PINK1 positively regulates HDAC3 to suppress dopaminergic neuronal cell death

Human Molecular Genetics, 2015, Vol. 24, No. 4 doi:10.1093/hmg/ddu526 Advance Access published on October 9, 2014 1127–1141 PINK1 positively regulates HDAC3 to suppress dopaminergic neuronal cell death Hyo-Kyoung Choi1,{, Youngsok Choi2,{, HeeBum Kang1, Eun-jin Lim3, Soo-Yeon Park1, Hyun-Seob Lee3, Ji-Min Park3, Jisook Moon3, Yoon-Jung Kim4, Insup Choi5, Eun-Hye Joe5, Kyung-Chul Choi6,∗ and Ho-Geun Yoon1,∗ 1 Received September 16, 2014; Revised September 16, 2014; Accepted October 8, 2014 Deciphering the molecular basis of neuronal cell death is a central issue in the etiology of neurodegenerative diseases, such as Parkinson’s and Alzheimer’s. Dysregulation of p53 levels has been implicated in neuronal apoptosis. The role of histone deacetylase 3 (HDAC3) in suppressing p53-dependent apoptosis has been recently emphasized; however, the molecular basis of modulation of p53 function by HDAC3 remains unclear. Here we show that PTEN-induced putative kinase 1 (PINK1), which is linked to autosomal recessive early-onset familial Parkinson’s disease, phosphorylates HDAC3 at Ser-424 to enhance its HDAC activity in a neural cell-specific manner. PINK1 prevents H2O2-induced C-terminal cleavage of HDAC3 via phosphorylation of HDAC3 at Ser424, which is reversed by protein phosphatase 4c. PINK1-mediated phosphorylation of HDAC3 enhances its direct association with p53 and causes subsequent hypoacetylation of p53. Genetic deletion of PINK1 partly impaired the suppressive role of HDAC3 in regulating p53 acetylation and transcriptional activity. However, depletion of HDAC3 fully abolished the PINK1-mediated p53 inhibitory loop. Finally, ectopic expression of phosphomometic-HDAC3S424E substantially overcomes the defective action of PINK1 against oxidative stress in dopaminergic neuronal cells. Together, our results uncovered a mechanism by which PINK1 –HDAC3 network mediates p53 inhibitory loop in response to oxidative stress-induced damage. INTRODUCTION HDAC3 belongs to a multimolecular complex that contains the NCoR and SMRT protein subunits, which are required for the physiological action of many nuclear hormone receptors (1,2). In addition, HDAC3 also functions as a corepressor for many sequence-specific transcription factors, including NF-kB, SMAD7 and c-Jun (3–5). Through a physical interaction with these transcription factors, HDAC3 is recruited to specific promoters, where this enzyme induces transcriptional repression through histone deacetylation. On the other hand, HDAC3 also deacetylates non-histone substrates, such as GATA-2, NF-kB, SRY and YY1 (6). For instance, HDAC3 regulates the duration of NF-kB action through deacetylation of RelA and promotion of its interaction with inhibitory-kBa (IkBa), thus leading to nuclear export of NF-kB and termination of NF-kB signaling, ∗ To whom correspondence should be addressed at: Department of Medicine, Graduate School, University of Ulsan College of Medicine, 388-1 Poongnapdong, Songpa-gu, Seoul 138-736, South Korea. Tel: +82 230102087; Fax: +82 230105307; Email: (K.-C.C.); Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, South Korea. Tel.: +82 222281683; Fax: +82 23125041; Email: (H.-G.Y.) † These authors contributed equally. # The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, South Korea, 2Fertility Center of CHA General Hospital, CHA Research Institute and 3Applied Bioscience, College of Life Science, CHA University, Seoul 135-081, South Korea, 4ILSONG Institute of Life Science, Hallym University, Rm 607, ILSONG Bldg, 1605-4 Gwanyangdong, Dongan-gu, Anyang, Gyonggi-do 431-060, South Korea, 5Department of Biomedical Sciences, Neuroscience Graduate Program, Ajou University School of Medicine, Suwon 443-380, South Korea and 6Department of Medicine, Graduate School, University of Ulsan College of Medicine, 388-1 Poongnap-dong, Songpa-gu, Seoul 138-736, South Korea 1128 Human Molecular Genetics, 2015, Vol. 24, No. 4 we show that PINK1-mediated phosphorylation of HDAC3 at Ser-424 enhances its association with p53. Finally, we demonstrated that HDAC3 is critical for PINK1-mediated effects on the p53 inhibitory loop. Our data reveal that HDAC3 is required for the antagonistic action of PINK1 against oxidative stress-induced DNA damage. RESULTS PINK1 phosphorylates Ser-424 of HDAC3 in the cytoplasm To gain insight into the functional role of HDAC3 in the regulation of p53-mediated apoptosis, a yeast two-hybrid assay was performed to identify HDAC3-interacting proteins (Supplementary Material, Table S1). Strains encoding PINK1, but not strains harboring the empty vector pGBKT7, displayed a strong interaction with HDAC3 (Supplementary Material, Fig. S1A). Endogenous co-immunoprecipitation (IP) analysis showed that of class I HDACs, only HDAC3 selectively interacts with PINK1 but not the PINK1-interacting proteins, Parkin and DJ-1 (Supplementary Material, Fig. S1B). Importantly, HDAC3 specifically interacted with an intact form of PINK1 (63 kDa) in the cytosolic fraction but not in the mitochondrial fraction. As expected, HDAC3 was also associated with NCoR in the nuclear fraction (Fig. 1A). GST pull-down analyses demonstrated that the C3 region of PINK1 (402 –510 amino acids) robustly interacted with N-terminal of HDAC3 (HDAC3-N2) (106 – 211 amino acids) (Supplementary Material, Fig. S1C and D). Consistent with recent observations that PINK1 has non-mitochondrial-associated functions (20,22 – 24), these results strongly suggest a possible role of the PINK1 – HDAC3 network in the cytoplasm. PINK1 is a serine/threonine kinase that phosphorylates several target proteins, including mitofusin and Parkin (25,26). Thus, we next examined whether PINK1 directly phosphorylates HDAC3 protein. An in vitro kinase assay demonstrated that wild-type PINK1 (PINK1WT), but not the inactive and pathogenic PINK1G309D mutant, phosphorylated the C-terminal domain of HDAC3 (Supplementary Material, Fig. S2A). To determine the amino acid residues essential for PINK1-mediated phosphorylation of HDAC3, we used the NetPhosK 1.0 program for sequence analyses to predict seven putative phosphorylation sites in the HDAC3 C-terminal domain. In vitro kinase assays following site-directed mutagenesis showed that mutation of S424A in HDAC3 resulted in the complete loss of phosphorylation, providing evidence that Ser-424 of HDAC3 is critical for PINK1-dependent phosphorylation (Supplementary Material, Fig. S2B). PINK1-mediated phosphorylation at Ser-424 of HDAC3 was further confirmed by detection of HDAC3 phosphorylation with the synthetic phospho-HDAC3S424 antibody. Phosphorylation of Flag-HDAC3WT, but not mutant Flag-HDAC3S424A, was detec (...truncated)