Siah-1 Facilitates Ubiquitination and Degradation of Synphilin-1*

Received for publication, June 16, 2003, and in revised form, September 8, 2003Published, JBC Papers in Press, September 23, 2003, DOI 10.1074/jbc.M306347200

Yoshito Nagano‡, Hiroshi Yamashita‡§, Tetsuya Takahashi‡, Shosei Kishida¶,Takeshi Nakamura‡, Eizo Iseki�, Nobutaka Hattori**, Yoshikuni Mizuno**,Akira Kikuchi¶, and Masayasu Matsumoto‡

From the Departments of ‡Clinical Neuroscience and Therapeutics and ¶Biochemistry, Hiroshima University GraduateSchool of Biomedical Sciences, Hiroshima 734-8551, �Department of Psychiatry, Yokohama City University, Yokohama236-0004, and **Department of Neurology, Juntendo University School of Medicine, Tokyo 113-0033, Japan

Parkinson’s disease is a common neurodegenerativedisorder characterized by loss of dopaminergic neuronsand appearance of Lewy bodies, cytoplasmic inclusionsthat are highly enriched with ubiquitin. Synphilin-1,�-synuclein, and Parkin represent the major compo-nents of Lewy bodies and are involved in the pathogen-esis of Parkinson’s disease. Synphilin-1 is an�-synuclein-binding protein that is ubiquitinated byParkin. Recently, a mutation in the synphilin-1 gene hasbeen reported in patients with sporadic Parkinson’s dis-ease. Although synphilin-1 localizes close to synapticvesicles, its function remains unknown. To investigatethe proteins that interact with synphilin-1, the presentstudy performed a yeast two-hybrid screening and iden-tified a novel interacting protein, Siah-1 ubiquitin li-gase. Synphilin-1 and Siah-1 proteins were endog-enously expressed in the central nervous system andwere found to coimmunoprecipitate each other in ratbrain homogenate. Confocal microscopic analysis re-vealed colocalization of both proteins in cells. Siah-1was found to interact with the N terminus of synphilin-1through its substrate-binding domain and to specificallyubiquitinate synphilin-1 via its RING finger domain.Siah-1 facilitated synphilin-1 degradation via the ubiq-uitin-proteasome pathway more efficiently than Parkin.Siah-1 was found to not facilitate ubiquitination anddegradation of wild type or mutant �-synuclein. Synphi-lin-1 inhibited high K�-induced dopamine release fromPC12 cells. Siah-1 was found to abrogate the inhibitoryeffects of synphilin-1 on dopamine release. Such find-ings suggest that Siah-1 might play a role in regulationof synphilin-1 function.

Parkinson’s disease (PD),1 which is characterized by tremor,bradykinesia, rigidity, and postural instability, represents the

second most common neurodegenerative disorder. PD is patho-logically characterized by loss of dopaminergic neurons in thesubstantia nigra pars compacta and appearance of Lewy bodies(LBs), cytoplasmic inclusions that are highly enriched withubiquitin (1–3). �-Synuclein is a presynaptic protein of unde-termined function that was found to be the main component ofLB (4, 5). Two rare missense mutations in the �-synuclein gene(A53T and A30P) cause autosomal dominant familial PD (6, 7).�-Synuclein has been implicated in the pathogenesis of severalneurodegenerative diseases, including PD, multiple system at-rophy, and dementia with LBs (8). In addition, �-synucleinknockout mice display increased dopamine release under stim-ulated conditions (9). Moreover, �-synuclein inhibits dopaminebiosynthesis (10), suggesting that �-synuclein is a negativeregulator of dopamine neurotransmission.

Synphilin-1 represents a cytoplasmic protein that interactswith �-synuclein (11) and localizes close to synaptic vesicles(12). Synphilin-1 has been found to constitute an intrinsiccomponent of LBs in PD, indicating that it might be involved inthe pathogenesis of PD (13). Synphilin-1 contains six ankyrin(ANK) repeats (Swiss Protein Database number Q9Y6H5), acoiled-coil domain, and an ATP/GTP-binding site (11). None-theless, the physiological function of synphilin-1 remains un-clear. Coexpression of synphilin-1 and �-synuclein in cells wasfound to lead to the deposition of eosinophilic inclusions thatresembled LBs (11, 14), supporting the hypothesis that theinteraction might be related to LB formation. Recently, a mu-tation in the synphilin-1 gene leading to an amino acid substi-tution of cysteine for arginine in position 621 was reported intwo apparently sporadic PD patients (15). The number of in-clusions in cells expressing this mutant synphilin-1 was signif-icantly reduced compared with wild type synphilin-1 (15).

Proteins fated to degrade in proteasomes are subjected tomodification by ubiquitin. Ubiquitination proceeds through asequential enzymatic reaction composed of ubiquitin-activatingenzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitinligase (E3). The exquisite specificity for proteins destined forubiquitination is usually determined by a diverse family of E3swith specific E2s. Parkin is a RING finger-type E3 that con-tains two RING finger domains and an IBR (in between RINGfingers) domain. One type of autosomal recessive juvenile par-kinsonism, which represents the major cause of juvenile PD,results from mutations of the Parkin gene (16). Autosomalrecessive juvenile parkinsonism-linked Parkin mutations havebeen demonstrated to disrupt E3 activity (17–19). Parkin hasbeen shown to interact with and ubiquitinate synphilin-1 (20).Coexpression of synphilin-1, �-synuclein, and Parkin elicits

* This work was supported by grants-in-aid for the Encouragement ofYoung Scientists (2001, 2002) and for Scientific Research (2003) fromthe Japanese Ministry of Education, Culture, Sports, Science, andTechnology. The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of ClinicalNeuroscience and Therapeutics, Hiroshima University GraduateSchool of Biomedical Sciences, 1-2-3 Kasumi, Hiroshima 734-8551, Ja-pan. Tel.: 81-82-257-5201; Fax: 81-82-505-0490; E-mail: yamashih@hiroshima-u.ac.jp.

1 The abbreviations use are used: PD, Parkinson’s disease; LB, Lewybody; ANK, ankyrin; E1, ubiquitin-activating enzyme; E2, ubiquitinconjugating enzyme; E3, ubiquitin ligase; ZF, zinc fingers; SBD, sub-strate-binding domain; Tet, tetracycline; Ub, ubiquitin; NRI, normalrabbit IgG; GST, glutathione S-transferase; DOPAC, dihydroxyphenyl-acetic acid; HVA, homovanilic acid; HPLC, high pressure liquid chro-

matography; IP, immunoprecipitation; WB, Western blotting; aa, aminoacids; HA, hemagglutinin; HEK, human embryonic kidney.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 51, Issue of December 19, pp. 51504–51514, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org51504

by guest on May 12, 2020

http://ww

w.jbc.org/

Dow

nloaded from

formation of ubiquitin-positive cytoplasmic inclusions thatresemble LBs, suggesting that synphilin-1 might link�-synuclein and Parkin to a common pathogenic mechanism(20). A recent report demonstrated that Dorfin, an E3 for mu-tant superoxide dismutase-1, also interacts with and ubiquiti-nates synphilin-1 (21). Like Parkin, Dorfin contains two RINGfinger domains and an IBR domain. Furthermore, Dorfin iscolocalized with ubiquitin in LBs of PD, suggesting that Dorfinis also implicated in the pathogenesis of PD.

Siah/Sina family proteins represent mammalian homologuesof the Drosophila Sina (seven in absentia) protein. Sina is also aRING finger-type E3 that is critically involved in neuronal de-velopment of the R7 photoreceptor cell in Drosophila (22). Sinafunctions downstream of the tyrosine kinase receptor Sevenlessand the Ras/Raf mitogen-activated protein kinase pathway (23–25). Sina is required for targeting the transcriptional repressorTramtrack for proteasome-mediated degradation, which is a req-uisite step for neuronal differentiation of R7 photoreceptor cells(26). In humans, there are two highly conserved Sina homo-logues, Siah-1 and Siah-2, which are abundantly expressed in thecentral nervous system, as well as other tissues (27, 28), and areassociated with synaptophysin (12). Like Parkin and Dorfin, bothSiah-1 and Siah-2 are also RING finger-type E3s. Siah proteinsfacilitate ubiquitination and proteasome-dependent degradationof multiple proteins, such as DCC (29), Nco-R (30), c-Myb, (31)BOB1/OBF1 (32, 33), Peg3/Pw1 (34), APC (35), Kid (36), Numb(37), synaptophysin (12), and group 1 metabotropic glutamatereceptors (38). The present report demonstrates that Siah-1 in-teracts with and ubiquitinates synphilin-1 in vivo, resulting infacilitation of synphilin-1 degradation via the ubiquitin-protea-some pathway.

EXPERIMENTAL PROCEDURES

Yeast Two-hybrid Screening—The full-length human synphilin-1cDNA was cloned by a library screening as described previously (39).The coding region of synphilin-1 cDNA was subcloned into the yeasttwo-hybrid vector pGBKT-7 (Clontech), which was in-frame fused to theGAL4-binding domain sequence. The recombinant plasmid was intro-duced into the yeast strain AH109. A rat brain cDNA library con-structed in pGAD10 (Clontech) was introduced into the yeast strainexpressing the synphilin-1 fusion protein, and �1.0 � 106 transfor-mants were screened for growth on SD plate media lacking tryptophan,leucine, histidine, and adenosine. Positive clones were detected by a�-galactosidase assay. An �-Synuclein construct in pGAD10 was usedas a positive control for the screening. To eliminate false positives,plasmid DNA from positive clones was purified, amplified, and retrans-formed into the yeast strain expressing synphilin-1 protein fused to theGAL4-binding domain. The positive clones in this second screeningwere subjected to DNA sequencing. A BLAST search revealed that oneof the isolated positive clones contained a fragment nearly identical tothe Siah-1a gene.

Vectors and Antibodies—Full-length human Siah-1 cDNA was am-plified from a human brain cDNA library (Stratagene) by PCR using aPfu DNA polymerase (Stratagene) and the following forward and re-verse primers: 5�-GAA TTC TCG AGA TGA GCC GTC AGA CTG CTAC-3� (F1) and 5�-GCG ATC TAG ATC AAC ACA TGG AAA TAG TTACAT TGA TGC C-3� (R1). The DNA fragment obtained from PCR wassubcloned in a pcDNA3 vector (Invitrogen) in-frame with the Myc tagsequence (pcDNA3-Myc-Siah-1). Human Siah-1 contains an N-terminalRING finger domain (amino acids (aa) 40–75), followed by a conservedcysteine/histidine-rich region (aa 98–152), which might represent twozinc fingers (ZF). Siah-1 contains a substrate-binding domain (SBD) (aa90–282) at the C terminus that interacts with a number of substrateproteins. The cDNA encoding Siah-1 mutants, such as RING fingerdomain-deleted Siah-1 (Siah�N) and SBD-deleted Siah-1 (Siah�C),were amplified by PCR using the following forward and reverse prim-ers: 5�-GAA TTC TCG AGA CAT GTT GTC CAA CTT GCC GG-3� (F2)and R1 for Siah�N and F1 and 5�-GCG ATC TAG ATC AGG TTG TAATGG ACT TAT GCT G-3� (R2) for Siah�C. The DNA fragment obtainedfrom PCR was subcloned into a pcDNA3 vector in-frame with the Myctag sequence (pcDNA3-Myc-Siah�N and pcDNA3-Myc-Siah�C). To con-struct the tetracycline (Tet)-repressible expression vectors for Siah-1and Siah�C, each cDNA encoding Siah-1 or Siah�C was inserted into a

pTet splice vector (Invitrogen) in-frame with the Myc tag sequence(pTet-splice-Myc-Siah-1 and pTet-splice-Myc-Siah�C). Ubiquitin (Ub)cDNA was amplified by PCR and subcloned into a pcDNA3 vectorin-frame with the FLAG tag sequence (pcDNA3-FLAG-Ub). The se-quences of all constructs were confirmed by DNA sequencing for bothcomplementary strands. The plasmid pcDNA3.1-Myc-Parkin vectorwas provided by Drs. N. Hattori and Y. Mizuno. The plasmidpcDNA-Myc-�-TrCP/FWD1 was provided by Drs. S. Kishida and A.Kikuchi. Expression vectors for HA-tagged synphilin-1 (pcDNA3-HA-synphilin-1) and HA-tagged �-synuclein (pcDNA3-HA-�-synuclein; wildtype, A53T, A30P) were generated as described previously (39, 40). Ananti-synphilin-1 polyclonal antibody was provided by Dr. E. Iseki. Ananti-�-synuclein monoclonal antibody was obtained from Pharmingen.An anti-FLAG monoclonal antibody (M2) was obtained from Sigma. Ananti-HA monoclonal antibody (F-7), an anti-HA polyclonal antibody(Y-11), an anti-Myc monoclonal antibody (9E10), and normal rabbit IgG(NRI) were obtained from Santa Cruz Biotechnology, Inc. Siah-1 anti-serum was raised against Siah-1 protein fused with glutathione S-transferase (GST) at the N terminus (GST-Siah). The antigen wasthoroughly mixed with Freund’s complete adjuvant to produce a sus-pension, which was intradermally injected into rabbits. Identical im-munogen was injected five times every week. Phlebotomy was ulti-mately performed to collect serum. Antiserum was filtered through aGST coupling Hitrap NHS activated HP column (Amersham Bio-sciences) to eliminate anti-GST antibodies. To purify anti-Siah-1 anti-bodies, pre-cleared serum was filtered through another column couplingwith GST-Siah-1. Siah-1 antibodies were subsequently eluted from thecolumn and dialyzed with phosphate-buffered saline.

Cell Culture and Transfection—HEK 293 and human dopaminergicneuroblastoma SH-SY5Y cells were obtained from American Type Cul-ture Collection. Cells were grown in Dulbecco’s modified Eagle’s me-dium containing 10% fetal bovine serum. Transfections of expressionvectors into HEK 293 cells were performed by FuGENE 6 (RocheApplied Science) according to the manufacturer’s instructions. PC12-Tet cells stably transfected with pTet-tTAk (Invitrogen) were grown inDulbecco’s modified Eagle’s medium containing 10% fetal bovine serum,5% horse serum, and 0.1 mg/ml hygromycin (Roche Applied Science). Toestablish a Tet-repressible Siah-1 expression system, PC12-Tet cellswere transfected with pTet-splice-Myc-Siah-1 or pTet-splice-Myc-Siah�C using LipofectAMINE 2000 (Invitrogen). After transfection,cells were grown in a medium containing 0.1 mg/ml hygromycin, 500ng/ml Tet (Sigma), and 400 �g/ml G418 (Sigma). For 3–4 weeks, colo-nies were selected by hygromycin and G418. Among the selected colo-nies, multiple monoclonal cell lines that exhibited Tet-repressible ex-pression of Siah-1 (PC12-Tet-Siah) or Siah�C (PC12-Tet-�C) wereestablished.

Immunoprecipitation—HEK 293 cells were transfected withpcDNA3-Myc-Siah-1, pcDNA3-Myc-Siah�N, pcDNA3-HA-synphilin-1,and pcDNA3-HA-�-synuclein. After 24 h, the cells were cultured with20 �M MG132 (Calbiochem) for 8 h and then washed with ice-coldphosphate-buffered saline and lysed in lysis buffer (1% Nonidet P-40, 50mM Tris-HCl, pH 7.4, 10% glycerol, 150 mM NaCl, 1 mM EDTA, pH 8.0)with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 �g/mlaprotinin, 10 �g/ml leupeptin). After the lysate protein contents werenormalized using a protein assay kit (Bio-Rad), the cell lysate (500�g/sample) was immunoprecipitated with NRI or anti-HA antibodiesusing protein G-Sepharose beads (Pierce). For immunoprecipitation ofthe endogenous proteins from rat brains, adult rat brains were homog-enized in 5 volumes of Tris-HCl (50 mM; pH 7.4), KCl (140 mM), EDTA(3 mM), and 0.5% Triton X-100 supplemented with protease inhibitors.The tissue homogenate was centrifuged at 5,000 � g at 4 °C for 20 min.The supernatant was used for immunoprecipitation with one of thefollowing antibodies: normal rabbit IgG, anti-GST, or anti-Siah-1 anti-bodies. Each immunoprecipitate was divided into two parts, separatedon SDS-PAGE, and transferred onto nitrocellulose membranes. Boundproteins were analyzed with immunoblotting.

Siah-1 Protein Expression in Rat Tissues—Rat brain protein lysatefrom various tissues (50 �g/lane) were separated on SDS-PAGE andimmunoblotted with anti-Siah-1, anti-synphilin-1, anti-�-synuclein,and anti-�-tubulin antibodies. To determine the specificity of anti-Siah-1 antibody, identical blots of rat protein lysate from various tis-sues were incubated with anti-Siah-1 antibody preabsorbed with anti-gen (GST-Siah-1).

Confocal Immunofluorescent Staining—Immunofluorescence wasperformed as described previously (41). Briefly, HEK 293 cells cotrans-fected with pcDNA3-HA-synphilin-1 and pcDNA3-Myc-Siah-1 or SH-SY5Y cells were seeded and grown on glass coverslips in medium.Adherent cells were fixed with neutral buffered 4% (w/v) paraformal-

Siah-mediated Ubiquitination of Synphilin-1 51505

by guest on May 12, 2020

http://ww

w.jbc.org/

Dow

nloaded from

dehyde and permeabilized with 0.2% Triton X-100. To assess the dis-tribution of endogenous Siah-1 and synphilin-1, untransfected SH-SY5Y cells were treated with anti-Siah-1 and anti-synphilin-1antibodies. Anti-Siah-1 and anti-synphilin-1 antibodies preabsorbedwith each antigen were also used. To assess the colocalization of syn-philin-1 and Siah-1, a double-labeling immunofluorescent staining wasperformed with a combination of anti-HA and anti-Myc antibodies.Anti-Siah-1, anti-synphilin-1, and anti-HA antibodies were visualizedby Alexa Fluor488 anti-rabbit antibody (Molecular Probes). Anti-Mycantibody was visualized by Alexa Fluor 568 anti-mouse antibody (Mo-lecular Probes). Immunostained preparations were examined with theZeiss LSM510 confocal microscope.

Pulse-Chase Assay—A pulse-chase assay was performed as describedpreviously (42, 43). HEK 293 cells were transfected with pcDNA3-HA-synphilin-1 and pcDNA3-Myc-Siah-1, pcDNA3-Myc-Siah�N, pcDNA3-Myc-Parkin, pcDNA-Myc-�-TrCP/FWD1, or empty pcDNA3-Myc vectors.After 24 h, cells were cultured with 20 �M MG132 for 8 h; cycloheximide(Sigma) was subsequently added to the medium to yield a final concen-tration of 40 �M, which would inhibit new synthesis of synphilin-1. Thecells were cultured for chase intervals of 0, 2, 4, 6, 8, 12, 18, 24, and 48 hand harvested in the lysis buffer after the appropriate chase time. Anequal amount of protein from each lysate was separated on SDS-PAGEand immunoblotted with an anti-HA antibody. The degree of synphilin-1expression was quantitated by densitometric analysis with NIH Imagesoftware.

Ubiquitination Assay—An in vivo ubiquitination assay was performedas described previously (12, 20, 44). HEK 293 cells were transfectedwith pcDNA3-Myc-Siah-1, pcDNA3-Myc-Siah�N, pcDNA3-Myc-Siah�C,pcDNA3-Myc-Parkin, pcDNA-Myc-�-TrCP/FWD1, pcDNA3-HA-synphilin-1, pcDNA3-HA-�-synuclein, or pcDNA3-FLAG-Ub. After 24 h,cells were cultured for 8 h with 20 �M MG132. Cells were lysed andimmunoprecipitated with an anti-HA antibody. Each precipitate was di-vided into two parts, separated on SDS-PAGE, and analyzed by immuno-blotting with anti-FLAG and anti-HA antibodies to detect ubiquitin-conjugated synphilin-1.

Preparation of GST Fusion-Siah Proteins and in Vitro Binding As-says—To generate Siah-1 protein fused with GST at the N terminus(GST-Siah), the coding region of Siah-1 cDNA was amplified by PCRusing the following forward and reverse primers: 5�-CTC GAA TTCATG AGC CGT CAG ACT GCT ACA G-3� (GF1) and 5�-CTC CTC GAGTCA ACA CAT GGA AAT AGT TAC ATT GAT GCC-3� (GR1). Toconstruct GST fusion proteins containing the RING finger domain andZF (aa 40–152; GST-RING-ZF), ZF alone (aa 75–152; GST-ZF), or SBD(aa 98–282; GST-SBD, aa 152–282, GST-SBD-S, and aa 180–240; GST-SBD-SS), the corresponding regions of Siah-1 cDNA were amplified by

PCR using the following forward and reverse primers: 5�-CTC GAATTC TTG GCG AGT CTT TTT GAG TGT C-3� (GF2) and 5�-CTC CTCGAG TCA GGT TGT AAT GGA CTT ATG CTG-3� (GR3) for GST-RING-ZF; 5�-CTC GAA TTC ACA TGT TGT CCA ACT TGC CGG-3� (GF3) andGR3 for GST-ZF; 5�-CTC GAA TTC TCA GTA CTT TTC CCC TGT AAATAT GCG-3� (GF4) and GR1 for GST-SBD; 5�-CTC GAA TTC CCC CATCTG ATG CAT CAG CAT AAG-3� (GF5) and GR1 for GST-SBD-S; and5�-CTG GAA TTC GCT GTT GAC TGG GTG ATG ATG-3� (GF6) and5�-CTC CTC GAG TCA AGG AGT CGC TTC CCA AGT CAA TC-3�(GR2) for GST-SBD-SS. DNA fragments obtained from PCR were sub-cloned into a pGEX6P-1 vector (Amersham Biosciences). The sequencesof all constructs were confirmed by DNA sequencing. GST fusion syn-philin-1 proteins that contained the N terminus (aa 1–202; GST-synph-N1), the ANK repeats 1–3 (aa 87–458; GST-synph-N2), the ANK re-peats 1–4 and the coiled-coil domain (aa 349–617; GST-synph-ANK), orthe ANK repeats 5–6 and the C terminus (aa 611–919; GST-synph-C),were constructed as described previously (39). GST fusion proteins wereproduced in Escherichia coli BL21 via isopropyl-�-D-thiogalactopyrano-side induction and purified using glutathione-Sepharose 4B (Amer-sham Biosciences) as described previously (8, 39).

For the precipitation assay, HEK 293 cells transfected withpcDNA3-HA-synphilin-1, pcDNA3-Myc-Siah-1, or pcDNA3-HA-�-synuclein were lysed and precipitated with various GST fusion proteinsor GST alone as described previously (39). Each binding assay wasconducted with 10 �g of GST fusion protein bound to glutathione-Sepharose 4B. After the lysate protein content was normalized andprecipitated with GST fusion proteins, bound proteins were separatedon SDS-PAGE and immunoblotted with anti-HA or anti-Myc antibod-ies. GST fusion proteins used for the binding assays were stained byCoomassie Brilliant Blue R250 (Sigma).

Dopamine Release Assay—PC12-Tet-Siah cells, PC12-Tet-�C cells,or PC12-Tet cells were plated onto 35-mm dishes at a density of 106 cellsper dish. In a standard experiment, cells were subsequently transfectedwith the indicated plasmid DNA using LipofectAMINE 2000 and grownwith or without Tet for 24 h. For a dopamine release assay, cells werewashed twice with a low K� solution (20 mM HEPES-NaOH, pH 7.4,140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM

KH2PO4, and 11 mM glucose) and incubated in the low K� solution for2 min. The medium was subsequently replaced with 1 ml of the low K�

solution or 1 ml of a high K� solution (20 mM HEPES-NaOH, pH 7.4, 85mM NaCl, 60 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4,and 11 mM glucose) for 0, 2, 4, 6, 10, and 16 min; the solution was thencollected. Concentrations of dopamine, dihydroxyphenylacetic acid(DOPAC), and homovanilic acid (HVA) were determined by high pres-sure liquid chromatography (HPLC) using a reverse-phase column and

FIG. 1. Siah-1 interaction with syn-philin-1. A, synphilin-1 and Siah-1 coim-munoprecipitation. Lysates preparedfrom HEK 293 cells transfected witheither empty pcDNA3-HA vector (HAvector) or pcDNA3-HA-synphilin-1 (HA-synph) and either pcDNA3-Myc-Siah-1(Myc-Siah) or pcDNA3-Myc-Siah�N (Myc-Siah�N) were immunoprecipitated (IP)with an anti-HA antibody or control nor-mal rabbit IgG (NRI). Immunoprecipitateswere divided into two parts and analyzedby Western blotting (WB) with anti-Mycand anti-HA antibodies. Molecular massmarkers are indicated on the right. B, co-immunoprecipitation of Siah-1 and synphi-lin-1 in rat brain homogenate. Rat brainhomogenate was subjected to IP with NRI,anti-GST, anti-synphilin-1, or anti-Siah-1antibodies followed by anti-synphilin-1 andanti-Siah-1 WB. Synphilin-1 migratesnear the 83-kDa marker. Molecular massmarkers are indicated on the right.

Siah-mediated Ubiquitination of Synphilin-151506

by guest on May 12, 2020

http://ww

w.jbc.org/

Dow

nloaded from

an electrochemical detector system (Eicom) as described previously(45). Dopamine, DOPAC, and HVA concentrations for each sample werenormalized for the total cell number for each condition. Dopaminerelease was calculated as a percentage of the total intracellular dopa-mine contents. Data were calculated from four independent experiments.Statistic analysis was performed with one-way analysis of variance.

RESULTS

Identification of Siah-1 as a Protein That Interacts withSynphilin-1—To identify proteins that interact with synphi-lin-1, yeast two-hybrid screening of a rat brain cDNA librarywas performed using full-length synphilin-1 as bait. From1.0 � 106 library transformants, a positive clone that containeda fragment nearly identical to the Siah-1a gene was obtained.Rat Siah-1a is a RING finger-type E3 that shares 99.6% aminoacid identity with human Siah-1. The full-length human Siah-1cDNA was isolated from a human brain cDNA library by PCR.

Interaction of Siah-1 with synphilin-1 was examined inmammalian cells using an immunoprecipitation assay. Fromlysates of HEK 293 cells coexpressing HA-tagged synphilin-1(HA-synphilin-1) and Myc-tagged Siah-1 (Myc-Siah-1), an an-ti-HA antibody immunoprecipitated Myc-Siah-1 (Fig. 1A). Inaddition, an anti-Myc antibody immunoprecipitated HA-syn-philin-1 in a similar manner (data not shown). It was concludedthat Siah-1 and synphilin-1 proteins interact with each other inmammalian cells. Similarly, HA-synphilin-1 and Myc-Siah�N,a deletion-mutant Siah-1 that lacks the RING finger domain,were also coimmunoprecipitated, suggesting that the RINGfinger domain of Siah-1 is not required for association withsynphilin-1 (Fig. 1A). To determine whether endogenous syn-philin-1 and Siah-1 interact in brain in vivo, an immunopre-cipitation assay using antibodies against Siah-1, synphilin-1,GST, or normal rabbit IgG was performed in rat brain homo-genate followed by immunoblotting with antibodies againstsynphilin-1 or Siah-1. Synphilin-1 and Siah-1 coimmunopre-

cipitated each other in vivo, suggesting physiologic relevance ofthe interaction (Fig. 1B).

Identification of Domains Involved in Siah-1-Synphilin-1 As-sociation—To define the specific domain of Siah-1 responsiblefor interaction with synphilin-1, a series of GST fusion proteinscontaining various truncations of the Siah-1-conserved regionwere generated. HEK 293 cells transfected with expressionvectors for HA-synphilin-1 were lysed and precipitated withvarious GST fusion Siah-1 proteins, such as GST-Siah, GST-RING-ZF, GST-ZF, GST-SBD, GST-SBD-S, and GST-SBD-SS,as well as GST alone as a control (Fig. 2A). Anti-HA immuno-blotting revealed that GST fusion Siah-1 proteins containingthe SBD exclusively precipitated synphilin-1 (Fig. 2B). Suchresults suggest that Siah-1 binds to synphilin-1 through itsSBD. Furthermore, the centrally located polypeptide in theSBD (aa 180–240; SBD-SS) was found to be necessary andsufficient for binding to synphilin-1 (Fig. 2B).

To identify the binding site of synphilin-1 to Siah-1, HEK 293cells transfected with expression vectors for Myc-Siah-1 werelysed and precipitated with various GST fusion synphilin-1proteins (Fig. 2A). Anti-Myc immunoblotting revealed that onlythe N-terminal residues 1–202 of synphilin-1 (synphilin-N1)could precipitate Siah-1 (Fig. 2C). In contrast, other regions ofsynphilin-1, including the ANK repeats or C-terminal domain,were unable to bind to Siah-1. It was concluded that the SBD ofSiah-1 and the N terminus of synphilin-1 interact witheach other.

Siah-1 and Synphilin-1 Proteins Are Widely Expressed in theBrain—To investigate expression of Siah-1, synphilin-1, and�-synuclein, rat protein lysate from various tissues were ana-lyzed by immunoblotting using antibodies against Siah-1, syn-philin-1, �-synuclein, or �-tubulin. Siah-1 was enriched inbrain, heart, pancreas, kidney, and skeletal muscle (Fig. 3).

FIG. 2. Identification of the bindingsites involved in Siah-1-synphilin-1interaction. A, schematic representationof the primary structures and constructsof Siah-1 and synphilin-1 utilized for thebinding assay. Siah-1 contains a RINGfinger domain, two zinc fingers, and asubstrate-binding domain (SBD). Synphi-lin-1 contains six ankyrin (ANK) repeats,a coiled-coil domain, and an ATP/GTP-binding site. B, mapping of the synphilin-1-binding site of Siah-1. HEK 293 cellstransfected with pcDNA3-HA-synphilin-1were lysed and precipitated (PT) with ei-ther the indicated GST fusion Siah pro-teins or GST alone as a control. Top,bound proteins were analyzed by WBwith an anti-HA antibody. Bottom, theamount of GST fusion proteins used forthe assay was detected by Coomassiestaining. C, identification of the Siah-binding site of synphilin-1. HEK 293 cellstransfected with pcDNA3-HA-Siah-1were lysed and precipitated with eitherthe indicated GST fusion synphilin-1 pro-teins or GST alone as a control. Top,bound proteins were analyzed by WBwith an anti-HA antibody. Bottom, theamount of GST fusion proteins used forthe assay was detected by Coomassiestaining.

Siah-mediated Ubiquitination of Synphilin-1 51507

by guest on May 12, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Siah-1 was expressed in all regions in the central nervoussystem, including the cerebral cortex, hippocampus, striatum,cerebellum, medulla, and spinal cord. In the rat brain, theanti-Siah-1 antibody recognized a single band of �35 kDa,similar to the predicted molecular mass of Siah-1 protein (32kDa), that disappeared when the antibody was preincubatedwith antigen (GST-Siah protein) (Fig. 3). Synphilin-1 was en-riched in all regions in the brain, whereas �-synuclein wasenriched in the cerebral cortex, hippocampus, and striatum.Such results demonstrate that both Siah-1 and synphilin-1 arecoexpressed in the brain.

Colocalization of Siah-1 with Synphilin-1 in the Cytoplasm—Because Siah-1 interacted with synphilin-1 in the brain, en-dogenous Siah-1 and synphilin-1 localization was investigatedwith an immunofluorescent study. Confocal microscopic anal-ysis revealed that Siah-1 localized in the cytoplasm of humandopaminergic SH-SY5Y cells. Synphilin-1 was observed as acytoplasmic ring-like appearance as described previously (11)(Fig. 4A). Such immunofluorescence disappeared when the an-tibodies were preincubated with each antigen. Furthermore, adouble-staining immunofluorescent study was performed todetermine whether Siah-1 colocalized with synphilin-1. Myc-Siah-1 was concomitantly expressed with HA-synphilin-1 bytransient transfection of HEK 293 cells. Confocal microscopicanalysis revealed that synphilin-1 formed cytoplasmic inclu-sions (Fig. 4B) and that Siah-1 was distributed peripheral tothe synphilin-1 inclusions. An overlay of Siah-1 and synphi-lin-1 staining demonstrated colocalization of both proteins.

Siah-1 Facilitates Synphilin-1 Degradation via the Ubiq-

uitin-Proteasome Pathway—Siah-1 has been demonstrated tospecifically mediate degradation of its substrate proteins. Ac-cordingly, the present study investigated whether Siah-1 me-diates synphilin-1 degradation. Myc-Siah-1 was concomitantlyexpressed with HA-synphilin-1 by transient transfection ofHEK 293 cells. Cell lysates were subjected to immunoblottingwith anti-HA or anti-Myc antibodies. In cells expressing syn-philin-1 alone, synphilin-1 was detected as an �102-kDa mo-lecular mass of protein. Nonetheless, in cells coexpressing syn-philin-1 and Siah-1, synphilin-1 was detected as a faint band(Fig. 5A). Moreover, synphilin-1 expression recovered with ad-dition of the potent proteasome inhibitor MG132 (20 �M) de-spite Siah-1 expression. On the other hand, with coexpressionof E3-inactive Siah�N, synphilin-1 expression was not altered(Fig. 5A). Such results suggest that Siah-1 might play a role insynphilin-1 degradation by the ubiquitin-proteasome pathway.In addition, Siah-1 expression was also remarkably increasedin the presence of MG132. To examine whether Siah-1 itself isalso ubiquitinated, HEK 293 cells were transfected with ex-

FIG. 3. Siah-1 protein expression in the central nervous sys-tem. Equal amounts of homogenates (50 �g protein) from the indicatedrat tissues were analyzed by WB with antibodies against Siah-1, syn-philin-1, �-synuclein, or �-tubulin. To determine the specificity ofSiah-1 antibody, an identical blot of rat protein lysate from varioustissues was incubated with anti-Siah-1 antibody preabsorbed with pu-rified Siah-1 protein. FIG. 4. Siah-1 and synphilin-1 colocalization. A, SH-SY5Y cells

were fixed, permeabilized, and labeled with either anti-Siah-1 or anti-synphilin-1 antibodies, which was followed by Alexa Fluor 488 anti-rabbit antibodies and then examined with a confocal microscope. Scalebar, 10 �m. Anti-Siah-1 or anti-synphilin-1 antibodies preabsorbedwith each antigen were also used for immunofluorescent stainingas a control (cont). B, HEK 293 cells were transfected withpcDNA3-Myc-Siah-1 and pcDNA3-HA-synphilin-1. Two days post-transfection, cells were fixed, permeabilized, labeled with anti-Myc(mouse) and anti-HA (rabbit) antibodies, which was followed by AlexaFluor 568 anti-mouse or Alexa Fluor 488 anti-rabbit antibodies, respec-tively, and then examined with a confocal microscope. Regions of over-lap between Siah-1 (red) and synphilin-1 (green) are shown in yellow.Scale bar, 10 �m.

Siah-mediated Ubiquitination of Synphilin-151508

by guest on May 12, 2020

http://ww

w.jbc.org/

Dow

nloaded from

pression vectors for FLAG-Ub and Myc-Siah-1, and the celllysates were immunoprecipitated with an anti-Myc antibody.The procedure was followed by immunoblotting with an anti-FLAG antibody to detect ubiquitin-conjugated Siah-1. In cellscoexpressing Myc-Siah-1 and FLAG-Ub, ubiquitinated highmolecular mass bands were detected in the anti-Myc immuno-precipitates, suggesting that Siah-1 was also ubiquitinated(Fig. 5B). Such results suggest that Siah-1 itself was alsodegraded through the ubiquitin-proteasome pathway as de-scribed previously (46).

In addition, to investigate whether Siah-1 would facilitatesynphilin-1 degradation, a pulse-chase assay was performed.In cells transfected with pcDNA3-HA-synphilin-1 andpcDNA3-Myc-Siah-1, the synphilin-1 degradation rate wasmuch higher than that of cells transfected with pcDNA3-HA-synphilin-1 and empty pcDNA3-Myc vectors. Moreover, thehalf-life of synphilin-1 was 8 h for cells cotransfected withexpression vectors for Siah-1, whereas that for cells cotrans-fected with empty vectors was 44 h (Fig. 5C). Nonetheless, thesynphilin-1 degradation rate in cells coexpressing Myc-Siah�Nwas slower than that in cells cotransfected with empty vectors,suggesting that synphilin-1 degradation was inhibited by adominant-negative effect of Siah�N on endogenous Siah-1 (Fig.5C). Such results are consistent with the notion that the RINGfinger domain of E3 is required for substrate ubiquitinationthat leads to degradation. As a negative control, �-TrCP/FWD1, an E3 for �-catenin, was utilized for the pulse-chaseassay. Both Siah-1 and �-TrCP/FWD1 independently facilitate�-catenin degradation (35, 47, 48). In cells transfected withHA-synphilin-1 and Myc-�-TrCP/FWD1, the synphilin-1 de-gradation rate was almost the same as that of cells transfectedwith empty vectors (Fig. 5C), suggesting that Siah-1 specifi-cally facilitated synphilin-1 degradation. Because Parkin hasalso been reported to regulate synphilin-1 degradation (20), thepresent study compared the effects of Siah-1 and Parkin on thesynphilin-1 degradation rate. It was noted that the synphilin-1degradation rate for cells coexpressing Myc-Siah-1 was higherthan that for cells coexpressing Myc-Parkin. Moreover, thehalf-life of synphilin-1 was 8 h for cells expressing Siah-1 and18 h for cells expressing Parkin (Fig. 5C). Siah-1 and Parkincellular expression levels were similar (data not shown). Suchresults suggest that Siah-1 facilitates synphilin-1 degradationmore efficiently than Parkin.

Siah-1 Ubiquitinates Synphilin-1 via Its RING Finger Do-main—Because proteasome-dependent proteolysis involvesubiquitination of target proteins, the present study investi-gated whether Siah-1 would facilitate synphilin-1 degradationby promoting synphilin-1 ubiquitination. HEK 293 cells weretransfected with expression vectors for HA-synphilin-1, FLAG-tagged ubiquitin (FLAG-Ub), and Myc-Siah-1 and subse-quently cultured with MG132. Cells were lysed and immuno-precipitated with an anti-HA antibody, followed byimmunoblotting with an anti-FLAG antibody to detect ubiq-uitin-conjugated synphilin-1. In cells coexpressing HA-synphi-lin-1 and FLAG-Ub, ubiquitinated high molecular mass pro-teins were detected in the anti-HA immunoprecipitates,suggesting that synphilin-1 was slightly ubiquitinated (Fig.6A). In cells coexpressing Myc-Siah-1, HA-synphilin-1, andFLAG-Ub, ubiquitin-conjugation of synphilin-1 was markedlyenhanced, suggesting that Siah-1 ubiquitinates synphilin-1(Fig. 6A). Conversely, coexpression of either Siah�C, a Siah-1deletion-mutant that lacks SBD, or Siah�N reduced synphi-lin-1 ubiquitination (Fig. 6A). Given the above, such resultsindicate that synphilin-1 acts as a substrate for Siah-1 ubiq-uitin ligase. Moreover, repeated ubiquitination assays revealedthat synphilin-1 ubiquitination levels achieved with Siah-1 arehigher than those achieved with Parkin, suggesting that Siah-1might ubiquitinate synphilin-1 more efficiently than Parkin(Fig. 6A). In addition, it was investigated whether �-TrCP/FWD1 ubiquitinates synphilin-1. Nonetheless, synphilin-1 wasnot ubiquitinated by �-TrCP/FWD1 (Fig. 6B), suggesting thatSiah-1 specifically ubiquitinates synphilin-1.

Siah-1 Fails to Ubiquitinate �-Synuclein—In addition, thepresent study investigated whether Siah-1 interacts with�-synuclein. Nonetheless, a ubiquitination assay revealed that�-synuclein was not ubiquitinated by Siah-1 (Fig. 7A). In addi-

FIG. 5. Siah-1-mediated facilitation of synphilin-1 degradationby the ubiquitin-proteasome pathway. A, HEK 293 cells were co-transfected with pcDNA3-HA-synphilin-1 and either pcDNA3-Myc-Siah-1 or pcDNA3-Myc-Siah�N. After 24 h, cells were cultured inthe presence or absence of MG132 (20 �M) for 8 h and subsequentlylysed. After the lysate protein content was normalized, an equalamount of protein from each lysate was analyzed by WB with anti-HAand anti-Myc antibodies. Molecular mass markers are indicated on theright. B, Siah-1 ubiquitination. Lysates prepared from HEK 293 cellscotransfected with pcDNA3-FLAG-ubiquitin and pcDNA3-Myc-Siah-1were immunoprecipitated with an anti-Myc antibody (Myc-IP). Immu-noprecipitates were divided into two parts and analyzed by WB withanti-FLAG and anti-Siah-1 antibodies. FLAG-Ub-conjugated proteinsin each total cell lysate were detected by WB with an anti-FLAGantibody. C, Siah-1-facilitated synphilin-1 degradation. HEK 293 cellswere cotransfected with pcDNA3-HA-synphilin-1 and an emptypcDNA3-Myc vector (vector), pcDNA3-Myc-Siah-1 (Siah), pcDNA3-Myc-Siah�N (Siah�N), pcDNA3.1-Myc-Parkin (Parkin), or pcDNA-Myc-�-TrCP/FWD1 (FWD1). After 24 h post-transfection, cells were treatedwith MG132 (20 �M) for 8 h; subsequently cycloheximide (40 �M) wasadded to the medium. Cells were harvested at the indicated time points,and an equal amount of protein from each lysate was analyzed by WBusing an anti-HA antibody. The degree of synphilin-1 expression wasquantitated by densitometric analysis with NIH image software andexpressed as a percentage of synphilin-1 expression at 0 h. Data arepresented as means � S.D. for four independent experiments.

Siah-mediated Ubiquitination of Synphilin-1 51509

by guest on May 12, 2020

http://ww

w.jbc.org/

Dow

nloaded from

tion, to investigate whether Siah-1 binds to �-synuclein, precip-itation assays were performed. HEK 293 cells transfected withexpression vectors for HA-�-synuclein wild type, A53T, or A30Pwere lysed and precipitated with GST fusion Siah-1 protein.Anti-HA immunoblotting revealed that �-synuclein and Siah-1did not bind to each other (Fig. 7B). It was also examinedwhether Siah-1 interacts with �-synuclein in mammalian cells.From lysates of HEK 293 cells cotransfected with expressionvectors for HA-�-synuclein and Myc-Siah-1, an anti-HA antibodywas found to not immunoprecipitate Myc-Siah-1 (data notshown). It was subsequently investigated whether Siah-1 medi-ates �-synuclein degradation. HEK 293 cells coexpressing HA-�-synuclein and Myc-Siah-1 were treated with MG132 for the in-dicated time. �-Synuclein expression levels were not altered bySiah-1 expression (Fig. 7C), suggesting that Siah-1 does notfacilitate �-synuclein degradation.

Effects of Siah-1 on Dopamine Release—Siah-1 is known tobe distributed in the central nervous system. In addition, re-cent papers have demonstrated that Siah-1 is involved in syn-aptic transmission (12, 38, 49). The present study examinedwhether Siah-1 affects dopamine release. Monoclonal cell lines,i.e. PC12-Tet-Siah (clones PS1–4) and PC12-Tet-�C (clonesPDC1–4), that have Tet-repressible expression systems forSiah-1 and Siah�C, respectively, were established. Immuno-blotting with an anti-Myc antibody demonstrated that Tet inthe culture medium could negatively regulate Siah-1 andSiah�C protein cellular expression (PS1 and PDC1). Expres-sions were found to be very low at 500 ng/ml of Tet (Siah� or�C�), moderate at 50 ng/ml of Tet (Siah�), and high in theabsence of Tet (Siah�� or �C��) (Fig. 8A). Siah-1 reduced the

total intracellular dopamine content without increasing do-pamine metabolites such as DOPAC or HVA in PC12-Tet-Siahcells (PS1). In contrast, the total intracellular dopamine con-tent in PC12-Tet-�C cells (PDC1) in the absence of Tet wassimilar to that in PC12-Tet cells, suggesting that Siah-1 mightinhibit dopamine biosynthesis (Fig. 8A). Similar to PC12-Tetcells, high K� (60 mM) stimulation induced dopamine releasefrom PC12-Tet-Siah (PS1) and PC12-Tet-�C (PDC1) cells,whereas low K� (4.7 mM) stimulation induced little dopaminerelease (Fig. 8B). High K�-induced dopamine release was rapidand reached a plateau at �6 min. Nonetheless, maximumextracellular dopamine levels were decreased in relation to thedecreased total intracellular dopamine contents resulting fromSiah-1 expression (Fig. 8B). Siah-1 did not affect dopaminerelease kinetics, which was calculated as a percentage of thetotal intracellular dopamine content. Independent from Siah-1expression, �80–90% of the total intracellular dopamine con-tent was released within 6 min (Fig. 8B). Similar results wereobtained from other monoclonal cell lines (PS1–4, PDC1–4)(Fig. 8C).

Synphilin-1 Inhibits High K�-induced Dopamine Release—The role of synphilin-1 in dopamine release is poorly under-stood. The present study also examined whether synphilin-1might be involved in dopamine release. PC12-Tet-Myc-Siah-1cells were transfected with empty expression vectors or expres-sion vectors for either synphilin-1 or �-synuclein, which weregrown in the presence of Tet to inhibit Siah-1 expression(Siah�) and then stimulated with high K� solution for 1 min.Synphilin-1 expression reduced the total intracellular dopa-mine content (Fig. 9A) without increasing dopamine metabo-

FIG. 6. Siah-1-mediated ubiquitina-tion of synphilin-1. A, lysates preparedfrom HEK 293 cells cotransfected withpcDNA3-HA-synphilin-1, pcDNA3-FLAG-ubiquitin, pcDNA3-Myc-Siah-1, pcDNA3-Myc-Siah�N, pcDNA3-Myc-Siah�C, orpcDNA3.1-Myc-Parkin were immunopre-cipitated with an anti-HA antibody (HA-IP). Immunoprecipitates were divided intotwo parts and analyzed by WB with anti-FLAG and anti-HA antibodies. Expressionof Myc-Siah-1, Myc-Siah�N, Myc-Siah�C,or Myc-Parkin in each total cell lysate wasdetected by WB with an anti-Myc antibody.FLAG-Ub-conjugated proteins in each totalcell lysate were detected by WB with ananti-FLAG antibody. Molecular massmarkers are indicated on the right. B, ly-sates prepared from HEK 293 cells co-transfected with pcDNA3-HA-synphilin-1,pcDNA3-FLAG-ubiquitin, and pcDNA-Myc-�-TrCP/FWD1 were subjected toHA-IP; the ubiquitination assay was per-formed in the same manner as A.

Siah-mediated Ubiquitination of Synphilin-151510

by guest on May 12, 2020

http://ww

w.jbc.org/

Dow

nloaded from

lites, such as DOPAC and HVA (data not shown). �-Synucleinexpression also reduced the total intracellular dopamine con-tent as described previously (10). Because the present worksuggests that Siah-1 facilitates synphilin-1 degradation, it canalso be said that Siah-1 might negatively regulate synphilin-1function. Upon Siah-1 coexpression with synphilin-1 and�-synuclein, a state attained by removing Tet from the medium(Siah�), the total intracellular dopamine content was furtherreduced (Fig. 9A). Expression of synphilin-1 and �-synucleininhibited the high K�-induced increase in extracellular dopa-mine levels, in proportion to the decreased total intracellulardopamine contents (Fig. 9B). With Siah-1 coexpression, thehigh K�-induced increase in extracellular dopamine levels wasfurther reduced. Nonetheless, when dopamine release kineticswere evaluated by percentage of dopamine release, high K�-induced dopamine release from cells expressing synphilin-1was depressed compared with controls, whereas release fromcells expressing �-synuclein was almost equal to controls (Fig.9C), suggesting that synphilin-1 might inhibit dopamine re-lease. Furthermore, when Siah-1 expression in the same cellswas induced by removing Tet from the medium (Siah�), dopam-ine release increased to control levels (Fig. 9C). Such resultssuggest that Siah-1 might abrogate the inhibitory effect of syn-philin-1 on dopamine release by facilitating synphilin-1 degrada-tion via the ubiquitin-proteasome pathway. Similar results wereobtained from other monoclonal cell lines (Fig. 9D).

DISCUSSION

The present study identified Siah-1 as a binding partner forsynphilin-1 that is implicated in the pathogenesis of Parkin-son’s disease. Siah/Sina family proteins are evolutionarily con-served E3 ubiquitin ligases that participate in regulating ubiq-uitination and proteasome-dependent degradation of multipleproteins. Siah-1 contains a RING finger domain at the N ter-minus that is required for interacting with E2s and a SBD atthe C terminus that is required for substrate binding. Bindingassays using GST fusion proteins indicated that Siah-1 alsobound to synphilin-1 via the SBD. In particular, amino acidresidues 180–240 in the SBD are necessary and sufficient forsynphilin-1 binding. Recently, a binding motif for Siah,RPVAXVXPXXR, was identified (50). Furthermore, the coresequence PXAXVXP was found in the Siah interacting proteinsSIP, OBF-1, DCC, and TIEG1, with more degenerate consensussequences found in NUMB, Vav, Kid, and N-CoR (50). The mostconserved residues in the motif appear to be VXP; mutagenesisof both of these residues reduced or abrogated Siah binding(50). The GST fusion protein binding assays of the presentstudy indicate that the SBD of Siah-1 binds to the N-terminalregion (aa 1–202) of synphilin-1, within which a consensussequence, PXXXVXP, is located at residues 74–80, suggestingthe presence of a binding motif.

Pulse-chase and ubiquitination assays demonstrated thatSiah-1 specifically facilitates synphilin-1 ubiquitination and

FIG. 7. Siah-1 did not facilitate �-synuclein ubiquitination and degradation. A, lysates prepared from HEK 293 cells cotransfected withpcDNA3-HA-�-synuclein, pcDNA3-FLAG-ubiquitin, and pcDNA3-Myc-Siah-1 or pcDNA3.1-Myc-Parkin were immunoprecipitated with an anti-HAantibody (HA-IP). Immunoprecipitates were divided into two parts and analyzed by WB with anti-FLAG and anti-HA antibodies. Expression ofMyc-Siah-1 or Myc-Parkin in each total cell lysate was detected by WB with an anti-Myc antibody. FLAG-Ub-conjugated proteins in each total celllysate were detected by WB with an anti-FLAG antibody. Molecular mass markers are indicated on the right. B, HEK 293 cells transfected withpcDNA3-HA-�-synuclein (wild type (WT), A53T, A30P) were lysed and precipitated (PT) with either GST fusion Siah protein or GST alone as acontrol. Top, the amount of GST fusion proteins used for the assay was detected by Coomassie staining. Bottom, bound proteins were analyzed byWB with an anti-HA antibody. C, HEK 293 cells transfected with pcDNA3-Myc-Siah-1 and pcDNA3-HA-�-synuclein were incubated with 20 �M

MG132 for the indicated times and then lysed. After the lysate protein content was normalized, an equal amount of protein from each lysate wasanalyzed by WB with anti-HA, anti-Myc, or anti-actin antibodies.

Siah-mediated Ubiquitination of Synphilin-1 51511

by guest on May 12, 2020

http://ww

w.jbc.org/

Dow

nloaded from

degradation. Although Parkin also exerts a similar function,synphilin-1 ubiquitination levels achieved with Siah-1 werehigher than those attained with Parkin. In addition, synphi-lin-1 degradation rates achieved with Siah-1 were also fasterthan those attained with Parkin. Such results suggest thatSiah-1 functions as a more efficient E3 for synphilin-1 thanParkin. Because Siah proteins interact with E2s, such asUbcH5, UbcH8, or UbcH9, and Parkin interacts with UbcH6,UbcH7, or Ubc12, interaction with different E2s might accountfor the distinct degradation rates. In addition to Siah-1 andParkin, a recent study demonstrated that Dorfin also ubiquiti-nates synphilin-1 (21). Accordingly, three RING finger-typeE3s, i.e. Siah-1, Parkin, and Dorfin, have been found to targetsynphilin-1 for ubiquitination and degradation.

The functional similarity among Siah-1, Parkin, and Dorfinraises important questions regarding redundancy and physio-logical significance of multiple pathways facilitating synphi-lin-1 degradation. Parkin and Dorfin bind the central portion of

synphilin-1, which contains ANK repeats, a coiled-coil domain,and an ATP/GTP-binding site. Nonetheless, Siah-1 was notfound to bind to that portion. As mentioned above, a specificpeptide motif that mediates interaction of each E3 with a rangeof substrate proteins has been elucidated. Accordingly, it re-mains possible that if a single protein contains multiple peptidemotifs for different E3s, such a protein could be ubiquitinatedby multiple E3s, leading to degradation by multiple, synergisticmechanisms. It appears uncertain whether three proteins cansimultaneously bind synphilin-1, but it remains possible thatSiah-1 might synergistically ubiquitinate synphilin-1 with Par-kin or Dorfin.

On the other hand, synphilin-1 is initially distributed in thecell bodies of immature neurons, subsequently becoming redis-tributed toward presynaptic nerve terminals during develop-ment after birth (51). It remains possible that Siah-1, Parkin,and Dorfin independently act as E3s for synphilin-1, dependingon the distribution or developmental stage, despite concurrent

FIG. 8. Effects of Siah expression on high K�-induced dopamine release. A, a PC12-Tet-Siah cell line, which exhibits a Tet-repressibleSiah-1 expression system, was grown in the presence of 0, 50, or 500 ng/ml of Tet for 24 h. PC12-Tet-�C cell line and PC12-Tet cell line (Cont andCont-T) were grown in the presence of 0 or 500 ng/ml of Tet. Anti-Myc WB demonstrated that Siah-1 or Siah�C protein expression is very low at500 ng/ml of Tet (Siah� or �C�), moderate at 50 ng/ml of Tet (Siah�), and high in the absence of Tet (Siah�� or �C��) (left). The totalintracellular dopamine, DOPAC, and HVA contents were measured by HPLC and normalized for total cell number (right). Data are presented asmeans � S.D. from four independent experiments. Statistical analysis was performed by one-way analysis of variance. *, p � 0.05. B, cells weretreated with either high K� solution (H) or low K� solution (L). Extracellular dopamine levels were measured at the indicated time points by HPLCand either normalized for total cell number (left) or calculated as a percentage of the total intracellular dopamine content (% dopamine release)(right). C, the total intracellular dopamine and % dopamine release at 1 min in the presence and absence of Tet (500 ng/ml) in other monoclonalcell lines (PS1–4 and PDC1–4). Data are presented as means from four independent experiments in each cell line.

Siah-mediated Ubiquitination of Synphilin-151512

by guest on May 12, 2020

http://ww

w.jbc.org/

Dow

nloaded from

expression of all three E3s in the adult brain. Future studieswill need to elucidate the reason underlying synphilin-1 ubiq-uitination by multiple E3s.

A previous study demonstrated that synphilin-1 stronglyassociated with synaptic vesicles; synphilin-1 was found to belocated close to synaptic vesicles using electron microscopy(51). The present study demonstrated that Siah-1 is distributedin the central nervous system, and recent papers have shownthat Siah-1 binds to group 1 metabotropic glutamate receptors(mGluR1 and mGluR5), which are involved in the regulation ofsynaptic transmission and regulates mGluR-mediated signal-ing (38). In addition, Siah-1 attenuates mGluR-mediated cal-cium current modulation at the synaptic terminal (49). Fur-thermore, it was demonstrated both that Siah-1 binds to

synaptophysin and that endogenous Siah-1 is localized on syn-aptic-like microvesicles in PC12 cells (12). Such findings sug-gest that Siah-1 might play a role in neurotransmitter releaseby facilitating ubiquitination of synaptic vesicle proteins, toinclude synphilin-1. In the present work, Siah-1 and synphi-lin-1 reduced intracellular dopamine content without increas-ing dopamine metabolites, suggesting that both proteins mightinhibit dopamine biosynthesis. Siah-1 did not affect high K�-induced dopamine release from PC12 cells. Synphilin-1 wasfound to moderately inhibit high K�-induced dopamine releasefrom cells, whereas coexpression of Siah-1 abrogated the inhib-itory effect of synphilin-1 on dopamine release. Such a resultsuggests that association of Siah-1 with synphilin-1 might beinvolved in the regulation of dopamine release. It must be said

FIG. 9. Synphilin-1-mediated inhi-bition of dopamine release was abro-gated by Siah-1. A, PC12-Tet, PC12-Tet-Siah, or PC12-Tet-�C cells weretransfected with empty expression vec-tors (Mock) or expression vectors for ei-ther HA-synphilin-1 (synph) or HA-�-synuclein (�S). Transfected cells weregrown in either the presence or absence of500 ng/ml of Tet to inhibit (Siah� andSiah�C�) or induce (Siah�� andSiah�C��) Siah-1 expression, respec-tively. The total intracellular dopaminecontents were measured by HPLC andnormalized for total cell number. B and C,cells were treated with high K� solutionfor 1 min. Extracellular dopamine levelswere measured by HPLC and either nor-malized for total cell number (B) or calcu-lated as a percentage of the total intracel-lular dopamine contents that were shownin A (C). Siah-1, Siah�C, synphilin-1, and�-synuclein expression was confirmed byWB with anti-Myc and anti-HA antibod-ies (C). Data are presented as means �S.D. from four independent experiments.Statistical analysis was performed byone-way analysis of variance. *, p � 0.05.D, dopamine release assays were per-formed using other monoclonal cell lines.The table presents the results of % do-pamine release at 1 min from multiplemonoclonal cell lines in the presence of 0or 500 ng/ml of Tet. Data are presented asmeans from four independent experi-ments in each cell lines. Statistical anal-ysis was performed by one-way analysisof variance. *, p � 0.05.

Siah-mediated Ubiquitination of Synphilin-1 51513

by guest on May 12, 2020

http://ww

w.jbc.org/

Dow

nloaded from

that the effects of Siah-1 and synphilin-1 on dopamine releaseis relatively small. Because multiple proteins close to synapticvesicles are involved in neurotransmitter release, our observa-tion might only represent one aspect of regulation of dopaminerelease. Further investigation should be performed to clarifythe role of Siah-1 and synphilin-1 in neurotransmission.

Recently, Siah-1 has been shown to form an SCF-type com-plex with Skp1, Ebi, SIP, and APC, to facilitate �-catenindegradation in a p53-dependent manner (35, 48, 52). A recentreport demonstrated that Parkin also functions in a multipleE3 complex that includes the F-box/WD repeat protein hSel-10and Cullin-1 (53). In the present study, Siah-1 was found todirectly bind to synphilin-1, but it remains possible that SCFbox-type E3s containing Siah-1 or Parkin also facilitate syn-philin-1 ubiquitination and affect synaptic transmission. It isbecoming increasingly clear that the ubiquitin-proteasomepathway is involved in synaptic function and neurodegenera-tion. Nonetheless, it is not understood how alteration of syn-philin-1 expression levels by multiple E3s relates to synapticfunction and PD pathogenesis. We are currently attempting toproduce Siah knockout mice, which should prove very useful forenhancement of the understanding of both Siah protein func-tion and the mechanisms by which Siah regulates synapticfunction through the ubiquitin-proteasome pathway.

Acknowledgments—We thank Y. Furuno for technical assistance andC. J. Hurt for manuscript corrections.

REFERENCES

1. Forno, L. S. (1996) J. Neuropathol. Exp. Neurol. 55, 259–2722. Pollanen, M. S., Dickson, D. W., and Bergeron, C. (1993) J. Neuropathol. Exp.

Neurol. 52, 183–1913. Galvin, J. E., Lee, V. M., Schmidt, M. L., Tu, P. H., Iwatsubo, T., and Tro-

janowski, J. Q. (1999) Adv. Neurol. 80, 313–3244. Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R.,

and Goedert, M. (1997) Nature 388, 839–8405. Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M., and Goedert, M.

(1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6469–64736. Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra,

A., Pike, B., Root, H., Rubenstein, J., Boyer, R., Stenroos, E. S., Chan-drasekharappa, S., Athanassiadou, A., Papapetropoulos, T., Johnson,W. G., Lazzarini, A. M., Duvoisin, R. C., Di Iorio, G., Golbe, L. I., andNussbaum, R. L. (1997) Science 276, 2045–2047

7. Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Pr-zuntek, H., Epplen, J. T., Schols, L., and Riess, O. (1998) Nat. Genet. 18,106–108

8. Takahashi, T., Yamashita, H., Nakamura, T., Nagano, Y., and Nakamura, S.(2002) Brain Res. 938, 73–80

9. Abeliovich, A., Schmitz, Y., Farinas, I., Choi-Lundberg, D., Ho, W. H., Castillo,P. E., Shinsky, N., Verdugo, J. M., Armanini, M., Ryan, A., Hynes, M.,Phillips, H., Sulzer, D., and Rosenthal, A. (2000) Neuron 25, 239–252

10. Perez, R. G., Waymire, J. C., Lin, E., Liu, J. J., Guo, F., and Zigmond, M. J.(2002) J. Neurosci. 22, 3090–3099

11. Engelender, S., Kaminsky, Z., Guo, X., Sharp, A. H., Amaravi, R. K., Kleider-lein, J. J., Margolis, R. L., Troncoso, J. C., Lanahan, A. A., Worley, P. F.,Dawson, V. L., Dawson, T. M., and Ross, C. A. (1999) Nat. Genet. 22,110–114

12. Wheeler, T. C., Chin, L. S., Li, Y., Roudabush, F. L., and Li, L. (2002) J. Biol.Chem. 277, 10273–10282

13. Wakabayashi, K., Engelender, S., Yoshimoto, M., Tsuji, S., Ross, C. A., andTakahashi, H. (2000) Ann. Neurol. 47, 521–523

14. McLean, P. J., Kawamata, H., and Hyman, B. T. (2001) Neuroscience 104,901–912

15. Marx, F. P., Holzmann, C., Strauss, K. M., Li, L., Eberhardt, O., Gerhardt, E.,Cookson, M. R., Hernandez, D., Farrer, M. J., Kachergus, J., Engelender, S.,Ross, C. A., Berger, K., Schols, L., Schulz, J. B., Riess, O., and Kruger, R.

(2003) Hum. Mol. Genet. 12, 1223–123116. Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura, Y.,

Minoshima, S., Yokochi, M., Mizuno, Y., and Shimizu, N. (1998) Nature 392,605–608

17. Shimura, H., Hattori, N., Kubo, S., Mizuno, Y., Asakawa, S., Minoshima, S.,Shimizu, N., Iwai, K., Chiba, T., Tanaka, K., and Suzuki, T. (2000) Nat.Genet. 25, 302–305

18. Imai, Y., Soda, M., and Takahashi, R. (2000) J. Biol. Chem. 275, 35661–3566419. Imai, Y., Soda, M., Inoue, H., Hattori, N., Mizuno, Y., and Takahashi, R. (2001)

Cell 105, 891–90220. Chung, K. K., Zhang, Y., Lim, K. L., Tanaka, Y., Huang, H., Gao, J., Ross,

C. A., Dawson, V. L., and Dawson, T. M. (2001) Nat. Med. 7, 1144–115021. Ito, T., Niwa, J. I., Hishikawa, N., Ishigaki, S., Doyu, M., and Sobue, G. (2003)

J. Biol. Chem.22. Carthew, R. W., and Rubin, G. M. (1990) Cell 63, 561–57723. Dickson, B., Sprenger, F., Morrison, D., and Hafen, E. (1992) Nature 360,

600–60324. Fortini, M. E., Simon, M. A., and Rubin, G. M. (1992) Nature 355, 559–56125. Neufeld, T. P., Tang, A. H., and Rubin, G. M. (1998) Genetics 148, 277–28626. Tang, A. H., Neufeld, T. P., Kwan, E., and Rubin, G. M. (1997) Cell 90,

459–46727. Holloway, A. J., Della, N. G., Fletcher, C. F., Largespada, D. A., Copeland,

N. G., Jenkins, N. A., and Bowtell, D. D. (1997) Genomics 41, 160–16828. Hu, G., Chung, Y. L., Glover, T., Valentine, V., Look, A. T., and Fearon, E. R.

(1997) Genomics 46, 103–11129. Hu, G., Zhang, S., Vidal, M., Baer, J. L., Xu, T., and Fearon, E. R. (1997) Genes

Dev. 11, 2701–271430. Zhang, J., Guenther, M. G., Carthew, R. W., and Lazar, M. A. (1998) Genes

Dev. 12, 1775–178031. Tanikawa, J., Ichikawa-Iwata, E., Kanei-Ishii, C., Nakai, A., Matsuzawa, S.,

Reed, J. C., and Ishii, S. (2000) J. Biol. Chem. 275, 15578–1558532. Boehm, J., He, Y., Greiner, A., Staudt, L., and Wirth, T. (2001) EMBO J. 20,

4153–416233. Tiedt, R., Bartholdy, B. A., Matthias, G., Newell, J. W., and Matthias, P. (2001)

EMBO J. 20, 4143–415234. Relaix, F., Wei, X., Li, W., Pan, J., Lin, Y., Bowtell, D. D., Sassoon, D. A., and

Wu, X. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2105–211035. Liu, J., Stevens, J., Rote, C. A., Yost, H. J., Hu, Y., Neufeld, K. L., White, R. L.,

and Matsunami, N. (2001) Mol. Cell 7, 927–93636. Germani, A., Bruzzoni-Giovanelli, H., Fellous, A., Gisselbrecht, S., Varin-

Blank, N., and Calvo, F. (2000) Oncogene 19, 5997–600637. Susini, L., Passer, B. J., Amzallag-Elbaz, N., Juven-Gershon, T., Prieur, S.,

Privat, N., Tuynder, M., Gendron, M. C., Israel, A., Amson, R., Oren, M.,and Telerman, A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 15067–15072

38. Ishikawa, K., Nash, S. R., Nishimune, A., Neki, A., Kaneko, S., and Nakanishi,S. (1999) Genes Cells 4, 381–390

39. Nagano, Y., Yamashita, H., Nakamura, T., Takahashi, T., Kondo, E., andNakamura, S. (2001) Neurosci. Lett. 316, 103–107

40. Nakamura, T., Yamashita, H., Nagano, Y., Takahashi, T., Avraham, S., Avra-ham, H., Matsumoto, M., and Nakamura, S. (2002) FEBS Lett. 521,190–194

41. Nakamura, T., Yamashita, H., Takahashi, T., and Nakamura, S. (2001) Bio-chem. Biophys. Res. Commun. 280, 1085–1092

42. Paolini, R., Molfetta, R., Piccoli, M., Frati, L., and Santoni, A. (2001) Proc.Natl. Acad. Sci. U. S. A. 98, 9611–9616

43. Nag, A., Bondar, T., Shiv, S., and Raychaudhuri, P. (2001) Mol. Cell. Biol. 21,6738–6747

44. Chin, L. S., Vavalle, J. P., and Li, L. (2002) J. Biol. Chem. 277, 35071–3507945. Takahashi, T., Yamashita, H., Zhang, Y. X., and Nakamura, S. (1996) Brain

Res. Bull. 41, 363–36746. Hu, G., and Fearon, E. R. (1999) Mol. Cell. Biol. 19, 724–73247. Kitagawa, M., Hatakeyama, S., Shirane, M., Matsumoto, M., Ishida, N., Hat-

tori, K., Nakamichi, I., Kikuchi, A., and Nakayama, K. (1999) EMBO J. 18,2401–2410

48. Matsuzawa, S. I., and Reed, J. C. (2001) Mol. Cell 7, 915–92649. Kammermeier, P. J., and Ikeda, S. R. (2001) BMC Neurosci. 2, 1550. House, C. M., Frew, I. J., Huang, H. L., Wiche, G., Traficante, N., Nice, E.,

Catimel, B., and Bowtell, D. D. (2003) Proc. Natl. Acad. Sci. U. S. A. 100,3101–3106

51. Ribeiro, C. S., Carneiro, K., Ross, C. A., Menezes, J. R., and Engelender, S.(2002) J. Biol. Chem. 277, 23927–23933

52. Matsuzawa, S., Li, C., Ni, C. Z., Takayama, S., Reed, J. C., and Ely, K. R.(2003) J. Biol. Chem. 278, 1837–1840

53. Staropoli, J. F., McDermott, C., Martinat, C., Schulman, B., Demireva, E., andAbeliovich, A. (2003) Neuron 37, 735–749

Siah-mediated Ubiquitination of Synphilin-151514

by guest on May 12, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Masayasu MatsumotoNakamura, Eizo Iseki, Nobutaka Hattori, Yoshikuni Mizuno, Akira Kikuchi and

Yoshito Nagano, Hiroshi Yamashita, Tetsuya Takahashi, Shosei Kishida, TakeshiSiah-1 Facilitates Ubiquitination and Degradation of Synphilin-1

doi: 10.1074/jbc.M306347200 originally published online September 23, 20032003, 278:51504-51514.J. Biol. Chem. 

  10.1074/jbc.M306347200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/278/51/51504.full.html#ref-list-1

This article cites 52 references, 21 of which can be accessed free at

by guest on May 12, 2020

http://ww

w.jbc.org/

Dow

nloaded from