using two-tailed unpaired t-test and statistical source data are provided as Supplementary Table 1. Results shown are representative of 2 (Supplementary Fig. 4j), 3 (Supplementary Fig. 4c, f, k), or 4 (Supplementary Fig. 4b) independent experiments. The experiment of Supplementary Fig. 4d was performed once.
Supplementary Figure 5 Suppressive role of MK2 in LPS-dependent macrophage necroptosis as well as in SM-induced autocrine TNF-dependent apoptosis.
a. p38MAPK/MK2 inhibition alone has no major impact on cell viability in LPS-treated macrophages. MK2/3 DKO + MK2 or MK2/3 DKO + vector macrophages were stimulated with LPS in presence of Nec-1s, the p38 inhibitor SB or the MK2 inhibitor PF where indicated. Cell death was quantified after 4 h using Sytox Green Nucleic Acid Stain. Results are expressed as means ± s.e.m. from n = 3 independent experiments. b. MK2 inhibition promotes LPS-induced necroptosis of macrophages. J774A.1 macrophages were stimulated with LPS in presence of Nec-1s, the MK2 inhibitor PF and zVAD where indicated. Cell death was quantified after 14 h. Results are expressed as means ± s.e.m. from n = 4 independent experiments (∗p = 0.0006, ∗∗p = 0.0012, ∗∗∗p = 0.0013, ∗∗∗∗p = 0.0004). c,d. MK2 inhibition triggers SM-induced apotosis of macrophages Immortalized BMDMs of the indicated genotypes were treated with 10 μM Smac mimetics (SM) with or without 1 h pretreatment with the caspase-inhibitor zVAD or the RIPK1 inhibitor Necrostatin-1 (Nec-1). After 7 h, cell viability was assessed by WST-1 assay in n = 3 independent wells (means ± s.d.,∗∗∗p = 0.0209) (in c). Immortalized macrophages as indicated in panel c were treated with LPS or SM with or without 1 h pretreatment with Nec-1 for the shown time points. Cell lysates were probed with the indicated antibodies (in d). The cleavage of RIPK1 in the course of apoptosis is reduced in MK2-positive cells and prevented by RIPK1 inhibition. EF2 is shown as loading control and MAPK activation (pERK1/2) for monitoring stimulation. Statistical analysis was performed using two-tailed unpaired t-test. The viability experiment of Supplementary Fig. 5c is representative of three independent experiments. Statistical source data and results of independent repeat experiments are provided in Supplementary Table 1. The immunoblot result of Supplementary Fig. 5d is representative of 2 independent experiments.
Supplementary Figure 6 Additive roles of the IKK- and p38MAPK/MK2-dependent checkpoints in the suppression of cytotoxic RIPK1 signaling.
Ligand binding induces recruitment of RIPK1 to the stimulated receptor and RIPK1 ubiquitination. The subsequent induction of TAK1 triggers downstream p38/MK2 activation, whereas the parallel recruitment of the IKK complex to poly-ubiquitinated RIPK1 mediates pro-survival NFκB activation and phosphorylation of receptor-associated RIPK1 (probably at murine S332 and human S331). MK2 activation downstream to TAK1-p38 signaling induces phosphorylation of the majority of cytosolic RIPK1 at S321/S336, which could further be recruited to the receptor complexes. These synergistic phosphorylation events by MK2 (cytosol, checkpoint 2) and IKK (receptor, checkpoint 1) keep RIPK1 activity and cytotoxicity under stringent control (left panel). When intermediate domain phosphorylation of RIPK1 is inhibited by YopP (inactivating TAK1 and IKK), or by pharmacological inhibition of p38/MK2 and IKK, RIPK1 undergoes activation and autophosphorylation. While activated RIPK1 dissociated from the receptor complex could seed for a cytosolic ripoptosome-like complex, non-phosphorylated RIPK1 in the cytosol could amplify the signal by enhancing cross autophosphorylation (in trans), which may subsequently promote the formation of fully functional RIPK1-containing complexes that elicit a strong pro-cytotoxic response (right panel).
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Menon, M., Gropengießer, J., Fischer, J. et al. p38MAPK/MK2-dependent phosphorylation controls cytotoxic RIPK1 signalling in inflammation and infection. Nat Cell Biol 19, 1248–1259 (2017). https://doi.org/10.1038/ncb3614
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