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The kinase Jnk2 promotes stress-induced mitophagy by targeting the small mitochondrial form of the tumor suppressor ARF for degradation

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A Corrigendum to this article was published on 18 June 2015

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Abstract

Mitophagy is essential for cellular homeostasis, but how mitophagy is regulated is largely unknown. Here we found that the kinase Jnk2 was required for stress-induced mitophagy. Jnk2 promoted ubiquitination and proteasomal degradation of the small mitochondrial form of the tumor suppressor ARF (smARF). Loss of Jnk2 led to the accumulation of smARF, which induced excessive autophagy that resulted in lysosomal degradation of the mitophagy adaptor p62 at steady state. Depletion of p62 prevented Jnk2-deficient cells from mounting mitophagy upon stress. Jnk2-deficient mice displayed defective mitophagy, which resulted in tissue damage under hypoxic stress, as well as hyperactivation of inflammasomes and increased mortality in sepsis. Our findings define a unique mechanism of maintaining immunological homeostasis that protects the host from tissue damage and mortality.

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Figure 1: Jnk2 is required for stress-induced mitophagy.
Figure 2: Loss of Jnk2 results in decreased steady-state levels of p62.
Figure 3: Loss of Jnk2 results in excessive steady-state autophagy.
Figure 4: Loss of Jnk2 stabilizes smARF.
Figure 5: Jnk2 promotes the ubiquitination and degradation of smARF.
Figure 6: Jnk2 prevents hypoxia-induced tissue damage.
Figure 7: Loss of Jnk2 enhances the activation of inflammasomes.

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  • 20 April 2015

    In the version of this article initially published, the staining intensity for the merged fluorescence microscopy did not match that in the single-staining panels in Figure 1c,e. The panels been replaced with the original staining intensities. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank M. Karin (University of California, San Diego) for Jnk2−/− mice; R. Youle (National Institute of Neurological Disorders and Stroke, National Institutes of Health) for the mCherry-parkin plasmid; Y. Cheng and M.A. Baker for help with the analysis of inflammasome activation; A. Yemelyanov for lentivirus preparation; and J. Hou for help with animal work. Supported by the US National Institutes of Health (HL114763 to J.L.) and the American Asthma Foundation (13-0114 to J.L.).

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Q.Z. and H.K. did experiments and analyzed the data; C.C., J.Y. and H.C.D.-U. did experiments; X.L., L.D., K.M.R. and N.S.C. provided reagents; and J.L. designed and supervised the study, did experiments, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Jing Liu.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Jnk2 is required for stress-induced mitophagy in vitro.

(a) WT and Jnk2-/- MEFs were treated with HBSS for indicated times. Degradation of Tom20 and hsp60 was assessed by immunoblotting. (b,c) WT MEFs were treated with hypoxia (b) or CCCP (c) in the absence or presence of bafilomycin A1. Degradation of Tom20 was determined. (d) Characterization of Jnk1-/- and Jnk2-/- MEFs. (e-g) Degradation of Tom20 in WT and Jnk1-/- MEFs treated with CCCP (e), or hypoxia (f), or HBSS (g). (h-j) Degradation of Tom20 in WT or Jnk2-/- MEFs transfected with expression vector encoding HA-JNK2 or empty vector followed by treatment with hypoxia (h), or CCCP (i), or HBSS (j). (k) Formation of LC3() in hypoxia- (left panel) or HBSS-treated (right panel) WT or Jnk2-/- MEFs. BA, bafilomycin A1. NS, non-specific. Data are representative of three independent experiments (a-k).

Supplementary Figure 2 Deletion of Jnk2 does not affect the stabilization of PINK1 or recruitment of parkin to mitochondria.

WT and Jnk2-/- MEFs transfected with mCherry-Parkin were treated with hypoxia or CCCP for indicated times. PINK1 protein leves were analyzed by immunoblotting (a,b). Mitochondria were immunostained for Tom20 (green). Colocalization between parkin and mitochondria was analyzed using fluorescence microscopy (c, left panel) and scored among 100 mCherry-parkin-positive cells (c, right panel). NS, non-significant. Data are representative of three independent experiments (a-c) (c; mean and s.e.m. of three independent experiments with biological duplicates in each).

Supplementary Figure 3 Jnk2 does not affect transcription of the gene encoding p62.

mRNA levels of p62 in WT and Jnk2-/- MEFs. NS, non-significant. Data are from three independent experiments with biological duplicates in each (mean and s.e.m. of n = 6(duplicates in three experiments)).

Supplementary Figure 4 smARF is stabilized in the absence of Jnk2.

(a) Expression of Beclin 1 and ATG5 in WT and Jnk2-/- MEFs. (b) Immunoblotting analysis of p19ARF and smARF in Jnk2-/- MEFs transfected with control siRNA or ARF siRNA. (c) Immunoblotting analysis of p19ARF and smARF in WT and Jnk2-/- MEFs with another ARF antibody (sc-32748, Santa Cruz Biotechnology). (d) MS/MS analysis of immunoprecipitated smARF from Jnk2-/- MEFs. (e) Expressions of p19ARF and smARF in WT MEFs transfected with control siRNA or JNK1 siRNA. (f) WT MEFs stably expressing Xpress-tagged smARF were transfected with control siRNA or JNK2 siRNA. Expression of Xpress-tagged smARF was assessed. (g) Expressions of p19ARF and smARF in WT or Jnk2-/- MEFs transfected with control siRNA or JNK1 siRNA. (h) Expression of p62 in WT, Jnk2-/- or Arf-/- MEFs transduced by control lentivirus or lentiviral vectors encoding Xpress-tagged p19ARF(M45I) as indicated. (i) WT or Arf-/- MEFs were transfected with control siRNA or JNK2 siRNA as indicated. Thirty-six h later, cells were treated with hypoxia or CCCP for indicated times. Expressions of Tom20 and p62 were determined. Data are representative of three independent experiments (a-i).

Supplementary Figure 5 Jnk2 facilitates the proteasomal degradation of smARF.

(a) WT or Jnk2-/- MEFs were treated with bafilomycin A1 for indicated times. Expressions of p19ARF and smARF were determined. (b) Expression of HIF-1α in hypoxia-treated WT or Jnk2-/- MEFs. (c) Expression of IκBα in TNF-treated WT or Jnk2-/- MEFs. (d) Co-immunoprecipitation of ectopic Xpress-tagged smARF and HA-JNK1 in Arf-/- MEFs. (e) WT and Jnk2-/- MEFs were fractionated into cytosolic or mitochondrial fractions. Expression of JNK2 was analyzed by immunoblotting. Tubulin and TIM23 were used as markers for the cytoplasm and mitochondria, respectively. (f) Co-immunoprecipitation of ectopic Xpress-tagged p19ARF and HA-JNK2 in Arf-/- MEFs. (g) Arf-/- MEFs were transfected with Xpress-tagged p19ARF and HA-ubiquitin together with control siRNA or JNK2 siRNA. Ubiquitination of Xpress-tagged p19ARF was determined by immunoprecipitation with anti-Xpress followed by immunoblotting with anti-HA. BA, bafilomycin A1. Data are representative of three independent experiments (a-g).

Supplementary Figure 6 p62 levels are reduced and smARF accumulates in the heart and lungs of Jnk2-deficient mice at steady state.

(a,b) Expressions of p62 (a), p19ARF and smARF (b) in the heart and lungs of WT and Jnk2-/- mice in the steady state. Data are representative of three independent experiments (a,b).

Supplementary Figure 7 Jnk2 suppresses the activation of inflammasomes.

(a) Activation of NF-κB (indicated by IκBα protein levels), p38 and ERK in LPS plus ATP-treated WT or Jnk2-/-BMDMs. (b) WT or Jnk2-/- BMDMs were transduced with control lentiviral vector or lentiviral vector encoding Xpress-tagged p62. Ninety-six h later, cells were treated with LPS plus ATP. Expression of Xpress-tagged p62 was determined. (c) Secretion of IL-1β and IL-18 in MH-S cells transfected with control siRNA or JNK2 siRNA followed by treatment with LPS plus ATP. (d) Secretion of IL-1β and IL-18 in WT or Jnk2-/- BMDMs treated with LPS plus Rotenone or Antimycin A. (e) Generation of mitochondrial ROS in Antimycin A-treated WT or Jnk2-/- BMDMs. (f) The steady-state expressions of p62, p19ARF and smARF in WT or Jnk2-/- BMDMs. (g) Hematological analyses of WT and Jnk2-/- mice (8-12 weeks old) were performed using a Hemavet Hematology Analyzer. RBC, red blood cells; HB, hemoglobin; HCT, hematocrit. (h,i) Ter119 (h) or Mitotracker Green (i) versus CD71 staining of RBCs from 3- or 8-week-old WT and Jnk2-/- mice. NS, non-specific. UT, untreated. Data are representative of three independent experiments (a ,b,d,e,f,h,i) (d; mean and s.e.m. of three mice per group with technical duplicates),or are from three independent experiments with biological duplicates in each (c; mean and s.e.m. of n = 6(duplicates in three experiments)), or are from ten mice per group (g; mean and s.e.m. of n = 10). *, P< 0.05 by unpaired Student’s t-test.

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Zhang, Q., Kuang, H., Chen, C. et al. The kinase Jnk2 promotes stress-induced mitophagy by targeting the small mitochondrial form of the tumor suppressor ARF for degradation. Nat Immunol 16, 458–466 (2015). https://doi.org/10.1038/ni.3130

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