Abstract
Autophagy is a lysosomal degradation pathway important for cellular homeostasis, mammalian development, cancer and immunity. Many molecular components of autophagy have been identified, but little is known about regulatory mechanisms controlling their effector functions. Here, we show that, in contrast to other p38 MAP kinase activators, the growth arrest and DNA damage 45 beta (Gadd45β)–MAPK/ERK kinase kinase 4 (MEKK4) pathway specifically directs p38 to autophagosomes. This process results in an accumulation of autophagosomes through p38-mediated inhibition of lysosome fusion. Conversely, autophagic flux is increased in p38-deficient fibroblasts and Gadd45β-deficient cells. We further identified the underlying mechanism and demonstrate that phosphorylation of the autophagy regulator autophagy-related (Atg)5 at threonine 75 through p38 is responsible for inhibition of starvation-induced autophagy. Thus, we show for the first time that Atg5 activity is controlled by phosphorylation and, moreover, that the spatial regulation of p38 by Gadd45β/MEKK4 negatively regulates the autophagic process.
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Abbreviations
- ASK1:
-
apoptosis signaling kinase 1
- Atg:
-
autophagy-related
- BMDM:
-
bone marrow-derived macrophage
- Gadd45β:
-
growth arrest and DNA damage 45 beta
- GFP:
-
green fluorescent protein
- JNK:
-
c-Jun N-terminal kinase
- LC3:
-
microtubule-associated protein 1 light chain 3
- LPS:
-
lipopolysaccharide
- MAPK:
-
mitogen-activated protein kinase
- M-CSF:
-
macrophage colony-stimulating factor
- MEF:
-
mouse embryonic fibroblast
- MEKK4:
-
MAPK/ERK kinase kinase 4
- MKK:
-
MAP kinase kinase
- PE:
-
phosphatidylethanolamine
- RFP:
-
red fluorescent protein
- TLR:
-
Toll-like receptor
- WT:
-
wild-type
References
Mizushima N, Komatsu M . Autophagy: renovation of cells and tissues. Cell 2011; 147: 728–741.
Levine B, Mizushima N, Virgin HW . Autophagy in immunity and inflammation. Nature 2011; 469: 323–335.
He C, Klionsky DJ . Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 2009; 43: 67–93.
Nishida Y, Arakawa S, Fujitani K, Yamaguchi H, Mizuta T, Kanaseki T et al. Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature 2009; 461: 654–658.
Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T et al. The role of autophagy during the early neonatal starvation period. Nature 2004; 432: 1032–1036.
Yousefi S, Perozzo R, Schmid I, Ziemiecki A, Schaffner T, Scapozza L et al. Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol 2006; 8: 1124–1132.
Lee IH, Finkel T . Regulation of autophagy by the p300 acetyltransferase. J Biol Chem 2009; 284: 6322–6328.
Lin SY, Li TY, Liu Q, Zhang C, Li X, Chen Y et al. GSK3-TIP60-ULK1 signaling pathway links growth factor deprivation to autophagy. Science 2012; 336: 477–481.
Alers S, Loffler AS, Wesselborg S, Stork B . Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol Cell Biol 2012; 32: 2–11.
Liebermann DA, Hoffman B . Myeloid differentiation (MyD) primary response genes in hematopoiesis. Oncogene 2002; 21: 3391–3402.
Papa S, Zazzeroni F, Bubici C, Jayawardena S, Alvarez K, Matsuda S et al. Gadd45 beta mediates the NF-kappa B suppression of JNK signalling by targeting MKK7/JNKK2. Nat Cell Biol 2004; 6: 146–153.
Takekawa M, Saito H . A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell 1998; 95: 521–530.
Lu B, Ferrandino AF, Flavell RA . Gadd45beta is important for perpetuating cognate and inflammatory signals in T cells. Nat Immunol 2004; 5: 38–44.
Gupta M, Gupta SK, Balliet AG, Hollander MC, Fornace AJ, Hoffman B et al. Hematopoietic cells from Gadd45a- and Gadd45b-deficient mice are sensitized to genotoxic-stress-induced apoptosis. Oncogene 2005; 24: 7170–7179.
Gupta SK, Gupta M, Hoffman B, Liebermann DA . Hematopoietic cells from gadd45a-deficient and gadd45b-deficient mice exhibit impaired stress responses to acute stimulation with cytokines, myeloablation and inflammation. Oncogene 2006; 25: 5537–5546.
Papa S, Zazzeroni F, Fu YX, Bubici C, Alvarez K, Dean K et al. Gadd45beta promotes hepatocyte survival during liver regeneration in mice by modulating JNK signaling. J Clin Invest 2008; 118: 1911–1923.
Mita H, Tsutsui J, Takekawa M, Witten EA, Saito H . Regulation of MTK1/MEKK4 kinase activity by its N-terminal autoinhibitory domain and GADD45 binding. Mol Cell Biol 2002; 22: 4544–4555.
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 2000; 19: 5720–5728.
Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 2008; 4: 151–175.
Kimura S, Noda T, Yoshimori T . Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 2007; 3: 452–460.
Adams RH, Porras A, Alonso G, Jones M, Vintersten K, Panelli S et al. Essential role of p38alpha MAP kinase in placental but not embryonic cardiovascular development. Mol Cell 2000; 6: 109–116.
Shaul YD, Seger R . The MEK/ERK cascade: from signaling specificity to diverse functions. Biochim Biophys Acta 2007; 1773: 1213–1226.
Zhang N, Ahsan MH, Zhu L, Sambucetti LC, Purchio AF, West DB . NF-kappaB and not the MAPK signaling pathway regulates GADD45beta expression during acute inflammation. J Biol Chem 2005; 280: 21400–21408.
Xu Y, Jagannath C, Liu XD, Sharafkhaneh A, Kolodziejska KE, Eissa NT . Toll-like receptor 4 is a sensor for autophagy associated with innate immunity. Immunity 2007; 27: 135–144.
Mizushima N, Levine B, Cuervo AM, Klionsky DJ . Autophagy fights disease through cellular self-digestion. Nature 2008; 451: 1069–1075.
Pattingre S, Bauvy C, Carpentier S, Levade T, Levine B, Codogno P . Role of JNK1-dependent Bcl-2 phosphorylation in ceramide-induced macroautophagy. J Biol Chem 2009; 284: 2719–2728.
Wei Y, Pattingre S, Sinha S, Bassik M, Levine B . JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy. Mol Cell 2008; 30: 678–688.
Comes F, Matrone A, Lastella P, Nico B, Susca FC, Bagnulo R et al. A novel cell type-specific role of p38alpha in the control of autophagy and cell death in colorectal cancer cells. Cell Death Differ 2007; 14: 693–702.
Haussinger D, Schliess F, Dombrowski F, Vom Dahl S . Involvement of p38MAPK in the regulation of proteolysis by liver cell hydration. Gastroenterology 1999; 116: 921–935.
Prick T, Thumm M, Kohrer K, Haussinger D, Vom Dahl S . In yeast, loss of Hog1 leads to osmosensitivity of autophagy. Biochem J 2006; 394: 153–161.
Tang G, Yue Z, Talloczy Z, Hagemann T, Cho W, Messing A et al. Autophagy induced by Alexander disease-mutant GFAP accumulation is regulated by p38/MAPK and mTOR signaling pathways. Hum Mol Genet 2008; 17: 1540–1555.
Webber JL, Tooze SA . Coordinated regulation of autophagy by p38alpha MAPK through mAtg9 and p38IP. EMBO J 2010; 29: 27–40.
Saitoh T, Fujita N, Hayashi T, Takahara K, Satoh T, Lee H et al. Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response. Proc Natl Acad Sci USA 2009; 106: 20842–20846.
Wang X, Mader MM, Toth JE, Yu X, Jin N, Campbell RM et al. Complete inhibition of anisomycin and UV radiation but not cytokine induced JNK and p38 activation by an aryl-substituted dihydropyrrolopyrazole quinoline and mixed lineage kinase 7 small interfering RNA. J Biol Chem 2005; 280: 19298–19305.
Ueffing N, Keil E, Freund C, Kuhne R, Schulze-Osthoff K, Schmitz I . Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment. Cell Death Differ 2008; 15: 773–782.
Porras A, Zuluaga S, Black E, Valladares A, Alvarez AM, Ambrosino C et al. P38 alpha mitogen-activated protein kinase sensitizes cells to apoptosis induced by different stimuli. Mol Biol Cell 2004; 15: 922–933.
McWhirter SM, Barbalat R, Monroe KM, Fontana MF, Hyodo M, Joncker NT et al. A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP. J Exp Med 2009; 206: 1899–1911.
Mak SK, Kultz D . Gadd45 proteins induce G2/M arrest and modulate apoptosis in kidney cells exposed to hyperosmotic stress. J Biol Chem 2004; 279: 39075–39084.
Gerwins P, Blank JL, Johnson GL . Cloning of a novel mitogen-activated protein kinase kinase kinase, MEKK4, that selectively regulates the c-Jun amino terminal kinase pathway. J Biol Chem 1997; 272: 8288–8295.
Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 1997; 275: 90–94.
Acknowledgements
We thank Stephanie Grosch, Birgit Lamik-Wolters, Moritz Lemke, Daniel Scholtyssik and Sabrina Schumann for their expert technical assistance. We wish to thank Dr Melanie Brinkmann, Dr Maximiliano Gutierrez and Alisha Walker for critically reading the manuscript as well as Marion Nissen and Dr Heinz Mehlhorn for help with electron microscopy. We are grateful to Drs Pär Gerwins, Antje Gohla, Dietmar Kültz, Stephan Ludwig, Hans-Uwe Simon, Björn Stork, Osamu Takeuchi, Sebastian Wesselborg and Tamotsu Yoshimori for various plasmids and to Dr Ute Fischer for recombinant caspase-3. We thank Dr Angel Nebreda for p38-deficient MEFs and Dr Noboru Mizushima for Atg5-deficient MEFs. This work was supported by grants GK1033, GK1302, SFB685 and SFB773 (to KS-O), Leibniz (to KP) and SCHM1586/3-1 (to IS) of the Deutsche Forschungsgemeinschaft and by the Fritz-Thyssen-Stiftung (to IS).
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Keil, E., Höcker, R., Schuster, M. et al. Phosphorylation of Atg5 by the Gadd45β–MEKK4-p38 pathway inhibits autophagy. Cell Death Differ 20, 321–332 (2013). https://doi.org/10.1038/cdd.2012.129
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DOI: https://doi.org/10.1038/cdd.2012.129
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