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302 nature genetics • volume 25 • july 2000

Familial Parkinson disease gene product, parkin, is aubiquitin-protein ligase

Hideki Shimura1,2, Nobutaka Hattori1, Shin-ichiro Kubo1, Yoshikuni Mizuno1, Shuichi Asakawa3,Shinsei Minoshima3, Nobuyoshi Shimizu3, Kazuhiro Iwai4, Tomoki Chiba2, Keiji Tanaka2 & Toshiaki Suzuki2

1Department of Neurology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan. 2Tokyo Metropolitan Institute of Medical Science and CREST,Japan Science and Technology Corporation (JST), Bunkyo-ku, Tokyo, Japan. 3Department of Molecular Biology, Keio University School of Medicine,Shinjuku-ku, Tokyo, Japan. 4Department of Molecular and System Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan. Correspondenceshould be addressed to K.T. (e-mail: tanakak@rinshoken.or.jp).

Autosomal recessive juvenile parkinsonism (AR–JP), one of themost common familial forms of Parkinson disease, is character-ized by selective dopaminergic neural cell death and the absenceof the Lewy body, a cytoplasmic inclusion body consisting ofaggregates of abnormally accumulated proteins1. We previouslycloned PARK2, mutations of which cause AR–JP (ref. 2), but thefunction of the gene product, parkin, remains unknown. Wereport here that parkin is involved in protein degradation as aubiquitin-protein ligase collaborating with the ubiquitin-conju-gating enzyme UbcH7, and that mutant parkins from AR–JPpatients show loss of the ubiquitin-protein ligase activity. Ourfindings indicate that accumulation of proteins that have yet tobe identified causes a selective neural cell death without forma-tion of Lewy bodies. Our findings should enhance the explo-ration of the molecular mechanisms of neurodegeneration inParkinson disease as well as in other neurodegenerative diseasesthat are characterized by involvement of abnormal protein ubiq-uitination, including Alzheimer disease, other tauopathies, CAGtriplet repeat disorders and amyotrophic lateral sclerosis3–10.Ubiquitin is a small, covalent modifier that forms a poly-ubiquitinchain on protein, which becomes a degradation signal attacked bythe 26S proteasome11–13. The poly-ubiquitin chain is synthesized bya cascade reaction involving three enzymes, E1 (ubiquitin-activat-

ing enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiqui-tin-ligating enzyme), acting as a substrate-recognition mole-cule11,12. A link between Parkinson disease and the ubiquitinsystem has been suggested in pathological studies and analyses oftwo gene products, UCH-L1 (ubiquitin carboxyl-terminal hydro-lase) and α-synuclein, whose mutations cause autosomal dominantfamilial Parkinson disease14–16. UCH-L1 is thought to produceubiquitin by both cleaving polymeric ubiquitin and releasing ubiq-uitin from small adducts such as glutathione and cellular amines17.α-Synuclein, one of the major components of Lewy bodies18, isdegraded by the 26S proteasome19, indicating that it is modified byubiquitin, and its mutation is known to extend the half-life of theprotein. With regard to parkin in AR–JP, the presence of carboxy-terminal two RING-finger motifs20, which are suggested to interactwith E2 (refs 21–27), prompted us to investigate its function as E3.

We first tested whether parkin interacts with a specific E2enzyme. We ectopically expressed Myc-tagged parkin togetherwith FLAG-, HA- or His-tagged Ubcs in human embryonic kidney293 (HEK293) cells and examined their interaction by immuno-precipitation (IP) with anti-Myc antibody. UbcH7 and, faintly,UbcH8 were co-immunoprecipitated with parkin, but no appre-ciable binding was observed for any other E2 enzymes examined,such as Ubc2, Ubc3, Ubc4, UbcH5A-C and UbcH6 (Fig. 1).

We next investigated the domain-structure of parkin to deter-mine the region that contributes to its interaction with UbcH7 (Fig.2). Parkin is divided into three parts: the amino-terminal Ubldomain, the carboxy-terminal RING-box and the linker region,which connects the former two segments. The C-terminal RING-box region consists of three domains termed RING1, RING2 andIBR (for in-between-RING; Fig. 2a; ref. 20). The C-terminal half ofparkin containing the RING-box is sufficient for the recruitment ofUbcH7, because deletion of the Ubl domain alone, as well astogether with the linker region, had no effect on binding withUbcH7 (Fig. 2b). Conversely, RING-box mutants lacking either theRING1 or RING2 domains showed loss of the ability to bind toUbcH7. In addition, when we examined two missense mutants ofAP–JP patients, parkinT240R, with a Thr→Arg mutation in RING1domain28, did not associate with UbcH7, whereas parkinR42P, witha mutation in Ubl-domain (N.H., unpublished data), was capableof binding UbcH7. Thus, the RING-box domain, but not the linkerregion or the Ubl domain, is used to associate with UbcH7.

To clarify the relationship between parkin and the ubiquitinsystem, we tested whether parkin traps target protein for ultimateubiquitination, as reported in several other dissimilar classes of E3(refs 21–24). When Myc-parkin and FLAG-ubiquitin were co-expressed in human dopaminergic neuroblastoma SH-SY5Y cells,we detected multiple ubiquitinated bands with high molecularmasses in the anti-Myc immunoprecipitates from cells pre-treatedwith the proteasome inhibitor MG-132 (Cbz-Leu-Leu-Leu-alde-

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Fig. 1 Association of parkin with UbcH7 in human embryonic kidney 293 cells.pcDNA3.1(+)Myc-parkin were co-transfected with the amounts (µg) indicated inparentheses of various expression vectors encoding FLAG-, His- or HA-Ubcs intoHEK293 cells. Cell extracts (top) and immunoprecipitates (middle) were analysedby immunoblotting (Blot) with various antibodies as indicated to detect taggedUbcs. Bottom, immunoprecipitated Myc-parkin. Note that when the expressionand detection levels of UbcH8 were increased, its binding to parkin was evident.Asterisks indicate non-specific bands containing the light chain of IgG.

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Fig. 2 Domain analysis ofparkin interacting withUbcH7. a, Schematic rep-resentation of wild-typeparkin and variousmutants of parkin. Thesingle-letter amino acidcode is used. The non-sense and missensemutants of parkin foundin AR–JP patients areshown as parkinQ311stop

and parkinT240R/parkinR42P,respectively. b, Associationof UbcH7 with variousmutant parkins. Theanalysis was similar to thatused in Fig. 1, except thatindicated amounts of vari-ous mutated parkins (µgin parentheses) and pcDNA3.1(+)FLAG-UbcH7 (7 µg) were used. Immunoblotting with anti-FLAG antibody was performed for crude extracts (top). Various Myc-parkinswere immunoprecipitated by anti-Myc antibody. Immunoblotting was performed with anti-Myc antibody to detect Myc-parkins (middle) or anti-FLAG antibody todetect co-immunoprecipitated UbcH7 (bottom). Asterisks indicate the heavy and light chains of IgG.

hyde; ref. 29), but no measurable ubiquitinated bands without thepretreatment of MG132 (Fig. 3). The poly-ubiquitin-tagged pro-teins associated with parkin increased time-dependently aftertreatment with MG-132, and some ubiquitinated proteins weresmaller than parkin. These results indicate that parkin is able tobind cellular protein that is ubiquitinated before degradation bythe 26S proteasome. Overexpression of UbcH7C86A defective forthioester linkage with ubiquitin prevented the accumulation ofubiquitinated proteins associated with parkin (data not shown),suggesting that ubiquitination is mediated by UbcH7. We did notobserve any appreciable ubiquitinated bands bound to parkin bythe same analysis for HEK293 cells (data not shown), indicatingthat the parkin function is cell-type specific.

We next examined the correlation between E2 binding of parkinand parkin-linked ubiquitination in SH-SY5Y neuronal cells. Dele-

tion or missense mutation in the RING-box, which resulted in fail-ure of recruitment of UbcH7, did not co-immunoprecipitate theseubiquitinated protein bands (Fig. 4), indicating that recruitment ofUbcH7 is necessary for this ubiquitination. The mutant parkinlacking the Ubl domain and naturally occurring parkinR42P failed toco-immunoprecipitate ubiquitinated proteins (Fig. 4). Thesemutated parkins, however, were able to bind UbcH7 (Fig. 2b), sug-gesting that the Ubl domain functions as a module necessary forbinding with the ubiquitinated proteins. These results indicate thatparkin is required for this ubiquitination, possibly functioning asan E3, and that the loss of its E3 function is the cause of AR–JP.

To demonstrate directly that parkin has E3 activity, we devisedan in vitro assay system for ubiquitination. First, immunoprecip-itated Myc-tagged parkin (IP-parkin) from MG132-treated SH-SY5Y cells was incubated with recombinant E1, E2 (UbcH7) and

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Fig. 3 Association of ubiquitinated cellular proteins with parkin after treat-ment with MG132 in human dopaminergic neuroblastoma SH-SY5Y cells.pcDNA3.1(+)Myc-parkin and pcDNA3.1(+)FLAG-ubiquitin (10 µg each) were co-transfected in SH-SY5Y cells. Forty-eight hours after transfection, all cells wereharvested. Note that in some experiments, treatment with MG132 was carriedout for 3, 8 or 24 h before harvesting the cells. Immunoprecipitates preparedby anti-Myc antibody were used for immunoblotting with anti-FLAG (top) andanti-Myc (bottom) antibodies. The high molecular-mass ubiquitinated proteinsare shown as (Ub)n on the right. Asterisk indicates the position of parkin. Onthe first lane of the lower panel, the faint immunoreactive band without trans-fection of pcDNA3.1(+)Myc-parkin represents the heavy chain of IgG of anti-Myc antibody treated as a control.

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Fig. 4 Domain analysis of parkin required for the binding of ubiquitinated pro-teins. Experimental conditions were similar to those of Fig. 3, except that theindicated amounts of expression vectors for various mutant Myc-parkins wereused and treatment with MG132 lasted 24 h before harvesting the cells. Forty-eight hours after transfection, the immunoprecipitates prepared by anti-Mycantibody were used for immunoblotting with anti-FLAG (top) or anti-Myc (bot-tom) antibodies. In the lower panel, asterisks indicate artefact bands. Note thatthe positions of wild-type and missense mutated parkins overlapped with theposition of heavy chain of IgG.

a b

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125I-ubiquitin in the presence of ATP. High molecular ubiquiti-nated signals were observed along with a signal at approximately30 kD (Fig. 5a), whereas no signal was noted in the absence of E1,E2 (UbcH7) or IP-parkin, confirming that parkin acts as E3. Thisubiquitinated 30-kD band, which is not UbcH7 (data notshown), is the major ubiquitinated protein yet to be identified.We next analysed the ubiquitination activity of each mutantparkin. None of the mutant IP-parkins supported this in vitroubiquitination (Fig. 5b), emphasizing the requirement for theUbl domain and RING-box region. In contrast to IP-parkin fromSH-SY5Y cells (Fig. 5c), we detected no ubiquitination activity inIP-parkin prepared in the same fashion from HEK293 cells (Fig.5c). When IP-parkin (HEK293) was mixed with extracts of SH-SY5Y cells and washed again before ubiquitination assay, wedetected ubiquitination activity (Fig. 5c). We obtained similarresults by using recombinant His-tagged parkin produced by thebaculovirus system (data not shown). These studies indicate thattarget protein for parkin or a critical factor collaborating withparkin is present in SH-SY5Y cells, but not in HEK293 cells.

Parkin consists of two functionally different domains (Fig. 6):the C-terminal RING-box, which recruits a specific E2 enzyme(UbcH7) and the N-terminal Ubl domain required for recogni-tion of target protein, designated ‘X’, for ubiquitination beforeproteasomal degradation. Whether parkin acts as a componentof a multi-subunit E3 complex remains to be determined, butour results show that parkin is essential for E3 activity. Althoughwe have identified the function of parkin, the mechanisms ofselective neural cell death in AR–JP remain unknown. To identifythese mechanisms and those of neurodegeneration, which result

in accumulation of proteins irrespective of the presence orabsence of aggregates, it is important to identify the unknowntarget ‘X’, whose accumulation may cause AR–JP.

MethodsExpression plasmids, transfection and cell culture. To prepare the pcD-NA3.1(+)Myc and pcDNA3.1(+)FLAG vector, we ligated oligo DNAsencoding N-terminal Myc and the FLAG epitope to the KpnI/BamHI site ofpcDNA3.1(+) (Invitrogen). We amplified the cDNAs of wild-type or N-ter-minal deletion mutants of parkin, Ubc2 (HHR6B), UbcH8 and ubiquitin byPCR with appropriate primers and ligated them into the BamHI site of thevectors. We prepared C-terminal deletion mutants and missense mutants ofparkin by mutating pcDNA3.1(+)Myc-parkin using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s pro-tocol. We cloned PCR-amplified, RGS-His-tagged Ubc3 into EcoRI site ofpCAGGS vector. pCGN-Ubc4 and other pcDNA3.1(+)FLAG-Ubcs weredonated by M. Nakao and H. Suzuki, respectively. We maintained HEK293cells and SH-SY5Y cells in DMEM supplemented with 10% fetal bovineserum, streptomycin (100 µg/ml) and penicillin (100 U/ml). Transfectionswere performed using the FuGENE6 transfection reagent according to themanufacturer’s instructions (Boehringer). In the case of inhibiting cellularproteasome activity, cells were pretreated with MG132 (50 µM; PeptideInstitute) for 12 h unless otherwise noted.

Immunological analysis. We performed IP as described using rabbit poly-clonal anti-Myc antibody (A-14; Santa Cruz Biotechnology; ref. 30). Cellextracts were prepared and IP with anti-Myc antibody was carried out 48 hafter transfection. We carried out immunoblotting using biotin-conjugat-ed mouse monoclonal anti-FLAG (M2) antibody (Sigma), mouse mono-clonal anti-Myc antibody (9E10; Santa Cruz Biotechnology), mouse mon-oclonal anti-HA antibody (HA.11; Berkeley Antibody Company), ormouse monoclonal anti-His antibody (RGS-His; Qiagen). To detectbiotin-conjugated anti-FLAG (M2) antibody, we used streptavidin-horse-radish peroxidase complex (Amersham Pharmacia Biotech).

In vitro ubiquitination assay. We prepared IP-parkin bound to anti-MycIgG-linked proteinA beads from extracts (1 mg protein) of MG132-treatedSH-SY5Y or HEK293 cells transfected with pcDNA3.1(+)Myc-parkin (10

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Fig. 5 Parkin exhibits ubiquitination activity in vitro. a, Detection of ubiquitina-tion activity in the immunoprecipitated (IP)-parkin from SH-SY5Y cells. SH-SY5Ycells were transfected with pcDNA3.1(+)Myc-parkin (10 µg) and treated withMG132. Forty-eight hours after transfection, IP-parkin from the extracts (1 mgprotein) was obtained as in Fig. 4, and incubated with ATP, 125I-ubiquitin andthe indicated combinations (+/–) of E1 or E2 (UbcH7) for the indicated period oftime. High molecular ubiquitinated signals were detected by autoradiographyand designated (Ub)n on the left. Asterisk indicates a major ubiquitin-ligatedband. b, Loss of in vitro ubiquitination activity in mutant IP-parkins. The ubiqui-tination assay was similar to that described in (a), except that various mutantMyc-tagged parkins were used. c, Absence of ubiquitination activity in IP-parkinfrom HEK293 cells. The ubiquitination activities of IP-parkin from SH-SY5Y cellsand of IP-parkin from HEK293 cells were examined as for (a) (left and middlelanes). In the right lane, IP-parkin from HEK293 cells was premixed with MG132-treated SH-SY5Y cell extracts (1 mg protein) and the washed IP-parkin was usedfor ubiquitination assay. In all experiments, one-third of IP-parkins used weredetected by immunoblotting with anti-Myc antibody (bottom).

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Fig. 6 Model of the parkin-directedubiquitination pathway. Ub, ubiqui-tin; E1, ubiquitin-activating enzyme;E2, ubiquitin-conjugating enzyme;Ubl, ubiquitin-like. ‘X’ is a presump-tive target protein. Note that parkinmay require other components toassociate with ‘X’.

a b c

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µg). Slurries of IP-parkin were incubated at 37 °C in reaction buffer (50 µl)containing ATP regenerating system30, recombinant His-tagged mouse E1(100 ng), His-UbcH7 (3 µg; E2) and 125I-labelled ubiquitin (2×105 cpm, 5µg). After terminating the reaction by the addition of 4×sample buffer (20µl) for SDS–PAGE, 20 µl boiled supernatant were separated by 10–20%SDS–PAGE and visualized by autoradiography.

AcknowledgementsWe thank E. Melamed for the DNA sample with a mutation in the Ubldomain.

Received 20 March; accepted 1 May 2000.

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