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• W2580495962 abstract "•PARIS is a PINK1 substrate•PINK1 phosphorylation of PARIS primes it for parkin ubiquitination and degradation•PINK1 controls PARIS-regulated PGC-1α levels and dopamine neuron survival•Conditional knockdown of PINK1 leads to PARIS-dependent loss of dopamine neurons Mutations in PTEN-induced putative kinase 1 (PINK1) and parkin cause autosomal-recessive Parkinson’s disease through a common pathway involving mitochondrial quality control. Parkin inactivation leads to accumulation of the parkin interacting substrate (PARIS, ZNF746) that plays an important role in dopamine cell loss through repression of proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α) promoter activity. Here, we show that PARIS links PINK1 and parkin in a common pathway that regulates dopaminergic neuron survival. PINK1 interacts with and phosphorylates serines 322 and 613 of PARIS to control its ubiquitination and clearance by parkin. PINK1 phosphorylation of PARIS alleviates PARIS toxicity, as well as repression of PGC-1α promoter activity. Conditional knockdown of PINK1 in adult mouse brains leads to a progressive loss of dopaminergic neurons in the substantia nigra that is dependent on PARIS. Altogether, these results uncover a function of PINK1 to direct parkin-PARIS-regulated PGC-1α expression and dopaminergic neuronal survival. Mutations in PTEN-induced putative kinase 1 (PINK1) and parkin cause autosomal-recessive Parkinson’s disease through a common pathway involving mitochondrial quality control. Parkin inactivation leads to accumulation of the parkin interacting substrate (PARIS, ZNF746) that plays an important role in dopamine cell loss through repression of proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α) promoter activity. Here, we show that PARIS links PINK1 and parkin in a common pathway that regulates dopaminergic neuron survival. PINK1 interacts with and phosphorylates serines 322 and 613 of PARIS to control its ubiquitination and clearance by parkin. PINK1 phosphorylation of PARIS alleviates PARIS toxicity, as well as repression of PGC-1α promoter activity. Conditional knockdown of PINK1 in adult mouse brains leads to a progressive loss of dopaminergic neurons in the substantia nigra that is dependent on PARIS. Altogether, these results uncover a function of PINK1 to direct parkin-PARIS-regulated PGC-1α expression and dopaminergic neuronal survival. Mutations in the E3 ligase PARKIN (Kitada et al., 1998Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism.Nature. 1998; 392: 605-608Crossref PubMed Scopus (4166) Google Scholar) or the serine-threonine kinase PINK1 (phosphatase and tensin [PTEN] homolog-induced putative kinase 1) (Valente et al., 2004Valente E.M. Abou-Sleiman P.M. Caputo V. Muqit M.M. Harvey K. Gispert S. Ali Z. Del Turco D. Bentivoglio A.R. Healy D.G. et al.Hereditary early-onset Parkinson’s disease caused by mutations in PINK1.Science. 2004; 304: 1158-1160Crossref PubMed Scopus (2682) Google Scholar) cause autosomal recessive Parkinson’s disease (PD) (Corti et al., 2011Corti O. Lesage S. Brice A. What genetics tells us about the causes and mechanisms of Parkinson’s disease.Physiol. Rev. 2011; 91: 1161-1218Crossref PubMed Scopus (431) Google Scholar, Martin et al., 2011Martin I. Dawson V.L. Dawson T.M. Recent advances in the genetics of Parkinson’s disease.Annu. Rev. Genomics Hum. Genet. 2011; 12: 301-325Crossref PubMed Scopus (319) Google Scholar). PINK1 and parkin interact in a poorly understood genetic pathway important for dopamine (DA) neuronal survival (Clark et al., 2006Clark I.E. Dodson M.W. Jiang C. Cao J.H. Huh J.R. Seol J.H. Yoo S.J. Hay B.A. Guo M. Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin.Nature. 2006; 441: 1162-1166Crossref PubMed Scopus (1389) Google Scholar, Park et al., 2006Park J. Lee S.B. Lee S. Kim Y. Song S. Kim S. Bae E. Kim J. Shong M. Kim J.M. Chung J. Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin.Nature. 2006; 441: 1157-1161Crossref PubMed Scopus (1332) Google Scholar, Yang et al., 2006Yang Y. Gehrke S. Imai Y. Huang Z. Ouyang Y. Wang J.W. Yang L. Beal M.F. Vogel H. Lu B. Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin.Proc. Natl. Acad. Sci. USA. 2006; 103: 10793-10798Crossref PubMed Scopus (667) Google Scholar). Several cosubstrates for PINK1 and parkin have been identified, tying these proteins to multiple aspects of mitochondrial quality control (Pickrell and Youle, 2015Pickrell A.M. Youle R.J. The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson’s disease.Neuron. 2015; 85: 257-273Abstract Full Text Full Text PDF PubMed Scopus (1307) Google Scholar, Scarffe et al., 2014Scarffe L.A. Stevens D.A. Dawson V.L. Dawson T.M. Parkin and PINK1: much more than mitophagy.Trends Neurosci. 2014; 37: 315-324Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, Winklhofer, 2014Winklhofer K.F. Parkin and mitochondrial quality control: toward assembling the puzzle.Trends Cell Biol. 2014; 24: 332-341Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). PARIS (ZNF746) is a pathologic parkin substrate, which is increased in sporadic and familial PD brains and is responsible for DA neuronal loss in mouse models of parkin inactivation (Shin et al., 2011Shin J.H. Ko H.S. Kang H. Lee Y. Lee Y.I. Pletinkova O. Troconso J.C. Dawson V.L. Dawson T.M. PARIS (ZNF746) repression of PGC-1α contributes to neurodegeneration in Parkinson’s disease.Cell. 2011; 144: 689-702Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, Siddiqui et al., 2015Siddiqui A. Bhaumik D. Chinta S.J. Rane A. Rajagopalan S. Lieu C.A. Lithgow G.J. Andersen J.K. Mitochondrial quality control via the PGC1α-TFEB signaling pathway is compromised by Parkin Q311X mutation but independently restored by rapamycin.J. Neurosci. 2015; 35: 12833-12844Crossref PubMed Scopus (96) Google Scholar, Siddiqui et al., 2016Siddiqui A. Rane A. Rajagopalan S. Chinta S.J. Andersen J.K. Detrimental effects of oxidative losses in parkin activity in a model of sporadic Parkinson’s disease are attenuated by restoration of PGC1alpha.Neurobiol. Dis. 2016; 93: 115-120Crossref PubMed Scopus (24) Google Scholar, Stevens et al., 2015Stevens D.A. Lee Y. Kang H.C. Lee B.D. Lee Y.I. Bower A. Jiang H. Kang S.U. Andrabi S.A. Dawson V.L. et al.Parkin loss leads to PARIS-dependent declines in mitochondrial mass and respiration.Proc. Natl. Acad. Sci. USA. 2015; 112: 11696-11701Crossref PubMed Scopus (170) Google Scholar). PARIS accumulation represses the transcriptional coactivator, peroxisome proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α), which is critical for DA neuron survival (Ciron et al., 2015Ciron C. Zheng L. Bobela W. Knott G.W. Leone T.C. Kelly D.P. Schneider B.L. PGC-1α activity in nigral dopamine neurons determines vulnerability to α-synuclein.Acta Neuropathol. Commun. 2015; 3: 16Crossref PubMed Scopus (61) Google Scholar, Jiang et al., 2016Jiang H. Kang S.U. Zhang S. Karuppagounder S. Xu J. Lee Y.K. Kang B.G. Lee Y. Zhang J. Pletnikova O. et al.Adult conditional knockout of PGC-1α leads to loss of dopamine neurons.eNeuro. 2016; 3: 1-8Crossref Scopus (67) Google Scholar, Mudò et al., 2012Mudò G. Mäkelä J. Di Liberto V. Tselykh T.V. Olivieri M. Piepponen P. Eriksson O. Mälkiä A. Bonomo A. Kairisalo M. et al.Transgenic expression and activation of PGC-1α protect dopaminergic neurons in the MPTP mouse model of Parkinson’s disease.Cell. Mol. Life Sci. 2012; 69: 1153-1165Crossref PubMed Scopus (237) Google Scholar, Zheng et al., 2010Zheng B. Liao Z. Locascio J.J. Lesniak K.A. Roderick S.S. Watt M.L. Eklund A.C. Zhang-James Y. Kim P.D. Hauser M.A. et al.Global PD Gene Expression (GPEX) ConsortiumPGC-1α, a potential therapeutic target for early intervention in Parkinson’s disease.Sci. Transl. Med. 2010; 2: 52ra73Crossref PubMed Scopus (600) Google Scholar). Because parkin and PINK1 are thought to regulate DA neuronal survival in a common pathway (Corti and Brice, 2013Corti O. Brice A. Mitochondrial quality control turns out to be the principal suspect in parkin and PINK1-related autosomal recessive Parkinson’s disease.Curr. Opin. Neurobiol. 2013; 23: 100-108Crossref PubMed Scopus (59) Google Scholar, Rochet et al., 2012Rochet J.C. Hay B.A. Guo M. Molecular insights into Parkinson’s disease.Prog. Mol. Biol. Transl. Sci. 2012; 107: 125-188Crossref PubMed Scopus (62) Google Scholar, Scarffe et al., 2014Scarffe L.A. Stevens D.A. Dawson V.L. Dawson T.M. Parkin and PINK1: much more than mitophagy.Trends Neurosci. 2014; 37: 315-324Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) and regulation of PARIS by parkin is critical for DA cell survival (Shin et al., 2011Shin J.H. Ko H.S. Kang H. Lee Y. Lee Y.I. Pletinkova O. Troconso J.C. Dawson V.L. Dawson T.M. PARIS (ZNF746) repression of PGC-1α contributes to neurodegeneration in Parkinson’s disease.Cell. 2011; 144: 689-702Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, Siddiqui et al., 2015Siddiqui A. Bhaumik D. Chinta S.J. Rane A. Rajagopalan S. Lieu C.A. Lithgow G.J. Andersen J.K. Mitochondrial quality control via the PGC1α-TFEB signaling pathway is compromised by Parkin Q311X mutation but independently restored by rapamycin.J. Neurosci. 2015; 35: 12833-12844Crossref PubMed Scopus (96) Google Scholar, Siddiqui et al., 2016Siddiqui A. Rane A. Rajagopalan S. Chinta S.J. Andersen J.K. Detrimental effects of oxidative losses in parkin activity in a model of sporadic Parkinson’s disease are attenuated by restoration of PGC1alpha.Neurobiol. Dis. 2016; 93: 115-120Crossref PubMed Scopus (24) Google Scholar, Stevens et al., 2015Stevens D.A. Lee Y. Kang H.C. Lee B.D. Lee Y.I. Bower A. Jiang H. Kang S.U. Andrabi S.A. Dawson V.L. et al.Parkin loss leads to PARIS-dependent declines in mitochondrial mass and respiration.Proc. Natl. Acad. Sci. USA. 2015; 112: 11696-11701Crossref PubMed Scopus (170) Google Scholar, Winklhofer, 2014Winklhofer K.F. Parkin and mitochondrial quality control: toward assembling the puzzle.Trends Cell Biol. 2014; 24: 332-341Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), we investigated whether PINK1 plays any role in the regulation of PARIS. Here we show that PINK1 is a priming kinase for parkin-mediated PARIS ubiquitination and clearance. PINK1 depletion in adult mouse brains leads to PARIS accumulation, PGC-1α repression, and progressive DA neuron loss that is PARIS dependent. Identification of PARIS as a PINK1 substrate provides a molecular mechanism linking PINK1 and parkin to DA neuronal loss in PD. Interaction of PINK1 and PARIS was first suggested by tandem affinity purification of PARIS from SH-SY5Y cells, which pulls down both endogenous parkin and PINK1 (Figure 1A). An in vitro pull-down assay using an anti-parkin antibody was conducted that showed coimmunoprecipitation of both PARIS and PINK1 (Shiba et al., 2009Shiba K. Arai T. Sato S. Kubo S. Ohba Y. Mizuno Y. Hattori N. Parkin stabilizes PINK1 through direct interaction.Biochem. Biophys. Res. Commun. 2009; 383: 331-335Crossref PubMed Scopus (59) Google Scholar, Shin et al., 2011Shin J.H. Ko H.S. Kang H. Lee Y. Lee Y.I. Pletinkova O. Troconso J.C. Dawson V.L. Dawson T.M. PARIS (ZNF746) repression of PGC-1α contributes to neurodegeneration in Parkinson’s disease.Cell. 2011; 144: 689-702Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). The addition of recombinant PINK1 enhances the association of these three proteins (Figure 1B). In the absence of parkin, an N-terminal V5-tagged recombinant PARIS (rV5-PARIS) pulls down glutathione S-transferase (GST)-tagged recombinant PINK1 (rGST-PINK1) (Figure 1C) suggesting that PINK1 directly interacts with PARIS. To further characterize this interaction, SH-SY5Y cells were transfected with N-terminal FLAG-tagged PARIS (FLAG-PARIS) or deletion mutants and N-terminal GFP-tagged PINK1 (GFP-PINK1). GFP-PINK1 coimmunoprecipitates PARIS, as well as the Krüppel-associated box (KRAB) domain containing N-terminal fragment (Figure S1A). PARIS coimmunoprecipitates both ∼65 and ∼55 kDa forms of PINK1 (Figure S1B), while PD-linked mutant L347P-PINK1 and kinase-inactive K219M-PINK1 exhibit reduced interaction with PARIS (Figure S1C). Coimmunoprecipitation of PARIS with deletion mutants of N-terminal GFP-tagged PINK1 (N, amino acids [aa] 1–270; C, aa 268–581) reveals that the N-terminal half of PINK1 is sufficient for the PARIS interaction (Figure S1D). Attempts to generate smaller fragments of GFP-PINK1 were hampered by protein instability. The interaction of PARIS with PINK1 was also evaluated in mouse brain. Immunoprecipitation of PARIS pulls down endogenous PINK1 (Figure 1D). Parkin is not required for the interaction of PARIS and PINK1, because immunoprecipitation of PARIS from both wild-type (WT) and parkin−/− brains pulls down PINK1 (Figure 1D). These experiments suggest a direct interaction of PINK1 with PARIS. Maltose-binding protein-fused Tribolium castaneum PINK1 (MaBP-TcPINK1) possesses both strong kinase activity and high homology with human PINK1 (Woodroof et al., 2011Woodroof H.I. Pogson J.H. Begley M. Cantley L.C. Deak M. Campbell D.G. van Aalten D.M. Whitworth A.J. Alessi D.R. Muqit M.M. Discovery of catalytically active orthologues of the Parkinson’s disease kinase PINK1: analysis of substrate specificity and impact of mutations.Open Biol. 2011; 1: 110012Crossref PubMed Scopus (73) Google Scholar). Wild-type TcPINK1 markedly increases immunopurified FLAG-PARIS phosphorylation as detected by 32P, whereas kinase-inactive TcPINK1 (D359A) fails to phosphorylate PARIS (Figure 2A). Moreover, despite lower kinase activity, recombinant human PINK1 purified from E. coli phosphorylates immunopurified FLAG-PARIS as detected by a phosphoserine antibody, while a kinase-inactive PINK1 (K219M) does not (data not shown). Consistent with these in vitro assays, PINK1 overexpression in SH-SY5Y cells phosphorylates FLAG-PARIS as detected by a phosphoserine immunoblot of FLAG immunoprecipitates (Figures 2B and 2C). The L347P, A217D, and G309D familial PD mutants of PINK1 and the K219M kinase-inactive PINK1 fail to phosphorylate FLAG-PARIS (Figures 2B and 2C). Phosphorylation of PARIS by PINK1 was identified by mass spectrometry at serine (S) 106, S322, S359, and S613 and at threonine (T) 603. Of these, S322 and S613 were conserved among mammals (human, monkey, chimpanzee, rat, and mouse) (Figures S2A–S2C). Site-directed mutagenesis of S322 or S613 to alanine (S322A or S613A) reduces the phosphorylation of PARIS by PINK1 (Figures 2D and 2E). To characterize the role of S613 phosphorylation of PARIS, we raised and purified rabbit antibodies against a PARIS peptide containing phosphoserine 613 (pS613). The purified rabbit antibody to pS613-PARIS is highly specific for S613 phosphorylation (Figures S2D and S2E). It appears that S322A substitution (S322A-PARIS) affects PARIS S613 phosphorylation, suggesting a synergistic interaction in the phosphorylation of these two serine residues (Figures S2D and S2E). Phosphorylation of PARIS S613 is markedly enhanced by PINK1 coexpression, whereas kinase-deficient PINK1 mutants fail to phosphorylate PARIS (Figures 2F and 2G). To determine whether PINK1 regulates the ubiquitination of PARIS by parkin, SH-SY5Y cells were transfected with PINK1, hemagglutinin (HA)-ubiquitin, and FLAG-PARIS. PINK1 enhances the endogenous ubiquitination of PARIS, while L347P and K219M PINK1 mutants have no effect on PARIS ubiquitination (Figure 3A), indicating the importance of PINK1 kinase activity. The HA-ubiquitin signal is not measurable in the absence of FLAG-PARIS expression, indicating that the ubiquitination is specific for FLAG-PARIS (Figure S3A). To determine whether endogenous PINK1 is required for PARIS ubiquitination, PINK1 was knocked down by short hairpin RNA (shRNA) (Figures 3B, S3B, and S3C), resulting in reduced ubiquitination of PARIS (Figure 3B). Ubiquitination is restored by expression of shRNA-resistant PINK1 (PINK1R) (Figures 3B, S3B, and S3C), thus controlling for potential off-target effects. To evaluate whether endogenous parkin contributes to the enhanced ubiquitination of PARIS following PINK1 phosphorylation, parkin was knocked down by shRNA, attenuating this enhancement (Figure 3C). To investigate the impact of PARIS phosphorylation on its proteasomal degradation, the tetracycline-sensitive PARIS expression system (TetP-PARIS-FLAG) was used to assess steady-state levels of PARIS-FLAG in PINK1 overexpression. Twenty-four hours after induction of PARIS-FLAG with tetracycline transactivator (tTA), doxycycline was added to block further PARIS-FLAG expression. PINK1 overexpression accelerates the degradation of PARIS, while kinase-deficient L347P and K219M PINK1 do not (Figure 3D). To evaluate the effect of endogenous PINK1 on endogenous PARIS levels, we knocked down PINK1 in SH-SY5Y cells, which led to a 3-fold increase in PARIS. The shRNA-resistant PINK1 (PINK1R) restores PARIS to baseline (Figure 3E). PARIS mRNA does not change in response to knockdown of PINK1 (Figure 3F). These results suggest that endogenous PINK1 even under basal conditions possesses sufficient catalytic activity to regulate its potential substrates. Supporting this notion, basal phosphorylation of both PARIS and PINK1 were detected at appreciable levels by phosphorylated protein enrichment (Figures S3E and S3F). The phosphorylation of PARIS and PINK1, measured by column binding, was enhanced by treatment with the mitochondrial depolarizing agent carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and abolished by phosphatase treatment of cell lysates, indicating that binding of proteins to the column requires phosphorylation (Figures S3E and S3F). Ubiquitin is the most extensively characterized PINK1 substrate (Fiesel et al., 2015Fiesel F.C. Ando M. Hudec R. Hill A.R. Castanedes-Casey M. Caulfield T.R. Moussaud-Lamodière E.L. Stankowski J.N. Bauer P.O. Lorenzo-Betancor O. et al.(Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation.EMBO Rep. 2015; 16: 1114-1130Crossref PubMed Scopus (107) Google Scholar, Kane et al., 2014Kane L.A. Lazarou M. Fogel A.I. Li Y. Yamano K. Sarraf S.A. Banerjee S. Youle R.J. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity.J. Cell Biol. 2014; 205: 143-153Crossref PubMed Scopus (818) Google Scholar, Kazlauskaite et al., 2014Kazlauskaite A. Kondapalli C. Gourlay R. Campbell D.G. Ritorto M.S. Hofmann K. Alessi D.R. Knebel A. Trost M. Muqit M.M. Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65.Biochem. J. 2014; 460: 127-139Crossref PubMed Scopus (556) Google Scholar, Koyano et al., 2014Koyano F. Okatsu K. Kosako H. Tamura Y. Go E. Kimura M. Kimura Y. Tsuchiya H. Yoshihara H. Hirokawa T. et al.Ubiquitin is phosphorylated by PINK1 to activate parkin.Nature. 2014; 510: 162-166Crossref PubMed Scopus (960) Google Scholar, Ordureau et al., 2014Ordureau A. Sarraf S.A. Duda D.M. Heo J.M. Jedrychowski M.P. Sviderskiy V.O. Olszewski J.L. Koerber J.T. Xie T. Beausoleil S.A. et al.Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis.Mol. Cell. 2014; 56: 360-375Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, Wauer et al., 2015aWauer T. Simicek M. Schubert A. Komander D. Mechanism of phospho-ubiquitin-induced PARKIN activation.Nature. 2015; 524: 370-374Crossref PubMed Scopus (292) Google Scholar). We used the phosphorylation of ubiquitin as a measure of PINK1 activity. Phosphorylated ubiquitin is detected in the postnuclear, mitochondrial, and cytoplasmic compartments, and the phosphorylation is enhanced by CCCP treatment (Figure S3G). Conversely, transfection of SH-SY5Y cells with small interfering RNA (siRNA) to PINK1 leads to a reduction in ubiquitin phosphorylation (Figure S3H). This would suggest that PINK1 is catalytically active under basal conditions. PINK1 knockdown also reduces PARIS phosphorylation in the cytoplasm (Figure S3H). FLAG-PARIS is phosphorylated at S613 by endogenous PINK1, because depletion of PINK1 reduces S613 phosphorylation (Figure 3G). Introduction of PINK1R, an shRNA-resistant PINK1, restores the phosphorylation of FLAG-PARIS, thus controlling for potential off-target effects (Figure 3G). Endogenous PARIS is phosphorylated by PINK1, because PINK1 knockdown reduces the relative phosphorylation of endogenous PARIS, leading to PARIS accumulation (Figure 3H). Altogether, these results demonstrate that PINK1 has basal catalytic activity in the absence of widescale mitochondrial depolarization and that PARIS is a physiologic phosphosubstrate of PINK1. PARIS ubiquitination and degradation are regulated by parkin (Shin et al., 2011Shin J.H. Ko H.S. Kang H. Lee Y. Lee Y.I. Pletinkova O. Troconso J.C. Dawson V.L. Dawson T.M. PARIS (ZNF746) repression of PGC-1α contributes to neurodegeneration in Parkinson’s disease.Cell. 2011; 144: 689-702Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). A ubiquitination assay in SH-SY5Y cells reveals that PARIS ubiquitination is markedly enhanced by parkin overexpression (Figure S4A). The role of PARIS phosphorylation by PINK1 in PARIS turnover was assessed using pulse-chase experiments. SH-SY5Y cells were transfected with TetP-PARIS-FLAG or TetP-PARIS-FLAG mutants (S322A, S613A, or S322/613A), HA-ubiquitin, parkin, and tTA. Parkin robustly ubiquitinates PARIS, but in S322A and S613A PARIS mutants, this effect is reduced (Figure 4A); meanwhile, S322/613A PARIS (serine 322/613 to alanine-double mutants [SA-DM]) is not ubiquitinated (Figure 4A), linking PARIS S322/S613 phosphorylation by PINK1 to subsequent ubiquitination by parkin. In addition, an in vitro ubiquitination reaction using recombinant ubiquitin, E1, UbcH7 as an E2, PARIS WT, or SA-DM, together with TcPINK1 and rat full-length parkin, showed increased activation of parkin by addition of TcPINK1 (Figure S4B), consistent with previous reports (Fiesel et al., 2015Fiesel F.C. Ando M. Hudec R. Hill A.R. Castanedes-Casey M. Caulfield T.R. Moussaud-Lamodière E.L. Stankowski J.N. Bauer P.O. Lorenzo-Betancor O. et al.(Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation.EMBO Rep. 2015; 16: 1114-1130Crossref PubMed Scopus (107) Google Scholar, Kazlauskaite et al., 2015Kazlauskaite A. Martínez-Torres R.J. Wilkie S. Kumar A. Peltier J. Gonzalez A. Johnson C. Zhang J. Hope A.G. Peggie M. et al.Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.EMBO Rep. 2015; 16: 939-954Crossref PubMed Scopus (144) Google Scholar, Kumar et al., 2015Kumar A. Aguirre J.D. Condos T.E. Martinez-Torres R.J. Chaugule V.K. Toth R. Sundaramoorthy R. Mercier P. Knebel A. Spratt D.E. et al.Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis.EMBO J. 2015; 34: 2506-2521Crossref PubMed Scopus (121) Google Scholar, Sauvé et al., 2015Sauvé V. Lilov A. Seirafi M. Vranas M. Rasool S. Kozlov G. Sprules T. Wang J. Trempe J.F. Gehring K. A Ubl/ubiquitin switch in the activation of Parkin.EMBO J. 2015; 34: 2492-2505Crossref PubMed Scopus (130) Google Scholar, Wauer et al., 2015aWauer T. Simicek M. Schubert A. Komander D. Mechanism of phospho-ubiquitin-induced PARKIN activation.Nature. 2015; 524: 370-374Crossref PubMed Scopus (292) Google Scholar, Yamano et al., 2015Yamano K. Queliconi B.B. Koyano F. Saeki Y. Hirokawa T. Tanaka K. Matsuda N. Site-specific interaction mapping of phosphorylated ubiquitin to uncover Parkin activation.J. Biol. Chem. 2015; 290: 25199-25211Crossref PubMed Scopus (47) Google Scholar). In contrast, phosphorylation mutant PARIS SA-DM fails to be phosphorylated and ubiquitinated by TcPINK1/parkin even in the presence of parkin activation (Figure S4B). In vitro studies have shown that TcPINK1 can phosphorylate ubiquitin and parkin, enhancing parkin activity (Fiesel et al., 2015Fiesel F.C. Ando M. Hudec R. Hill A.R. Castanedes-Casey M. Caulfield T.R. Moussaud-Lamodière E.L. Stankowski J.N. Bauer P.O. Lorenzo-Betancor O. et al.(Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation.EMBO Rep. 2015; 16: 1114-1130Crossref PubMed Scopus (107) Google Scholar, Kane et al., 2014Kane L.A. Lazarou M. Fogel A.I. Li Y. Yamano K. Sarraf S.A. Banerjee S. Youle R.J. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity.J. Cell Biol. 2014; 205: 143-153Crossref PubMed Scopus (818) Google Scholar, Kazlauskaite et al., 2014Kazlauskaite A. Kondapalli C. Gourlay R. Campbell D.G. Ritorto M.S. Hofmann K. Alessi D.R. Knebel A. Trost M. Muqit M.M. Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65.Biochem. J. 2014; 460: 127-139Crossref PubMed Scopus (556) Google Scholar, Kazlauskaite et al., 2015Kazlauskaite A. Martínez-Torres R.J. Wilkie S. Kumar A. Peltier J. Gonzalez A. Johnson C. Zhang J. Hope A.G. Peggie M. et al.Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.EMBO Rep. 2015; 16: 939-954Crossref PubMed Scopus (144) Google Scholar, Koyano et al., 2014Koyano F. Okatsu K. Kosako H. Tamura Y. Go E. Kimura M. Kimura Y. Tsuchiya H. Yoshihara H. Hirokawa T. et al.Ubiquitin is phosphorylated by PINK1 to activate parkin.Nature. 2014; 510: 162-166Crossref PubMed Scopus (960) Google Scholar, Kumar et al., 2015Kumar A. Aguirre J.D. Condos T.E. Martinez-Torres R.J. Chaugule V.K. Toth R. Sundaramoorthy R. Mercier P. Knebel A. Spratt D.E. et al.Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis.EMBO J. 2015; 34: 2506-2521Crossref PubMed Scopus (121) Google Scholar, Ordureau et al., 2014Ordureau A. Sarraf S.A. Duda D.M. Heo J.M. Jedrychowski M.P. Sviderskiy V.O. Olszewski J.L. Koerber J.T. Xie T. Beausoleil S.A. et al.Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis.Mol. Cell. 2014; 56: 360-375Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, Sauvé et al., 2015Sauvé V. Lilov A. Seirafi M. Vranas M. Rasool S. Kozlov G. Sprules T. Wang J. Trempe J.F. Gehring K. A Ubl/ubiquitin switch in the activation of Parkin.EMBO J. 2015; 34: 2492-2505Crossref PubMed Scopus (130) Google Scholar, Wauer et al., 2015aWauer T. Simicek M. Schubert A. Komander D. Mechanism of phospho-ubiquitin-induced PARKIN activation.Nature. 2015; 524: 370-374Crossref PubMed Scopus (292) Google Scholar, Wauer et al., 2015bWauer T. Swatek K.N. Wagstaff J.L. Gladkova C. Pruneda J.N. Michel M.A. Gersch M. Johnson C.M. Freund S.M. Komander D. Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis.EMBO J. 2015; 34: 307-325Crossref PubMed Scopus (210) Google Scholar, Yamano et al., 2015Yamano K. Queliconi B.B. Koyano F. Saeki Y. Hirokawa T. Tanaka K. Matsuda N. Site-specific interaction mapping of phosphorylated ubiquitin to uncover Parkin activation.J. Biol. Chem. 2015; 290: 25199-25211Crossref PubMed Scopus (47) Google Scholar). We observe strong phosphorylation of both PARIS and parkin by TcPINK1 in vitro (Figures S4C–S4E). Phosphorylation-deficient ubiquitin (S65A-Ub) and constitutively active parkin (rat parkin fragments 219–465) were used to examine the effect of PARIS phosphorylation on ubiquitination independent of parkin or ubiquitin phosphorylation. Rat parkin enhances PARIS ubiquitination but fails to ubiquitinate the PARIS phosphorylation mutant (Figure 4B), demonstrating the necessity of PARIS S322/S613 phosphorylation for parkin-mediated ubiquitination. The impact of PARIS phosphorylation on its steady-state levels was assessed using TetP-PARIS-FLAG or TetP-PARIS-FLAG mutants (S322A or S613A). PINK1 overexpression reduces steady-state levels of PARIS-FLAG by greater than 50%, whereas S322A PARIS-FLAG is only reduced by about 30% and S613A PARIS-FLAG is not reduced (Figure 4C). We examined whether this difference might be due to a change in the binding of mutant PARIS for N-terminal V5-tagged parkin (V5-parkin). S322A or S613A mutations diminished the parkin interaction, while double mutation (S322/613A) abolished PARIS’s interaction with parkin (Figure 4D), suggesting that phosphorylation of PARIS by PINK1 promotes parkin interaction. To mimic phosphorylation of PARIS, plasmids with serine-to-aspartate substitution at S322 and/or S613 (S322D, S613D, or S322/613D [SD-DM]) were generated and the stability of the proteins was assessed. Cellular levels of S322D, S613D, and SD-DM are significantly lower than WT PARIS (Figure S4F), and SD-DM PARIS was stabilized by the proteasome inhibitor, MG132, or parkin knockdown (Figure 4E). These changes in protein expression are not due to transcriptional alterations, because mCherry, expressed from the same transcript via the internal ribosomal entry site (IRES), did not show noticeable differences among the groups (Figures 4E and S4G). The physiologic role of endogenous parkin and PINK1 in PARIS clearance was assessed using a tetracycline-responsive PARIS expression system (TetP-PARIS-FLAG) to monitor steady-state levels of PARIS-FLAG after PINK1 or parkin knockdown. With control shRNA, PARIS-FLAG decrease by ∼60% by 24 hr after doxycycline treatment (Figure 4F). PINK1 knockdown delays PARIS degradation by ∼50%, while parkin knockdown almost prevents PARIS clearance (Figure 4F), demonstrating that PARIS clearance is regulated by both endogenous PINK1 and parkin. Furthermore, parkin knockdown prevents the reduction in PARIS se" @default.
• W2580495962 created "2017-02-03" @default.
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• W2580495962 date "2017-01-01" @default.
• W2580495962 modified "2023-10-02" @default.
• W2580495962 title "PINK1 Primes Parkin-Mediated Ubiquitination of PARIS in Dopaminergic Neuronal Survival" @default.
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