Effect of Ser-129 Phosphorylation on Interaction of�-Synuclein with Synaptic and Cellular Membranes*□S

Received for publication, April 20, 2011, and in revised form, August 11, 2011 Published, JBC Papers in Press, August 17, 2011, DOI 10.1074/jbc.M111.253450

Naomi P. Visanji‡1,2, Sabine Wislet-Gendebien§1,3, Loren W. Oschipok¶, Gang Zhang‡, Isabelle Aubert�,Paul E. Fraser‡**, and Anurag Tandon‡ ‡‡4

From the ‡Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario M5S 3H2, Canada, §GIGANeurosciences, University of Liege, 1 Avenue de l’Hopital, 4000 Liege, Belgium, the ¶Brain Research Centre, University of BritishColumbia, British Columbia V6T 2B5, Canada, the �Sunnybrook Research Institute and Department of Laboratory Medicine andPathobiology, University of Toronto, Toronto, Ontario M4N 3M5, Canada, and the Departments of **Medical Biophysics and‡‡Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Background: The majority of �-synuclein is phosphorylated at serine 129 in Lewy bodies.Results: The membrane association of PD-linked mutant �-synuclein, but not wild-type �-synuclein, was increased by serine129 phosphorylation.Conclusion: Pathological serine 129 phosphorylation regulates membrane accumulation of mutant �-synuclein.Significance:The relationship of serine 129 phosphorylation to pathogenic aggregation of normal andmutant�-synucleinmaybe governed by distinct effects on phosphoprotein membrane accumulation.

In the healthy brain, less than 5% of �-synuclein (�-syn) isphosphorylated at serine 129 (Ser(P)-129). However, withinParkinson disease (PD) Lewy bodies, 89% of�-syn is Ser(P)-129.The effects of Ser(P)-129 modification on �-syn distributionand solubility are poorly understood. As �-syn normally existsin both membrane-bound and cytosolic compartments, weexamined the binding and dissociation of Ser(P)-129 �-syn andanalyzed the effects of manipulating Ser(P)-129 levels on �-synmembrane interactions using synaptosomal membranes andneural precursor cells from �-syn-deficient mice or transgenicmice expressing human �-syn. We first evaluated the recoveryof the Ser(P)-129 epitope following either �-syn membranebinding or dissociation. We demonstrate a rapid turnover ofSer(P)-129 during both binding to and dissociation from synap-tic membranes. Although the membrane binding of WT �-synwas insensitive to modulation of Ser(P)-129 levels by multiplestrategies (the use of phosphomimic S129D and nonphosphory-lated S129A �-syn mutants; by enzymatic dephosphorylation ofSer(P)-129 or proteasome inhibitor-induced elevation in Ser(P)-129; or by inhibitionor stable overexpressionofPLK2), PDmutantSer(P)-129 �-syn showed a preferential membrane associationcomparedwithWTSer(P)-129�-syn. Collectively, these data sug-gest that phosphorylation at Ser-129 is dynamic and that the sub-cellular distribution of �-syn bearing PD-linked mutations, A30Por A53T, is influenced by the phosphorylation state of Ser-129.

Parkinson disease (PD)5 is a progressive neurodegenerativedisorder characterized by a loss of dopaminergic neurons in thesubstantia nigra pars compacta and depletion of dopamine inthe striatum. The major pathological hallmark in affectedbrains is the presence of �-synuclein (�-syn)-positive Lewybodies (LBs) in surviving neurons. Normally, �-syn is an abun-dant presynaptic protein thought to play a role in synaptic plas-ticity (1, 2).However,multiplication of the gene encoding�-synor missense mutations (A53T, A30P, or E46K) are known tocause inherited forms of PD (3). In vitro studies suggest that�-syn is natively unfolded in aqueous solution and that expo-sure to lipids stabilizes the amino terminus in an amphipathic�-helix that aligns polar and nonpolar residues into opposingorientations (4–6). This secondary structure confers the lipid-binding properties for direct membrane interaction such thatpurified recombinant�-syn canbind to small diameter artificialvesicles rich in acidic phospholipids (7, 8), to purified synapticvesicles and membranes (9–11), and to membranes withinintact cells (12).

�-syn also has the propensity to aggregate and can form amy-loid-like fibrils, which are the main component of LBs (13).Increasing evidence suggests that phosphorylation may be animportant regulator of �-syn oligomerization, fibrillogenesis,LB formation, and neurotoxicity in vivo (14). Immunohisto-chemical and biochemical studies suggest that the majority of�-syn within LBs from patients with PD and related synucle-inopathies is phosphorylated at serine residue 129 (Ser(P)-129)(15–18). Moreover, alteration of Ser-129 to the negativelycharged amino acid aspartate, to mimic phosphorylation, sig-nificantly enhanced �-syn toxicity in Drosophila, although itstoxicity in studies with rodents has been equivocal (14, 19, 20).Several kinases, including casein kinases 1 and 2 (21–23) andmembers of the G-protein-coupled receptor kinase family (24),

* This work was supported in part by Canadian Institutes of Health ResearchOperating Grants MOP 84501 (to A. T.), FRN 93603 (to I. A.), and MOP 84527(to P. E. F.) and an Alzheimer Association grant (to I. A.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1.

1 Both authors contributed equally to this work.2 Supported by a fellowship from the Parkinson Society of Canada.3 Supported by a fellowship from the Canadian Institutes of Health Research.4 To whom correspondence should be addressed: Centre for Research in Neu-

rodegenerative Diseases, University of Toronto, 6 Queen’s Park CrescentWest, Toronto, Ontario M5S 3H2, Canada. Tel.: 416-978-8880; Fax: 416-978-1878; E-mail: a.tandon@utoronto.ca.

5 The abbreviations used are: PD, Parkinson disease; �-syn, �-synuclein;ANOVA, analysis of variance; LB, Lewy body; NPC, neural precursor cells;CIP, calf intestinal phosphatase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 41, pp. 35863–35873, October 14, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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This article has been withdrawn by Naomi Visanji, Sabine Wislet-Gendebien, Loren Oschipok, Isabelle Aubert, Paul Fraser, and Anurag Tandon. Gang Zhang was not reachable. The pS129 immunoblot in Fig. 1 was used for both membrane and cytosol fractions. In Fig. 4B, the pS129 blots from representative WT and A30P tissues are the same.

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are capable of phosphorylating �-syn at Ser-129. However,recent evidence suggests that a member of the polo-like kinasefamily, PLK2, is the prime contributor to phosphorylation of�-syn at Ser-129 (25, 26). Inhibitory RNA-induced knockdownof PLK2 in human cortical cultures or the knock-out of the plk2gene in mice reduced the levels of Ser(P)-129 �-syn by �70%,indicatingthatPLK2isresponsible for themajorityofphosphor-ylation of �-syn at Ser-129 in vivo. Nevertheless, Ser-129 phos-phorylation reduced �-syn aggregation in vitro (27), andpharmacological inhibition of kinases associated with phos-phorylation of Ser-129 failed to reduce �-syn aggregation in acellular model (28), suggesting that �-syn aggregation is eitherinhibited or is independent of Ser-129 phosphorylation.Because inclusion formation may be linked to nucleation-

elongation process initiated on membranes (29), we hypothe-sized that phosphorylation of�-syn at Ser-129may affectmem-brane binding. Therefore, we took advantage of in vitro bindingand dissociation assays (11, 30) to analyze the effect of phos-phorylation at Ser-129 on the interaction of�-synwith synapticmembranes. We then tested the effects of manipulating thelevel of Ser-129 phosphorylation, both pharmacologically andby virus-mediated expression of PLK2, on membrane bindingof �-syn in neural precursor cells prepared from transgenicmice expressing human �-syn.

EXPERIMENTAL PRECEDURES

Synaptosomal Membrane Preparation

Synaptosomal membranes were prepared as described pre-viously (31). Briefly, murine cerebral cortices were homoge-nized in ice-cold buffer A (320 mM sucrose, 1 mM EGTA, and 5mM HEPES (pH 7.4)) and centrifuged (1000 � g, 10 min). Thesupernatant was centrifuged (24,000 � g, 10 min), and theresulting pellet (P2) was resuspended in buffer A. The P2 frac-tion was loaded onto a discontinuous Ficoll gradient (13, 9, and5% in buffer A) and centrifuged (35,000 � g, 35 min). The 13 to9% interface, containing intact synaptosomes, was resuspendedin buffer B (140 mM NaCl, 5 mM KCl, 20 mM HEPES, 5 mM

NaHCO3, 1.2 mMNa2HPO4, 1 mMMgCl2, 1 mM EGTA, and 10mM glucose) and centrifuged (24,000 � g, 10 min). Synapto-somes were lysed in buffer C (10mMHEPES, 18mMKOAc (pH7.2)), centrifuged (24,000 � g, 10 min), and resuspended inbuffer D (25 mM HEPES, 125 mM KOAc, and 2.5 mM MgCl2).

Cytosol Preparation

Brains from �-syn KO mice and mice overexpressing WT,A30P, or A53T�-syn were homogenized in 85mM sucrose, 100mM KOAc, 1 mM MgOAc, and 20 mM HEPES (pH 7.4).The homogenate was centrifuged (15,000 � g, 10 min), and theresulting supernatant was centrifuged (100,000 � g, 1 h). Thesupernatant was dialyzed for 4 h in 145 mM KOAc and 25 mM

HEPES (pH 7.2). Protein concentration was determined byBCA protein assay (Pierce).

Binding/Dissociation Experiments

Synaptosomal membranes were incubated with cytosol for10min at 37 °C in the absence or presence of a serine/threoninephosphatase inhibitor mixture (phosphatase inhibitor mixture

3 purchased from Sigma) and 1 �M okadaic acid. Membraneand supernatant were separated by centrifugation (24,000 � g,10 min), and �-syn levels in the membrane and supernatantfractions were quantified by Western blotting.

Western Blotting

Proteins were separated by electrophoresis using 12% Tris-glycine polyacrylamide gels. Proteins were transferred to nitro-cellulose membranes (Life Sciences) and probed with antibod-ies against �-syn (Syn-1, BD Transduction Laboratories,1:1000), Ser(P)-129 �-syn (clone 11A5, 1:500, Elan Pharmaceu-ticals), GAPDH (Sigma 1:1000), syntaxin (Sigma, 1:1000), tetra-His (Qiagen, 1:1000), and ubiquitin (Dako, 1:1000). BoundHRP-conjugated anti-mouse or anti-rabbit IgG (Sigma) wasrevealed by chemiluminescence using ECL Plus (GE Health-care) and quantified with a Storm 860 fluorescent imager andImageQuant software (GE Healthcare).

Isolation of Mouse Whole Brain Neural Precursor Cells

Mouse whole brain neural precursor cells (NPC) were pre-pared according to themethodof Ray andGage (32). Briefly, thebrains of six mice, overexpressing human WT, A30P, or A53T�-syn under the control of the hamster prion protein promoter(see Ref. 30) were chopped into pieces (�2 mm3) before beingincubated at 37 °C in 10 volumes (g/ml) of DMEM containingpapain (2.5 units/ml), protease type 1 (1 unit/ml), and DNase I(250 units/ml). The tissue was triturated every 5 min until asmooth consistency was achieved (�30 min). Following diges-tion, the cell suspension was mixed with an equal volume ofDMEM/F-12/N2 containing 10% FBS. The suspension wasthen passed through a sterilized 15-�m nylon mesh filter andcentrifuged (1000� g for 3min). The pellet was resuspended inDMEM/F-12/N2 containing 10% FBS and mixed with an equalvolume of Percoll (9:1 v/v Percoll/PBS) and centrifuged(20,000 � g for 30 min at 18 °C). NPC were harvested from thelow buoyancy fraction (�5 ml above the red blood cell layer).Cells were washed in cold PBS containing 1� antibiotic/anti-mycotic solution (containing penicillin, streptomycin, andamphotericin B) before plating in DMEM/F-12/N2 containing1� antibiotic/antimycotic solution, �FGF (20mg/ml), EGF (20ng/ml), and heparin (5 �g/ml). Proliferating NPC began togrow in clusters�2–3weeks post-isolation. During this period,half the media were changed weekly, and �FGF (20 mg/ml),EGF (20 ng/ml), and heparin (5 mg/ml) were added every 3–4days.

Tissue Culture

SHSY5Y Cells—SHSY5Y cells (ATCC) were maintainedunder an atmosphere of air and 5% CO2 at 37 °C. The mediumconsisted of DMEM containing 4.5 g/liter glucose, 0.01 mM

sodium pyruvate, 1 �M nonessential amino acids, and 10% fetalbovine serum (Multicell). Cells were cultured in 10-cm platesand passaged at �75% confluence.Maintenance of NPC—NPC were maintained under an

atmosphere of air and 5% CO2 at 37 °C. The medium consistedof DMEM/F-12/N2 (Invitrogen) containing �FGF (20 mg/ml,Invitrogen), EGF (20 ng/ml, Invitrogen), and heparin (5mg/ml,

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Sigma). Cells were cultured in T75 flasks and passaged at�75%confluence.Drug Treatment—BI2536 (AxonMedchem, Groningen, The

Netherlands) was dissolved in PBS (10 mM) and stored at�20 °C until use. Epoxomicin (Peptides International, Louis-ville, KY) was dissolved in PBS (1 mM) and stored at �20 °Cuntil use. Both BI2536 and epoxomicin were added to cells inculture for 24 h prior to harvesting.Nucleofection—pcDNA3WT, S129A, and S129D �-syn plas-

mids were prepared as described previously (33). 4 � 106SHSY5Y cells were resuspended in 100�l of transfection buffer(Amaxa Biosystems, Gaithersburg, MD). 4 �g of DNA wasadded, and the cell/DNA mixture was transferred to 1-cmtransfection cuvettes (Amaxa Biosystems, Gaithersburg, MD).Nucleoporation was carried out using the Amaxa nucleofectorset to a predefined program (A023). Following electroporation,cells were cultured for 48 h prior to harvest.

Virus Production and Infection

Snk/PLK2 was subcloned from pcDNA3.1/V5-His Snk/PLK2 (Addgene plasmid 16015 (34)) into a pWPI Bicistroniclentiviral vector pWPI/hPLK2WT/Neo. A mutant pWPI/hPLK2K111M/Neo was created using site-directed mutagene-sis (Stratagene). Based on sequence homology, the K111MPLK2 mutation corresponds to the K82M PLKI sequence lack-ing kinase activity (35). Viral production was mediated byHEK293T cells co-transfected with 3 �g of the envelopeplasmid pMD2.G (Addgene plasmid 12259), 5 �g of packag-ing plasmid psPAX2 (Addgene plasmid 12260), and 10 �gof either pWPI/�/Neo, pWPI/hPLK2WT/Neo, or pWPI/hPLK2K111M/Neo using Lipofectamine 2000 (Invitrogen) (36,37). The lentivirus was harvested 48 h post-transfection,cleared by centrifugation (3000 rpm, 15 min) and filteredthrough 0.45-�m pore size cellulose acetate filters. NPC weredissociated and incubated with lentivirus at for 5 h. Cells werethen rinsed in DMEM/F-12/N2 and cultured in DMEM/F-12/N2 containing �FGF (20 mg/ml), EGF (20 ng/ml), and hep-arin (5 mg/ml). 48 h post-infection, the infected NPC wereselected with G418 (50 �g/ml) for 2 weeks (38, 39).

Cellular Fractionation

Cells were hypotonically lysed in 10mMHEPES/KoAc buffercontaining protease inhibitor (Sigma) and phosphatase inhibi-tor mixture (Sigma). Lysates were centrifuged (20,000 � g, 5min at 4 °C). The soluble fraction was removed and centrifuged(100,000 � g 15 min). Proteins in the remaining pellet wereextracted in 100 mMNaCl, 50 mM Tris, 1 mM EDTA, 1% TritonX-100 containing protease and phosphatase inhibitors and cen-trifuged (20,000 � g, 5 min at 4 °C) to produce the membrane-bound fraction. Protein levels were quantified byWestern blot-ting of equal volumes of the membrane and soluble fractions.

Immunofluorescence Microscopy

Cells were fixed with 4% formaldehyde for 15 min, permea-bilized with 0.3% Triton X-100, blocked in 100% HHG (1 mM

HEPES, 2%horse serum, 10%goat serum inHanks’ buffered saltsolution), and incubated with primary antibodies (11A5 1:500and anti-His 1:500) overnight at 4 °C. Cells were then incubated

with fluorescent secondary antibodies (Alexa Fluor 488 or 5551:500) for 1 h at room temperature before mounting withProLong� Gold DAPI mounting media (Invitrogen).

Statistical Analysis

Statistical comparisons were carried out using GraphPadPrizm version 4.0 forWindows. In all cases either a one-way ortwo-way ANOVA, followed by a Bonferroni post hoc multiplecomparison test, was used.

RESULTS

Total�-syn and Ser(P)-129�-syn in Synaptosomes—The dis-tribution of total �-syn and Ser(P)-129 �-syn was examined insynaptosomes prepared from transgenic mice expressinghuman (WT, A30P, or A53T) but not endogenous murine�-syn (Fig. 1). The total �-syn expression relative to GAPDHwas similar between the WT, A30P, and A53T isoforms. How-ever, total A30P Ser(P)-129 �-syn was lower than the corre-sponding levels of bothWT and A53T Ser(P)-129 �-syn. A30PSer(P)-129 �-syn was reduced in both the membrane-boundand cytosolic fractions. Thus, similar to unphosphorylated�-syn, Ser(P)-129 �-syn can also exist as both membrane-bound and cytosolic forms.Does Phosphorylation at Ser-129 Regulate Binding or Disso-

ciation of �-syn to/from Synaptic Membranes?—The effect ofSer-129 phosphorylation on �-syn membrane interaction ispoorly understood, and despite the small proportion of Ser(P)-129 �-syn in normal brains, it remains possible that Ser(P)-129represents an intermediate step for �-syn membrane associa-tionordissociation.Therefore, todeterminewhetherphosphor-ylation or dephosphorylation at Ser-129 may play a role in reg-ulating �-syn exchange between membrane and cytosoliccompartments, we measured the amount of Ser(P)-129 �-synbefore and after membrane binding or dissociation in vitro asdescribed previously (11, 30).Binding of �-syn to synaptic membranes was determined by

incubating �-syn�/� synaptic membranes with brain cytosolprepared from mice overexpressing the human form of �-syn(WT, A30P, or A53T) (11). The recovery of total �-syn andSer(P)-129 �-syn in the membrane and cytosolic fractions wascomparedwith the startingmaterial. Following incubationwithWT �-syn cytosol, 30 � 3% of total �-syn was recovered in themembrane fraction, and 67 � 2% remained in the cytosol (Fig.2A). Using cytosol prepared from A30P or A53T mice, slightlylower amounts, 10 � 4 and 12 � 5% respectively, were recov-ered onmembranes, and the corresponding 90� 2 and 88� 3%remained in the cytosol (Fig. 2, B and C). Therefore, recovered

FIGURE 1. Levels of �-syn and Ser(P)-129 �-syn in synaptosomes. Intactsynaptosomes were isolated from the brains of transgenic mice expressingthe human �-syn (WT, A30P, or A53T). The levels of total and Ser(P)-129 �-synwere visualized by Western blot in intact synaptosomes (Total), the mem-brane fraction (Membrane), and cytosolic fraction (Cytosol).

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�-syn matched the quantity of �-syn in the starting material.Surprisingly, however, the net recovery of Ser(P)-129 �-syn inthe same experiments was substantially less than the total inputamount. Only 3 � 1 and 63 � 2% of Ser(P)-129WT �-syn wererecovered in the membrane and cytosolic fractions, respec-tively (Fig. 2D), totaling 66%of initial Ser(P)-129WT�-syn, andsuggesting a loss of 34% of Ser(P)-129 WT �-syn. Similarly, netrecovery of both A30P and A53T Ser(P)-129 �-syn was alsoreduced after binding. Cytosolic Ser(P)-129 �-syn was 55� 5 and65 � 5%, respectively, and the level recovered on the membranewas only 10�3 and4�1%of the starting input amounts (Fig. 2,EandF).Thesedata suggestnet lossofphosphorylated�-synduringthe process of binding with the synaptic membrane.Using another cell-free assay tomeasure�-synmovement off

membranes (30), we then investigated changes to Ser(P)-129�-syn during dissociation from synaptic membranes preparedfrommice overexpressingWT, A30P, or A53T�-syn into cyto-sol prepared from �-syn�/� mice. As illustrated in Fig. 3A, fol-lowing incubation with membranes fromWT mice, 7 � 1% of�-syn dissociated to the cytosol and 92 � 2% remained mem-

brane-bound. A similar pattern was observed with membranesprepared frombothA30P andA53Tmice, with 10� 2 and 12�1% found in the cytosol and 88 � 2 and 88 � 2% remainingmembrane-bound, respectively (Fig. 3, B and C). Thus, follow-ing incubation with �-syn�/� cytosol, a small percentage ofWT, A30P, and A53T �-syn disassociates into the cytosol, withthe majority remaining membrane-bound. We then measuredthe disassociation of Ser(P)-129 �-syn from synaptic mem-branes using the same assay. As illustrated in Fig. 3D, followingexposure to �-syn�/� cytosol the level of membrane-boundSer(P)-129 �-syn was reduced to 52 � 4% of starting material.However, only 2� 1%ofWTSer(P)-129�-synwas found in thecytosol. Thus, �46% of Ser(P)-129 �-syn was lost during theincubation. A similar loss of Ser(P)-129 was observed with bothA30P and A53T �-syn with membrane-bound Ser(P)-129�-syn reduced to 40 � 4 and 45 � 6%, respectively, and theamount recovered in the cytosol was only 5 � 1 and 6 � 2%,respectively (Fig. 3, E and F). These data also suggest a net lossof phosphorylated �-syn during its dissociation from the syn-aptic membrane.

FIGURE 2. Ser(P)-129 �-syn binding to synaptic membranes. Cytosol obtained from the brains of transgenic mice expressing the human form of WT, A30P,or A53T �-syn was incubated with �-syn�/� synaptic membranes for 10 min at 37 °C. The percentage of membrane-bound and cytosolic �-syn (WT, A30P, andA53T, A–C, respectively) and Ser(P)-129 �-syn (WT, A30P, and A53T, D–F, respectively) relative to starting material are displayed.

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The experiments described above required the co-incuba-tion of membrane and cytosol to assess the transfer of �-synbetween fractions. To test whether the loss of the Ser(P)-129signal is due to degradation or dephosphorylation during incu-bation, we first compared the effects of incubating membraneand cytosol separately. These control experiments demon-strated that there was no loss of either cytosolic or membrane-bound Ser(P)-129�-syn during incubation. Following a 10-minincubation at 37 °C, the level ofmembrane and cytosolic Ser(P)-129 was 123 � 13 and 122 � 24% of starting material, respec-tively (p� 0.05; n� 6) (supplemental Fig. 1A). Thus, loss of theSer(P)-129 epitope occurred only during co-incubation ofmembrane and cytosol, suggesting either that componentsfrom both fractions are required for the loss of the Ser(P)-129epitope or that dephosphorylation occurred during the transferof �-syn from one fraction to another. We also assessedwhether the loss of Ser(P)-129 �-syn could be mitigated byblockade of endogenous phosphatases in the binding reactionmixture. As shown in supplemental Fig. 1B, a serine/threoninephosphatase inhibitor mixture did not affect the recovery ofSer(P)-129 �-syn on membranes or in cytosol. Therefore, wecannot rule out that some of the Ser(P)-129 �-syn may be pref-erentially degraded when membrane and cytosol fractions arecombined. Alternatively, it remains possible that the phospha-tase involved may be insensitive to the inhibitor mixture.

Does Enzymatic Dephosphorylation or Mutation of Ser-129Affect �-syn Interaction with Synaptic Membranes?—Theresults above suggest that although Ser(P)-129 �-syn levels canbe rapidly modulated, phosphorylation does not directly con-trol�-synmembrane interaction because Ser(P)-129�-syn lossoccurred during both the binding and dissociation events andSer(P)-129 �-syn was not preferentially associated with eitherthemembrane or cytosolic compartments. Therefore, we askedwhether a change in phosphorylation state may be requiredprior to binding to or dissociation from synaptic membranes.We first measured the effects of enzymatic dephosphory-

lation of �-syn prior to binding to synaptic membranes. Wepreincubated cytosol prepared from mice overexpressing WT,A30P, or A53T �-syn with calf intestinal phosphatase (CIP) todephosphorylate proteins.We then incubated this untreated orCIP-treated cytosol with �-syn-deficient membranes and com-pared the binding capacity of the phosphorylated and dephos-phorylated �-syn. As we reported previously (11), WT andA53T �-syn showed equivalent binding to synaptic mem-branes, and both were higher than A30P �-syn. However, nosignificant difference in binding capacity was evident betweeneach phosphorylated versus the dephosphorylated�-syn (Fig. 4,A and B, p � 0.05, two-way ANOVA).

Second, we assessed whether membrane distribution of�-syn mutants that mimic dephosphorylated (S129A) or phos-

FIGURE 3. Ser(P)-129 �-syn dissociation from synaptic membranes. Synaptic membranes isolated from the brains of transgenic mice expressing the humanform of WT, A30P, or A53T �-syn were incubated with �-syn�/� cytosol for 10 min at 37 °C. The percentage of membrane-bound and cytosolic �-syn (WT, A30P,and A53T, A–C, respectively) and Ser(P)-129 �-syn (WT, A30P, and A53T, D–F, respectively) relative to starting material are displayed.

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phorylated (S129D) �-syn differed in SHSY5Y cells. Cells weretransfected with WT, S129A, or S129D �-syn, and following a48-h period were fractionated, and the level of membrane-bound �-syn was assessed by Western blot (Fig. 4, C and D).The level of membrane binding was similar forWT (47� 10%),S129A (50 � 9%), and S129D (50 � 7%) �-syn (p � 0.05, one-way ANOVA). These data also support the data above that thephosphorylation state of �-syn at Ser-129 does not influencesteady-state membrane binding of �-syn.Does Manipulation of Phosphorylation at Ser-129 Affect the

Membrane Binding of Endogenous �-syn?—As an alternativeapproach,wemanipulated the Ser-129 phosphorylation state of�-syn acutely or chronically in NPC isolated from miceexpressing WT �-syn using inhibitors of either PLK2 or theproteasome or by overexpression of PLK2.Treatment with the PLK2 inhibitor BI2536 (1.0 �M) signifi-

cantly reduced the level of Ser(P)-129�-syn relative to GAPDH�6-fold from 7.3 � 2.5 to 1.2 � 0.7% (p � 0.05, one-wayANOVA, see Fig. 5A), but it had no effect on the total amount ofmembrane-bound �-syn (Fig. 5B). Conversely, exposure to theproteasome inhibitor epoxomicin (20 nM) significantlyincreased the level of Ser(P)-129 �-syn�50-fold from 1.3� 0.6to 65� 18% (p� 0.05 one-wayANOVA, see Fig. 6A) but had noeffect on the levels of membrane-bound �-syn (Fig. 6B). Inaddition, the epoxomicin-induced increase in Ser(P)-129 �-synwas completely blocked by BI2536, independently of theincreased protein ubiquitination and with no effect on mem-brane binding of �-syn (Fig. 6, A–C), indicating that the accu-mulation of Ser(P)-129 �-syn was dependent of PLK2-medi-ated phosphorylation. Following treatment with epoxomicin,Ser(P)-129 levels were elevated such that we could readilydetect membrane-bound levels of Ser(P)-129. Of the total

Ser(P)-129 �-syn, �40% was associated with the membranes(data not shown).To study the long term effects of alterations in levels of

Ser(P)-129 �-syn onmembrane binding of �-syn, we generatedNPC stably expressing His6-tagged human PLK2, which phos-phorylates �-syn only at Ser-129 (26), either asWT or a kinase-dead version bearing a K111M mutation. Expression of theepitope-tagged protein was verified WT and K111M cells byboth immunofluorescence (Fig. 7A) andWestern blotting (Fig.7C). To confirm the kinase activity of the exogenous PLK2expression, Ser(P)-129 �-syn was assessed by immunofluores-cence in control, WT, and K111M cells (Fig. 7A) and Westernblotting (Fig. 7, B and C). Compared with the empty vectorcontrols, Ser(P)-129 �-syn was elevated by WT PLK2 andreduced by K111M PLK2 (p � 0.01 and p � 0.05, respectively,one-wayANOVA).Nevertheless, despite the changes in Ser(P)-129, the proportion of membrane-bound �-syn was similar forthe empty vector control (48 � 1.4%), WT PLK2 (43 � 3.4%),and K111M PLK2 (42 � 2.7%) (p � 0.05, one-way ANOVA)(Fig. 7D). Thus, both stable increases and reductions in theextent of �-syn Ser-129 phosphorylation had no effect on themembrane binding capacity of �-syn.Differential Effect of Ser-129 Phosphorylation on the Mem-

brane Binding of PD-linked �-syn Mutants—Our data aboveindicate that manipulating Ser-129 phosphorylation has littleeffect on the binding properties of WT �-syn, suggesting thatSer(P)-129 is likely not a critical, though short lived, modifica-tion during membrane binding or dissociation. In previousstudies, we showed that both A30P and A53T �-syn have fastermembrane dissociation kinetics than WT �-syn and that bothmutants also display more transient association upon bindingto synaptic membranes (11, 30). Therefore, we asked whether

FIGURE 4. Binding of dephosphorylated or phosphomimic �-syn to synaptic membranes. A, cytosol obtained from the brains of transgenic mice express-ing the human form of WT, A30P or A53T �-syn was incubated with or without CIP for 1 h at 37 °C. The CIP-treated cytosol was then incubated with �-syn�/�

synaptic membranes for 10 min at 37 °C, and the level of membrane-bound �-syn was quantified. B, representative Western blots showing the level of total (T),cytosolic (C), and membrane-bound (M) �-syn and Ser(P)-129 �-syn are shown. C, SHSY5Y cells were transfected with either WT, S129A, or S129D �-syn, and thelevel of membrane-bound �-syn was quantified 48 h post-transfection. D, representative Western blots showing the level of membrane-bound (M) andcytosolic (C) �-syn and syntaxin in untransfected (UT) and transfected (WT, S129A, and S129D) lysates are shown.

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Ser(P)-129 might have any differential effects on the mem-brane-binding properties of either A30P or A53T �-syn.Because of the relatively low endogenous levels of Ser(P)-129�-syn, we used epoxomicin to increase acutely the levels of thephosphoprotein. Treatment of NPC expressing WT, A30P, orA53T �-syn with 20 nM epoxomicin for 24 h elevated totalSer(P)-129 �-syn levels but had no effect on total �-syn expres-sion (data not shown). Surprisingly, there was a striking differ-ence in the distribution of WT and mutant phosphoproteins(Fig. 8A). Although the proportion of Ser(P)-129�-syn that wasmembrane-bound in WT �-syn cells was 38 � 1.6%, it wassignificantly higher in A30P andA53T�-syn cells (79� 5.6 and81 � 2.3%, respectively (both p � 0.001, one-way ANOVA))(Fig. 8B). These results suggest that increased Ser-129 phos-phorylation may have differential effects on �-syn dependingon the presence of PD-linked mutations.

DISCUSSION

Phosphorylation of �-syn at Ser-129 has been observed inpost-mortem brain tissue from patients with PD, multiple sys-

tem atrophy, and neurodegeneration with brain iron accumu-lation type 1 (15). Under normal physiological conditions,�4%of �-syn is phosphorylated at Ser-129, whereas �89% of �-synis phosphorylated at this residue in LB (15, 40). However, theeffects of phosphorylation on the physiological and pathogenicproperties of �-syn in vivo remain unknown. In this study, weused twocell-freeassays tomeasurechanges to�-synphosphor-ylation during binding to or dissociation from synaptic mem-branes (11, 30). We also analyzed the effects of manipulatingSer(P)-129 �-syn levels by enzymatic, pharmacological, orgenetic approaches to assess the impact of Ser-129 phosphory-lation on �-syn membrane binding. Our data suggest that thephosphorylation state of�-syn at Ser-129 is highly dynamic, butdoes not appear to influence the binding ofWT �-syn to mem-branes. We also compared the effects of modifying Ser(P)-129levels in neural cells expressing PD-linked �-syn bearing A30Por A53T mutations. When Ser-129 phosphorylation in thesecells was increased by treatment with the proteasome inhibitorepoxomicin, the phosphorylated �-syn PD mutants showed anincreased association with membrane fractions as comparedwith WT �-syn, suggesting the possibility that the Ser(P)-129modification may differentially affect �-syn conformationdepending on mutations in the amino-terminal membranebinding domain.We initially investigated the distribution of�-syn and Ser(P)-

129 �-syn in brain synaptosomes prepared from transgenicmice expressing humanWT, A30P, or A53T �-syn but not theendogenous murine �-syn. Consistent with our previous find-ings (30), we found that the relative amount of synaptosomal�-syn in themembrane-bound and cytosolic fractions was sim-ilar for WT, A30P and A53T �-syn. Experiments in yeast havesuggested that A30P �-syn has a lower membrane bindingcapacity than WT and A53T �-syn (41, 42). However, we havepreviously shown that cytosolic factors expressed in brain,which yeast may lack, influence the membrane binding abilityof recombinant �-syn and can indeed increase the bindingcapacity of A30P �-syn (11). We also demonstrate that in syn-aptosomes, there was a lower amount of A30P Ser(P)-129�-syn, as compared with WT and A53T, in both the mem-brane-bound and cytosolic fractions. Indeed, these data sup-port those of Zabrocki et al. (29) who also suggest that in yeastreduced Ser(P)-129 A30P �-syn correlates with reduced mem-brane binding.As the binding of �-syn to membranes has been shown to be

dynamic (30, 43), we investigated changes in the phosphoryla-tion of �-syn at serine 129 during exchange between the mem-brane and cytosolic compartments. We found that followingbinding to synaptosomalmembranes, therewas a loss of Ser(P)-129 �-syn, for WT, A30P, and A53T. A relative loss of Ser(P)-129 �-syn was also observed during dissociation from syn-aptosomal membranes, suggesting that degradation ordephosphorylation of�-synmay be involved in the binding anddissociation of �-syn. Separate incubations of synaptic mem-branes or cytosol did not induce a loss of the Ser(P)-129 epitope,arguing that the loss of the phospho-epitope requires both frac-tions to mix to initiate degradation or dephosphorylation. Toshed some light on a role for potential phosphatases, we alsoincluded a serine/threonine phosphatase inhibitor mixture in

FIGURE 5. Effect of reducing Ser(P)-129 �-syn on membrane binding of�-syn in WT NPC. WT NPC were treated with BI2536 (0.01–10 mM) for 24 h.A, total Ser(P)-129 �-syn was quantified relative to GAPDH (*, p � 0.05 cf. 0 mM

BI2536). B, level of membrane-bound �-syn was quantified and expressed asa percent of membrane-bound �-syn in untreated cells. Representative West-ern blots showing the level of membrane- bound (M) and cytosolic (C) �-synand syntaxin are shown.

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the binding and dissociation assays. The phosphatase inhibitormixture failed to block the loss of the Ser(P)-129 signal, raisingthe possibility that Ser(P)-129 �-syn may undergo preferentialdegradation or that the phosphatase responsible is incom-pletely inactivated by the inhibitors.We examined the ability of enzymatically dephosphorylated

�-syn to interact with synaptic membranes. There was no sig-nificant difference in the membrane binding capacity of �-synfrom control cytosol or that containing no Ser(P)-129 �-synfollowing exposure to CIP, suggesting that phosphorylation atSer-129 does not directly affect the ability of �-syn to interactwith synaptic membranes. This is further supported by ourresults that there is no difference in the membrane bindingcapacity of WT �-syn or two Ser-129 mutants of �-syn, onewith Ser-129 replaced with alanine (S129A), to prevent phos-phorylation, and one with Ser-129 replaced with aspartate(S129D), to mimic the negative electrostatic charge of Ser(P)-129. It has previously been shown that S129A �-syn increasesaggregate formation in Drosophila (14). However, more recentstudies comparing the two Ser-129 mutants by adenoviralexpression in rat brain had differing results. One reported thatbothmutants had similar aggregation profiles and toxicity (19),and the other suggested that S129A�-syn inducedmore degen-eration than the S129D �-syn, but with no significant differ-ences in the morphology of �-syn aggregates betweenWT andS129A/D mutants (20). These latter findings are consistent

with our results that the membrane binding of S129A andS129D �-syn is similar, although controversy remains regard-ing the mechanism of toxicity of the S129A and S129D �-synmutants.Interestingly, we were unable to find evidence of expression

of PLK2 in synaptosomal preparations nor indeedwas there anyeffect of the polo-like kinase inhibitor BI2536 (44) on Ser(P)-129�-syn levels in synaptosomes (data not shown).Wehypoth-esized that the proteins present in synaptosomes may not rep-resent the full complement of proteins required for modeling�-syn phosphorylation and binding dynamics. Therefore, wemade use of NPC isolated from the brains of mice expressingWT �-syn to investigate the effects of manipulating levels ofSer(P)-129 �-syn on membrane binding. Consistent with pre-vious reports (25, 28), 1.0 �M BI2536 inhibited levels of Ser(P)-129 �-syn by�87% in cultured cells. This significant inhibitionof Ser(P)-129 �-syn levels over a 24-h period demonstrates thatthe phosphorylation of �-syn at Ser-129 is an ongoing process.However, we demonstrate that the membrane-bound �-synpool in NPC was insensitive to the loss of Ser-129 phosphory-lation by BI2536. We further investigated the role of �-synphosphorylation at Ser-129 in membrane binding by treatingNPC with a proteasome inhibitor that has been reported toincrease levels of Ser(P)-129 �-syn via stabilization of PLK2levels (28). Significant increases in Ser(P)-129 �-syn levels bythis method also had no effect on membrane binding over a

FIGURE 6. Effect of increasing the level of Ser(P)-129 �-syn on membrane binding of �-syn in WT NPC. WT �-syn expressing NPC were treated with BI2536(1 mM) and/or epoxomicin (EPOX) (20 nM) for 24 h. A, level of Ser(P)-129 �-syn relative to GAPDH was quantified (left panel; *, #, and $ � p � 0.05 ofEPOX-BI2536�, EPOX-BI2536�, and EPOX� BI2536�, respectively) and a representative Western blot (right panel). B, level of membrane-bound �-syn wasquantified and expressed as a percent of membrane-bound �-syn in untreated cells. Representative Western blots showing the level of membrane bound (M)and cytosolic (C) �-syn and syntaxin are shown. C, representative Western blot showing the total level of ubiquitinated protein.

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24-h period. Moreover, additional studies using NPC with sta-ble expression of either WT PLK2 or a K111M mutant withreduced kinase activity produced cell lines with significantlyincreased or reduced Ser(P)-129 levels, albeit without effects on�-syn membrane binding. Thus, it seems that the membranebinding capacity of �-syn is independent of either acute orchronic increases or decreases in Ser-129 phosphorylation.Although the early structures of inclusion formation are

poorly understood and there remains controversy as towhether�-syn is phosphorylated before or after aggregation (27, 28), it isnotable that themajority of aggregated�-syn inLB is phosphor-ylated at Ser-129. Therefore, we also examined the membranebinding capacity of Ser(P)-129 �-syn itself. Because existinglevels of Ser(P)-129�-synwere very low inNPC,we took advan-tage of the fact that a short term exposure to proteasome inhib-

itors induces a substantial increase in Ser(P)-129 modificationas shown in Fig. 6. Following epoxomicin treatment of NPCexpressing WT, A30P, or A53T �-syn, we were able to moreclearly compare the membrane binding of WT, A30P, andA53T Ser(P)-129 �-syn. Interestingly, membrane binding ofphosphorylated A30P and A53T �-syn was significantly higherthan for WT Ser(P)-129 �-syn. It is known that both A30P andA53Tmutant forms of�-syn have a higher propensity to aggre-gate (45, 46), and indeed these mutants have been known to bephosphorylated at Ser-129 in Lewy bodies of familial forms ofPD (18). Our finding that both A53T and A30P �-syn preferen-tially accumulate on membranes may underlie the enhancedpropensity of these familial PD-related mutants to aggregate.As phosphorylation of WT �-syn at Ser-129 appears not to

mediate its membrane binding or toxicity, per se, the following

FIGURE 7. Expression of WT and mutant PLK2 in WT NPC. WT NPC stably expressing PLK2 WT or human PLK2 K111M were cultured. A, immunocytochemistryfor tetra-His (red) and Ser(P)-129 �-syn (green) was performed in empty vector, PLK2 WT or PLK2 K111M cells (panels i–iii, respectively). B, level of Ser(P)-129�-syn relative to GAPDH was quantified. C, representative Western blots showing the total levels of tetra-His, Ser(P)-129 �-syn, and GAPDH are shown. (*, p �0.05; **, p � 0.01 cf. vector-infected cells). D, level of membrane-bound �-syn was quantified. Representative Western blots showing the level of membrane-bound (M) and cytosolic (C) �-syn and syntaxin are shown.

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questions arise. Why does this residue undergo phosphoryla-tion and dephosphorylation and why is the majority of �-syn inLB phosphorylated at Ser-129? Given that Ser-129 is quite dis-tal to the amino-terminal membrane binding region, one pos-sible role for modification of this site may be to regulate inter-actionswith binding partners on synaptic vesicles. For example,the site of binding of �-syn to vesicular synaptobrevin-2 over-laps with the Ser-129 residue (47). Modulation of protein-pro-tein interactions by Ser-129 phosphorylation could therebyimpact vesicle mobilization without a direct effect on �-synmembrane binding. With respect to pathological Ser(P)-129�-syn accumulation, one hypothesis is that �-synmay be phos-phorylated after aggregation as it has a higher affinity forkinases and a lower affinity for phosphatases in this alteredconformation (28). Furthermore, studies in transgenic miceand cultured cells suggest that cellular toxicity, including pro-teasomal dysfunction, increases both casein kinase 2 activityand PLK2 leading to enhanced phosphorylation of�-syn at Ser-129 (23, 25).In conclusion, our data demonstrate that phosphorylation of

�-syn at Ser-129 is a continuous cellular process. Althoughblocking �-syn phosphorylation at Ser-129 or enhancingSer(P)-129 levels, by enzymatic, pharmacological, and molecu-lar biological means, had little effect on the membrane bindingof WT �-syn under either acute or chronic conditions, weobtain contrasting results with the pathogenic �-syn mutants,indicating thatmembrane association of A30P andA53T �-synwas differentially up-regulated by Ser-129 phosphorylation.This work raises the possibility that the relationship of Ser-129

phosphorylation to the pathogenic aggregation of WT andmutant �-syn may be governed by distinct effects on phospho-protein accumulation on intracellular membranes.

Acknowledgment—We are grateful to John P. Anderson (Elan Phar-maceuticals) for the generous provision of antibodies recognizingSer(P)-129 �-syn.

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Aubert, Paul E. Fraser and Anurag TandonNaomi P. Visanji, Sabine Wislet-Gendebien, Loren W. Oschipok, Gang Zhang, Isabelle

Cellular Membranes-Synuclein with Synaptic andαEffect of Ser-129 Phosphorylation on Interaction of

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VOLUME 286 (2011) PAGES 35863–35871DOI 10.1074/jbc.W120.015778

Withdrawal: Effect of Ser-129 phosphorylation oninteraction of a-synuclein with synaptic and cellularmembranes.Naomi P. Visanji, Sabine Wislet-Gendebien, Loren W. Oschipok, Gang Zhang,Isabelle Aubert, Paul E. Fraser, and Anurag Tandon

This article has been withdrawn by Naomi Visanji, Sabine Wislet-Gendebien, Loren Oschipok, Isabelle Aubert, Paul Fraser, and AnuragTandon. Gang Zhang was not reachable. The pS129 immunoblot inFig. 1 was used for both membrane and cytosol fractions. In Fig. 4B,the pS129 blots from representative WT and A30P tissues are thesame.

J. Biol. Chem. (2020) 295(39) 13695–13695 13695© 2020 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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