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Analysis of neuronal phosphoproteome reveals PINK1 regulation of BAD function and cell death

Cell Death & Differentiationvolume 25pages904917 (2018) | Download Citation

Abstract

PINK1 mutations that disrupt its kinase activity cause autosomal recessive early onset Parkinson’s disease (PD). Although research in recent years has elucidated a PINK1-Parkin pathway of mitophagy activation that requires PINK1 kinase activity, mitophagy-independent functions of PINK1 and their possible roles in PD pathogenesis have been proposed. Using an unbiased quantitative mass spectrometry approach to analyze the phosphoproteome in primary neurons from wild type and Pink1 knockout mice after mitochondrial depolarization, we uncovered PINK1-regulated phosphorylation sites, which involve coordinated activation of multiple signaling pathways that control cellular response to stress. We further identified the pro-apoptotic protein BAD as a potential mitochondrial substrate of PINK1 both in vitro and in vivo, and found that cells more susceptible to a12poptosis induced by mitochondrial damage can be rescued by phosphorylation mimic BAD. Our results thus suggest that PINK1 kinase activity is important for pro-apoptotic protein function in regulation of cell death.

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References

  1. 1.

    Valente EM, Abou-Sleiman PM, Caputo V, Muqit MMK, Harvey K, Gispert S, et al. Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 2004;304:1158.

  2. 2.

    Abeliovich A. Parkinson’s disease: Pro-survival effects of PINK1. Nature 2007;448:759–60.

  3. 3.

    Pridgeon JW, Olzmann JA, Chin LS, Li L. PINK1 Protects against oxidative stress by phosphorylating mitochondrial chaperone TRAP1. PLoS Biol 2007;5:e172.

  4. 4.

    Lazarou M, Sliter DA, Kane LA, Sarraf SA, Wang C, Burman JL, et al. The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 2015;524:309–14.

  5. 5.

    Pickrell AM, Youle RJ. The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson’s disease. Neuron 2015;85:257–73.

  6. 6.

    Arena G, Valente EM. PINK1 in the limelight: multiple functions of an eclectic protein in human health and disease. J Pathol 2017;241:251–63.

  7. 7.

    Voigt A, Berlemann LA, Winklhofer KF. The mitochondrial kinase PINK1: functions beyond mitophagy. J Neurochem 2016;139:232–9. Suppl 1

  8. 8.

    Van Laar VS, Arnold B, Cassady SJ, Chu CT, Burton EA, Berman SB. Bioenergetics of neurons inhibit the translocation response of Parkin following rapid mitochondrial depolarization. Hum Mol Genet 2011;20:927–40.

  9. 9.

    Cai Q, Zakaria HM, Simone A, Sheng Z-H. Spatial parkin translocation and degradation of damaged mitochondria via mitophagy in live cortical neurons. Current Biol 2012;22:545–52.

  10. 10.

    Rakovic A, Shurkewitsch K, Seibler P, Grünewald A, Zanon A, Hagenah J, et al. Phosphatase and Tensin Homolog (PTEN)-induced Putative Kinase 1 (PINK1)-dependent Ubiquitination of Endogenous Parkin Attenuates Mitophagy: Study in Human Primary Fibroblasts and Induced Pluripotent Stem Cell-Derived Neurons. J Biol Chem 2013;288:2223–37.

  11. 11.

    Morais VA, Haddad D, Craessaerts K, De Bock PJ, Swerts J, Vilain S, et al. PINK1 loss-of-function mutations affect mitochondrial complex I activity via NdufA10 ubiquinone uncoupling. Science 2014;344:203–7.

  12. 12.

    Qin X, Zheng C, Yates JR 3rd, Liao L. Quantitative phosphoproteomic profiling of PINK1-deficient cells identifies phosphorylation changes in nuclear proteins. Mol Biosyst 2014;10:1719–29.

  13. 13.

    Moon Y, Lee KH, Park JH, Geum D, Kim K. Mitochondrial membrane depolarization and the selective death of dopaminergic neurons by rotenone: protective effect of coenzyme Q10. J Neurochem 2005;93:1199–208.

  14. 14.

    Narendra DP, Jin SM, Tanaka A, Suen DF, Gautier CA, Shen J, et al. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol 2010;8:e1000298.

  15. 15.

    Dayon L, Hainard A, Licker V, Turck N, Kuhn K, Hochstrasser DF, Burkhard PR, Sanchez J-C. Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags. Anal Chem 2008;80:2921–31.

  16. 16.

    Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protocols 2008;4:44–57.

  17. 17.

    Futschik LKaM. Mfuzz: A software package for soft clustering of microarray data. Bioinformation 2007;2:3.

  18. 18.

    Mi H, Muruganujan A, Thomas PD. PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res 2013;41:D377–D386. (Database issue)

  19. 19.

    Schwartz D, Gygi SP. An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat Biotechnol 2005;23:1391–8.

  20. 20.

    Horn H, Schoof EM, Kim J, Robin X, Miller ML, Diella F, et al. KinomeXplorer: an integrated platform for kinome biology studies. Nat Methods 2014;11:603–4.

  21. 21.

    Deng H, Wu Y, Jankovic J. The EIF4G1 gene and Parkinson’s disease. Acta Neurol Scand 2015;132:73–78.

  22. 22.

    Dhungel N, Eleuteri S, Li L-b, Kramer NJ, Chartron J, Spencer B, et al. Parkinson’s disease genes VPS35 and EIF4G1 interact genetically and converge on α–synuclein. Neuron 2015;85:76–87.

  23. 23.

    Gao F, Chen D, Si J, Hu Q, Qin Z, Fang M, et al. The mitochondrial protein BNIP3L is the substrate of PARK2 and mediates mitophagy in PINK1/PARK2 pathway. Hum Mol Genet 2015;24:2528–38.

  24. 24.

    Zhang T, Xue L, Li L, Tang C, Wan Z, Wang R, et al. BNIP3 Protein Suppresses PINK1 Kinase Proteolytic Cleavage to Promote Mitophagy. J Biol Chem 2016;291:21616–29.

  25. 25.

    Gozuacik D, Bialik S, Raveh T, Mitou G, Shohat G, Sabanay H, et al. DAP-kinase is a mediator of endoplasmic reticulum stress-induced caspase activation and autophagic cell death. Cell Death Differ 2008;15:1875–86.

  26. 26.

    Vaux DL, Korsmeyer SJ. Cell Death in Development. Cell 1999;96:245–54.

  27. 27.

    Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 Not BCL-XL. Cell 1996;87:619–28.

  28. 28.

    Woodroof HI, Pogson JH, Begley M, Cantley LC, Deak M, Campbell DG, et al. Discovery of catalytically active orthologues of the Parkinson’s disease kinase PINK1: analysis of substrate specificity and impact of mutations. Open Biol 2011;1:110012.

  29. 29.

    Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 Not BCL-XL. Cell 1996;87:619–28.

  30. 30.

    Hirai I, Wang HG. Survival-factor-induced phosphorylation of Bad results in its dissociation from Bcl-x(L) but not Bcl-2. Biochem J 2001;359(Pt 2):345–52.

  31. 31.

    Desai S, Pillai P, Win-Piazza H, Acevedo-Duncan M. PKC-iota promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway. Biochim Biophys Acta 2011;1813:1190–7.

  32. 32.

    Tan Y, Demeter MR, Ruan H, Comb MJ. BAD Ser-155 phosphorylation regulates BAD/Bcl-XL interaction and cell survival. J Biol Chem 2000;275:25865–9.

  33. 33.

    Datta SR, Katsov A, Hu L, Petros A, Fesik SW, Yaffe MB, et al. 14-3-3 Proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol Cell 2000;6:41–51.

  34. 34.

    Datta SR, Brunet B, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev 1999;13:2905–27.

  35. 35.

    Darzynkiewicz Z, Galkowski D, Zhao H. Analysis of apoptosis by cytometry using TUNEL assay. Methods 2008;44:250–4.

  36. 36.

    Triplett JC, Zhang Z, Sultana R, Cai J, Klein JB, Büeler H, Butterfield DA. Quantitative expression proteomics and phosphoproteomics profile of brain from PINK1 knockout mice: insights into mechanisms of familial Parkinson’s disease. J Neurochem 2015;133:750–65.

  37. 37.

    Kondapalli C, Kazlauskaite A, Zhang N, Woodroof HI, Campbell DG, Gourlay R, et al. PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65. Open Biol 2012;2:120080.

  38. 38.

    Shlevkov E, Kramer T, Schapansky J, LaVoie MJ, Schwarz TL. Miro phosphorylation sites regulate Parkin recruitment and mitochondrial motility. Proc Natl Acad Sci U S A 2016;113:E6097–E6106.

  39. 39.

    Kostic M, Ludtmann MHR, Bading H, Hershfinkel M, Steer E, Chu CT, et al. PKA Phosphorylation of NCLX reverses mitochondrial calcium overload and depolarization, promoting survival of PINK1-Deficient dopaminergic neurons. Cell Rep 2015;13:376–86.

  40. 40.

    Bhujabal Z, Birgisdottir ÅB, Sjøttem E, Brenne HB, Øvervatn A, Habisov S, et al. FKBP8 recruits LC3A to mediate Parkin-independent mitophagy. EMBO Rep 2017;18:947–61.

  41. 41.

    Chu CT, Ji J, Dagda RK, Jiang JF, Tyurina YY, Kapralov AA, et al. Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells. Nat Cell Biol 2013;15:1197–205.

  42. 42.

    Strappazzon F, Nazio F, Corrado M, Cianfanelli V, Romagnoli A, Fimia GM, et al. AMBRA1 is able to induce mitophagy via LC3 binding, regardless of PARKIN and p62/SQSTM1. Cell Death Differ 2015;22:419–32.

  43. 43.

    Plun-Favreau H, Klupsch K, Moisoi N, Gandhi S, Kjaer S, Frith D, et al. The mitochondrial protease HtrA2 is regulated by Parkinson’s disease-associated kinase PINK1. Nat Cell Biol 2007;9:1243–52.

  44. 44.

    Chao DT, Korsmeyer SJ. BCL-2 FAMILY: Regulators of cell death. Annu Rev Immunol 1998;16:395–419.

  45. 45.

    Datta SR, Ranger AM, Lin MZ, Sturgill JF, Ma Y-C, Cowan CW, et al. Survival Factor-Mediated BAD phosphorylation raises the mitochondrial threshold for apoptosis. Dev Cell 2002;3:631–43.

  46. 46.

    Wang IHaH-G. Survival-factor-induced phosphorylation of Bad results in its dissociation from Bcl-xL but not Bcl-2. Biochem J 2001;359:345–52.

  47. 47.

    Wang Y, Nartiss Y, Steipe B, McQuibban GA, Kim PK. ROS-induced mitochondrial depolarization initiates PARK2/PARKIN-dependent mitochondrial degradation by autophagy. Autophagy 2012;8:1462–76.

  48. 48.

    Padman BS, Bach M, Lucarelli G, Prescott M, Ramm G. The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells. Autophagy 2013;9:1862–75.

  49. 49.

    Tang F-L, Liu W, Hu J-X, Erion Joanna R, Ye J, Mei L, et al. VPS35 Deficiency or Mutation causes dopaminergic neuronal loss by impairing mitochondrial fusion and function. Cell Rep 2015;12:1631–43.

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Acknowledgements

The authors would like to acknowledge financial support from the Shanghai Pujiang Talent Project (14PJ1402900), and ECNU National “985” Project grant.

Author's contributions

LL, ZZ, and LX conceived the project; HW, QZ performed the experiments. BT, HW, and LL analyzed data; LL, BT, and HW wrote the paper.

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Author notes

  1. Edited by N. Chandel

Affiliations

  1. Key Laboratory of Brain Functional Genomics of Ministry of Education, Shanghai Key Laboratory of Brain Functional Genomics, and Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, China

    • Huida Wan
    • , Bin Tang
    • , Qiufang Zeng
    •  & Lujian Liao
  2. Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610041, China

    • Xun Liao
  3. Institute of Precision Medicine, State key laboratory of Medical Genetics, the Xiangya Hospital and the Xiangya Medical School, Central South University, Changsha, Hunan, 410078, China

    • Zhuohua Zhang

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The authors declare that they have no competing interests.

Corresponding authors

Correspondence to Bin Tang or Lujian Liao.

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DOI

https://doi.org/10.1038/s41418-017-0027-x