Phosphorylation of Synucleins by Members of the Polo-like Kina Family*□S
Received for publication,November3,2009Published,JBC Papers in Press,November4,2009,DOI10.1074/jbc.M109.081950
Martial K.Mbefo‡1,Katerina E.Paleologou‡1,Ahmed Boucharaba§,Abid Oueslati‡,Heinrich Schell¶,
Margot Fournier‡,Diana Olschewski‡,Guowei Yinʈ,Markus Zweckstetterʈ‡‡2,Eliezer Masliah**,Philipp J.Kahle¶3, Harald Hirling§,and Hilal A.Lashuel‡4
From the‡Laboratory of Molecular Neurobiology and Neuroproteomics and the§Laboratory of Cellular Neurobiology,Brain Mind Institute,Ecole Polytechnique Federale de Lausanne,CH-1015Lausanne,Switzerland,the¶Laboratory for Functional Neurogenetics,Department of Neurodegeneration,Hertie Institute for Clinical Brain Rearch,University Clinics Tu¨bingen,
D-72076Tu¨bingen,Germany,theʈDepartment of NMR-bad Structural Biology,Max Planck Institute for Biophysical Chemistry, 37077Go¨ttingen,Germany,the‡‡Deutsche Forschungsgemeinschaft Rearch Center for the Molecular Physiology of the Brain
D-37073Go¨ttingen,Germany,and the**Department of Neurosciences,University of California,San Di
ego,La Jolla,California
Phosphorylation of␣-synuclein(␣-syn)at Ser-129is a hallmark of Parkinson dia and related synucleinopathies.However,the identity of the natural kinas and phosphatas responsible for regulating␣-syn phosphorylation remain unknown.Here we dem-onstrate that three cloly related members of the human Polo-like kina(PLK)family(PLK1,PLK2,and PLK3)phosphorylate␣-syn and-syn specifically at Ser-129and Ser-118,respectively.Unlike other kinas reported to partially phosphorylate␣-syn at Ser-129 in vitro,phosphorylation by PLK2and PLK3is quantitative(>95% conversion).Only PLK1and PLK3phosphorylate-syn at Ser-118, whereas no phosphorylation of␥-syn was detected by any of the four PLKs(PLK1to-4).PLK-mediated phosphorylation was greatly reduced in an isolated C-terminal fragment(residues103–140)of␣-syn,suggesting substrate recognition via the N-terminal repeats and/or the non-amyloid component domain of␣-syn.PLKs specifically co-localized with phosphorylated Ser-129(Ser(P)-129)␣-syn in various subcellular compartments(cytoplasm,nucleus,and membranes)of mammalian cell lines and primary neurons as well as in␣-syn transgenic mice,especially cortical brain areas involved in synaptic plasticity.Furthermore,we report that the levels of PLK2are significantly incread in brains of Alzheimer dia and Lewy body dia patients.Taken together,the results provide biochemica
l and in vivo evidence of␣-syn and-syn phosphorylation by specific PLKs.Our results suggest a need for further studies to elucidate the potential role of PLK-syn interactions in the normal biology of the proteins as well as their involvement in the pathogenesis of Parkinson dia and other synucleinopathies.
Increasing evidence suggests that phosphorylation may play an important role in the oligomerization and fibrillogenesis(1),Lewy body formation(1,2)and neurotoxicity of␣-synuclein(␣-syn)5in vivo(3).The majority of␣-syn within Lewy bodies(LBs)in dis-ead human brains and animal models of Parkinson dia(PD) and related synucleinopathies is phosphorylated at Ser-129 (Ser(P)-129)(1,2,4–7).Although recent studies support the notion that phosphorylation at Ser-129is related to pathology and blocks␣-syn fibrillization in vitro(8,9),the exact mechanisms by which phosphorylation at Ser-129modulates␣-syn aggregation and toxicity in vivo remain elusive.Unraveling the role of phos-phorylation in modulating the physiological and pathogenic activ-ities of␣-syn requires identification of the kinas and phospha-tas involved in regulating its phosphorylation in vivo. Several kinas that phosphorylate␣-syn at rine and tyro-sine residues,primarily in its C-terminal region,have been identified using in vitro kina assays and co-transfection stud-ies.Cain kina I and II,G-protein-coupled receptor kinas (GRK1,GRK2,GRK5,and GRK6),and calmodulin-dependent kina II(10–1
2)phosphorylate␣-syn at Ser-129.Ser-87is the only residue outside the C-terminal region reported to undergo phosphorylation by cain kina I(12)and the dual specificity tyrosine-regulated kina1A(13).Tyrosine phosphorylation has also been reported at Tyr-125by Fyn(14),Syk(15),Lyn (15),c-Frg(15),and Src(16)tyrosine kinas,with Syk(15)also phosphorylating at Tyr-133and Tyr-136.
The Polo-like kinas(PLKs)compri a family of conrved Ser/Thr protein kinas that play key roles in cell cycle regula-tion,cellular respon to stress,and carcinogenesis.In mam-malian cells,the PLK family consists of three cloly related kinas PLK1,PLK2/Snk(rum-inducible kina),PLK3/Fnk (fibroblast growth factor-inducible kina)also designated Prk (proliferation-related kina),and a distant member PLK4/Sak (Snk akin kina)(17–21).The four PLKs share a conrved quence motif characterized by two regions:a highly con-rved N-terminal rine/threonine catalytic domain and a
*This work was supported by the Swiss Federal Institute of Technology Lau-sanne and grants to the Laboratory of Molecular Neurobiology and Neu-roproteomics from the Swiss National Science Foundation(Grant310000-110027)and the Michael J.Fox Foundation.
□S The on-line version of this article(available at www.jbc)contains
supplemental Figs.1–3.
1Both of the authors contributed equally to this work.
2Supported by German Science Foundation Grant Foundation Grants ZW 71/2-1and3-1.
3Supported by the Helmholtz Alliance“Aging Brain”and the Hertie Foundat. 4To whom correspondence should be addresd.Tel.:41-21-69-39691;Fax: 41-21-693-96-65;E-mail:hilal.lashuel@epfl.ch.5The abbreviations ud are:␣-,-,and␥-syn,␣-,-,and␥-synuclein,respec-tively;LB,Lewy body;PD,Parkinson dia;PLK,Polo-like kina;PBD,
Polo box domain;AD,Alzheimer dia;LBD,Lewy body dia;WT,wild老是焦虑怎么办
type;MALDI,matrix-assisted lar desorption ionization;TOF,time-of-
flight;HSQC,heteronuclear single quantum coherence;siRNA,small inter-
炸胡萝卜丸子
ference RNA;MOPS,3-(N-morpholino)propanesulfonic acid;TBS,Tris-buff-
ered saline;NAC,non-amyloid component.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.285,NO.4,pp.2807–2822,January22,2010©2010by The American Society for Biochemistry and Molecular Biology,Inc.Printed in the U.S.A.
at CHINA MEDICAL UNIVERSITY LIBRARY, on March 30, 2013www.jbc Downloaded from
www.jbc/content/suppl/2009/11/04/M109.081950.DC1.html Supplemental Material can be found at:
C-terminal non-catalytic termed the Polo box domain (PBD)(Fig.1)(22,23).The PBD plays important roles in regulating substrate interactions,targeting,subcellular localization,and autoinhibition of the PLKs (24).The PBD of PLK1,PLK2,and PLK3contain two tandem Polo boxes (ϳ80residues in length)that associate to form a phosphopeptide binding site,whereas PLK4posss only a single PB,which mediates the dimeriza-tion of PLK4,resulting in a structure that rembles the PBD of the other PLKs (24–26).
Recent reports demonstrate that ␣-syn is phosphorylated by PLK2(27,28).In this report,we confirm the find-ings and demonstrate for the first time,using in vitro kina assays,co-transfection,and small interference RNA
(siRNA)-mediated knockdown of PLKs,that ␣-and -,but not ␥-syn are phosphorylated by specific members of the PLK family.PLK phosphorylation of synucleins occurs specifically at Ser-129in ␣-syn and Ser-118in -syn and appears to be mediated by specific interactions between the PLKs and the N-terminal region (residues 1–95)of syn.The findings were validated by colocalization of ␣-syn and PLKs in different subcellular compartments and co-transfection studies as well as siRNA-mediated knockdown of PLKs in mammalian cells and primary neurons.PLK2and PLK3partly co-localized with Ser(P)-129␣-syn in primary hip-pocampal neurons as well as in cor-tical brain areas of ␣-syn transgenic mice.Furthermore,we demonstrate that the level of neuronal PLK2is elevated in the Alzheimer dia (AD)and Lewy body dia (LBD)brains and correlates with the incread levels of Ser(P)-129in the brains compared with healthy controls.Together,our findings point to PLK2and PLK3as the pri-mary PLKs responsible for ␣-syn phosphorylation and highlight the importance of further studies to elu-cidate the potential role of the inter-actions between the PLKs and ␣-syn and their implications for ␣-syn aggregation and toxicity in PD and other synucleinopathies.
罗伯罗夫斯基仓鼠EXPERIMENTAL PROCEDURES Cloning,Expression,and Purifica-tion of Proteins —pCMV6-Entry-PLK1(PLK1),pCMV6-Entry-PLK2
(PLK2),pCMV6-Entry-PLK3(PLK3)and pCMV6-Entry-PLK4(PLK4)open reading frame clones of hu-
man PLKs were from OriGENE and provided as Myc/DDK-tagged plasmids.pAAV-CMV-␣-syn wild type (WT)(␣-syn)was kindly pro-vided by Patrick Aebischer’s laboratory (Brain Mind Institute,Ecole Polytechnique Federale de Lausanne).The ␣-syn deletion mutant (73–83⌬)and the /␣-syn chimera (-syn-(1–72)/␣-syn-(73–83)/-syn-(73–134))were kindly provided by Prof.Michel Goedert (Medical Rearch Council Laboratory of Molecular Biology,Cambridge,UK)(29).All constructs were confirmed by DNA quencing (Mycrosynth,Switzer-land)using the primers provided by the supplier.Recombi-nant PLK enzymes were purchad from Intvitrogen and Cell Signaling Technologies.All recombinant ␣-syn proteins and mutants ud in this study were expresd as described previously (8).
Unless otherwi specified,the following antibodies were ud in this study:anti-human PLK1(sc-5585),anti-human/rodent PLK2H-90(sc-25421),and anti-human ␣-syn (clone 211)antibodies (Santa Cruz Biotechnology,Inc.,Santa Cruz,CA).Anti-human PRK (PLK3)(BD 556518)were purchad from BD Pharmigen.Anti-Ser(P)-129␣-syn was from Wako,-actin antibody was from Abcam (Cambridge,MA),and goat anti-mou Cy3was from (GE Healthcare).
FIGURE 1.Schematic depiction of the quences of synucleins and PLK1s-4.A ,schematic depictions illus-trating the quence similarities and differences among the four PLKs and members
of the synuclein family of proteins.The four members of the PLK family share a conrved N-terminal kina domain.The PBD of PLK1,
PLK2,and PLK3contain two tandem Polo boxes (ϳ80residues in length)that associate to form a phosphopep-tide binding site,whereas PLK4posss only a single PB,which mediates the dimerization of PLK4.B ,quence alignment of the human WT ␣-,-,and ␥-syn generated by the Clustal 2.0.8multiple quence
alignment program.Ser-129in ␣-syn and Ser-118are highlighted within the square .aa ,amino acids.
Synucleins Phosphorylation by PLKs
at CHINA MEDICAL UNIVERSITY LIBRARY, on March 30, 2013汽车保养维修
www.jbc Downloaded from
Preparation of␣-Syn C-terminal Fragment—WT␣-syn in50 m M Tris,150m M sodium chloride,pH7.6,was trypsinized at a concentration of3mg/ml and a mass ratio of␣-syn/trypsin (Promega Corp.,Madison,WI)of100:1.The reaction was incu-bated at37°C for24h to generate the C-terminal fragment (residues103–140).The C-terminal fragment was then purified by size exclusion ch
romatography and using a Superdex75HR 10/30column(GE Healthcare).
In Vitro Kina Assay—WT or mutant␣-syn was phosphory-lated by PLK1to-4(Cell Signaling Technologies and Invitro-gen)at a concentration of1.44mg/ml(100M),unless other-wi stated.The phosphorylation reactions were carried out in the prence of1.09m M ATP(Sigma),1ϫreaction solution(20
m M HEPES,10m M MgCl
2,2m M DDT,pH7.4),and1g of
PLK,144g of␣-syn at30°C for15min to24h.The reactions were quenched with EDTA disodium salt(25m M final concen-tration)(Axon Lab,AG,Le Mont-Sur-Lausanne,Switzerland). The progress of the reaction was monitored by matrix-assisted lar desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)(30).
Real-time NMR Spectroscopy—NMR samples contained
ϳ0.1m M15N-labeled␣-syn in200m M HEPES,10m M MgCl
2,2
m M dithiothreitol,and1.09m M ATP,pH6.9.The real-time assay was started by the addition of kina into the NMR sam-ple,with a protein/kina ratio of100:0.5g.NMR experiments were acquired on a Bruker Avance600-MHz NMR spectrom-eter.NMR data were procesd by Bruker TOPSPIN version 2.0.To allow for efficient phosphorylation,the temperature was t to303K.To reduce the impact of signal broadening due to amide proton exchange,the temperature was lowered to288K during the measurement of1H,15N heteronuclear single quan-tum coherence(HSQC)spectra.In the ca of PLK3,the phos-phorylation reaction proceeded very rapidly at303K.There-fore,we also followed the kinetics of phosphorylation of␣-syn by PLK3at293and288K usingϳ0.075m M␣-syn(protein/ kinaϭ100:0.5g).Backbone resonance assignments of WT and phosphorylated␣-syn were obtained previously.The degree of phosphorylation at Ser-129was calculated from the intensity of the NMR signals of phosphorylated and unphos-phorylated Ser-129.Errors were estimated bad on the signal/ noi ratio in the NMR spectra.
MALDI-TOF MS—MALDI calibrants were from Sigma,tri-fluoroacetic acid was from Pierce,and sinapinic acid was from Fluka.3-l aliquots of in vitro PLK-phosphorylated␣-syn were rerved for the MALDI analysis.The samples were diluted 1:10in0.1%trifluoroacetic acid and further mixed with an equal volume of14mg/ml sinapinic acid solution in0.1%tri-fluoroacetic acid,ACN(1:1).0.5l of this sol
ution was depos-ited on the mirror-polished target and analyzed with a4700 MALDI-TOF/TOF instrument(Applied Biosystems Inc.,Fos-ter City,CA).The spectra were calibrated on the pudomo-lecular peak of cytochrome c,apomyoglobin,ubiquitin,and ␣-syn prepared with the same matrix and deposited clo to the samples.
Cell Culture and Transient Transfection—Human Embry-onic Kidney cells(HEK293T)and immortal cell lines from HeLa were grown in Dulbecco’s modified Eagle’s medium (Invitrogen AG,Bal,Switzerland)supplemented with10%fetal bovine rum and5%penicillin/streptomycin in a
humidified incubator,5%CO
2
,37°C.For HEK293T cells, transient transfection was performed on6-well plates at a cell confluence of70–80%,using the standard calcium phosphate
我爱红领巾(CaPO
4
)
transfection protocol.For HeLa cells,transient trans-fection was performed employing Lipofectamine TM2000 (Invitrogen AG,Bal,Switzerland)according to the manufac-turer’s instructions.The overall amount of plasmid ud was
4g/well.
PLK2and PLK3Small Interfering RNA(siRNA)Silencing—
ON-TARGET siRNA quence targeting human PLK2
5Ј-GGACATGGCTGTGAATCAG-3Ј(57)and ON-TARGET siRNA quence targeting human PLK35Ј-CGGCCTCAT-GCGCACATCC-3Ј(58)as well as non-targeting(ON-TARGET siControl)were purchad from Dharmacon(Lafay-ette,CO).HeLa cells were grown at a density of200,000cells/
well and transfected the day after with incread concentration
of each siRNA independently using Lipofectamine TM2000 according to the manufacturer’s instructions.Cells were treated with siRNA for48h followed by transient transfection
with␣-syn WT using Lipofectamine TM2000for an additional
24h before analysis by Western blot and immunofluorescence.
For silencing in primary neurons,hippocampal neurons were prepared from P0Sprague-Dawley rats(Charles River/Iffa Credo,L’Arbresle Cedex,France)as described previously by Steiner et al.(31)and were plated at a density of200,000cells/
35-mm dish(Falcon,BD Biosciences)in growth medium(min-imum Eagle’s medium,20m M gluco,0.5m M glutamine,100 units/ml penicillin,100g/ml streptomycin,10%hor rum). Neuron’s medium is kept,and the cells were transfected for2h
at day12with a60n M concentration of each murine PLK2and
PLK3siRNA(sc-39153and sc-39151,respectively,Santa Cruz Biotechnology,Inc.,Santa Cruz,CA)using Lipofectamine TM 2000reagent(Invitrogen)following the manufacturer’s instructions.Neurons were then reincubated in their own and previous media for72h.For biochemical analysis by Western blotting,the cells were lyd with in a buffer containing Hepes (0.02M),EGTA(0.02M),EDTA(0.02M),KCl(0.1M),dithio-threitol(0.1M),and1%Triton X-100.The lysate was centri-fuged at20,000ϫg for15min,and the supernatant was recov-
ered and analyzed immediately by Western blotting or stored immediately atϪ80°C.
Subcellular Fractionation—HEK293T cells were transiently transfected with PLK1to-3and␣-syn WT for24h.Subcellular fractionation was carried out employing the subcellular pro-teome extraction kit(Calbiochem)according to the manufac-turer’s instructions.The fractions were analyzed by Western blot,and the purity was evaluated using control antibodies pro-vided as subcellular markers of cell compartment,including cytosolic,membrane/particulate,and nucleus(anti-Hsp90,
anti-calnexin,and anti-PARP-1,respectively).
SDS-PAGE and Immunoblot—Cells were harvested24h
post-transfection,and total homogenate was prepared by lysis
in a buffer containing150m M NaCl,1m M EDTA,20m M Tris,
pH7.40,0.5%Nonidet P-40,enriched with a1:200protea inhibitor mixture(Sigma),1m M phenylmethylsulfonyl fluoride (Sigma),and1M okadaic acid(Acros).Cleared lysates were obtained by centrifugation at14,000rpm,4°C for20min.10g
Synucleins Phosphorylation by PLKs
at CHINA MEDICAL UNIVERSITY LIBRARY, on March 30, 2013www.jbc Downloaded from
of total protein diluted in loading buffer were parated on 1-mm,12%SDS-PAGE.The proteins were then transferred to nitrocellulo membrane using the iBlot TM dry blotting system (Invitrogen AG,Bal,Switzerland)for7min.The membrane was then probed overnight with the primary antibody after30 min of blocking in Odysy blocking buffer(Li-Cor Biosciences GmbH)diluted1:3in phosphate-buffered saline(Sigma).After four times washes with PBST(phosphate-buffered saline,0.01% (v/v)Tween20(Sigma)),the membrane was incubated for1h with condary antibody(goat or rabbit Alexa Fluor680IgG) protected from light at room temperature.The immunoblot was finally washed four times with PBST and three times with phosphate-buffered saline and scanned in a Li-COR scanner at a wavelength of700nm.
Subcellular Localization and Immunocytochemistry in Pri-mary Neurons—For immunocytochemistry,hippocampal rat neurons were prepared as described(32).Hippocampal neu-rons(200,000cells in a35-mm2dish)were cultured on cover-slips(12mm;VWR)coated with poly-D-lysine(BD Biosciences) and laminin mou(Invitrogen)and fixed for12min at room temperature in a freshly prepared solution(pH7.4)containing 4%paraformaldehyde and4%sucro in phosphate-buffered saline.Neurons were then washed three times in MTBS buffer (66m M NaCl,100m M Tris-HCl,pH7.4)and then incubated overnight with the first antibody in buffer A containing5%nor-mal goat
rum,5%normal hor rum,3%bovine rum albu-min,0.3%Triton X-100in MTBS for overnight at4°C.The following day,cultures were rind three times in MTBS and incubated in the buffer A containing goat anti-mou Cy3cou-pled(1:1000;Jackson ImmunoRearch,West Grove,PA)or goat anti-rabbit Alexa Fluor488coupled as condary anti-body(IF1:500;Molecular Probes Inc.)for2h at room temper-ature.After three washes in MTBS,coverslips were mounted on glass slides with4Ј,6-diamidino-2-phenylindole-Vectashield medium(Vector)and imaged with a Zeiss LSM510confocal microscope.
Immunostaining of Mou Brain Sections—(Thy1)-[A30P]␣-syn mice were sacrificed by cervical dislocation.Brains were discted and fixed in4%paraformaldehyde in phosphate buffer(pH7.4).The fixed brains were embedded in paraffin, and4-m-thick ctions were cut on a microtome.Serial c-tions were directly transferred onto SuperFrost Plus coverslips, incubated overnight at60°C,and paraffin-decorated with xylene.Rehydration was performed over a descending ethanol ries:100,95,and75%Tris-buffered saline,pH7.4(TBS).Per-
oxida activity was eliminated by treatment with1%H
2O
2
in
TBS for30min,followed by three washes in TBS.Antigen retrieval was carried out by heating the ctions up to90°C in citrate buffer(pH6.0)for30min,followed by a cooling step of 15min on ice.Blocking was done in5%goat antirum in TBS for30min at room temperature.Incubation with rabbit mono-clonal antibody against human Ser(P)-129␣-syn(1:500; Abcam)and anti-PLK2or anti-PLK3(1:50;both from Santa Cruz Biotechnology,Inc.)was performed in TBS containing2% goat antirum at4°C overnight.Subquently,the ctions were washed and incubated at room temperature with biotiny-lated goat anti-rabbit antibody followed by the avidin-biotin complex(VECTASTAIN ABC kit).For visualization,Vector SG-Blue was ud followed by nuclear staining with Nuclear
Fast Red for5min and short rinsing under tap water.Sections
were then dehydrated(ethanol75%,95%,100%,and xylene)and
mounted with Pertex.Photomicrographs were taken with an
长期以往
Axioplan2imaging microscope(Carl Zeiss)and procesd with
AxioVision version4.3imaging software.
Human Samples and Tissue Processing—Analysis of␣-syn phosphorylation was performed with post-mortem human
temporal cortex samples.The human samples were obtained
from the Alzheimer’s Dia Rearch Center at the University
of California San Diego.A total of18cas were included,of
which six were non-demented controls,six were diagnod as
AD,and six were diagnod as LBD.Cas of LBD were defined
bad on the clinical prentation of dementia and the patho-
logical findings of LBs in the locus coeruleus,substantia nigra,
or nucleus basalis of Meynert as well as in cortical and subcor-
tical regions.LBs were detected using hematoxylin/eosin anti-
褒义词四字成语ubiquitin and anti-␣-syn antibodies as recommended by the Consortium on LBD.In all cas,the brains were procesd
within8h after death.Brains were divided sagitally,and the
right hemibrain was rially ctioned and prerved atϪ70°C
for Western blot analysis.To analyze the distribution and levels
of␣-syn,PLK2,and PLK3in the brains of patients with AD and LBD,samples from the temporal cortex were homogenized and
fractioned into cytosolic and membrane fractions by ultracen-
trifugation.Approximately20g of protein/well were loaded into4–12%BisTris gels with MOPS/SDS buffer and blotted
onto polyvinylidene difluoride membranes.Blots were incu-
bated overnight with antibodies against PLK2and PLK3
(1:1000;Invitrogen),Ser(P)-129␣-syn(WAKO),and␣-syn (1:1000;Chemicon,Temecula,CA),followed by condary
antibodies tagged with horradish peroxida(1:5000;Santa
Cruz Biotechnology,Inc.).Immunoreactivity was visualized by
enhanced chemiluminescence and analyzed with a Versadoc
XL imaging apparatus(Bio-Rad).Analysis of actin levels was
ud as a loading control.
RESULTS
To determine whether␣-syn is a substrate for members of the PLK family of kinas(PLK1,PLK2,PLK3,and PLK4),we
performed in vitro kina assays using purified recombinant ␣-syn together with each of the four PLK
s,and co-transfection experiments in mammalian cells(human embryonic kidney
HEK293T,HeLa and SH-SY5Y neuroblastoma cells).
dvd影碟机PLK1to-3Phosphorylate␣-Syn Specifically at Ser-129in Vitro—To asss the phosphorylation of␣-syn by the PLKs,␣-syn(100M)was incubated with either of the four kinas at 1g of PLK,144g of␣-syn.The reactions were incubated at 30°C,and the extent of phosphorylation was monitored by MALDI-TOF mass spectrometry.Upon incubation with PLK1, PLK2,and PLK3,we obrved a shift in the molecular mass of ␣-syn by80Da(from14,462to14,542Da)for PLK1,-2,and-3, which corresponds to the addition of one phosphate group(Fig.
2).A clor examination of Fig.2A demonstrates that PLK2,
and PLK3phosphorylate␣-syn quantitatively,whereasϳ60–70%conversion was obrved for PLK1(Fig.2A).No other kina has been obrved(data not shown)or reported to phos-phorylate␣-syn with the same efficiency.The MALDI-TOF
Synucleins Phosphorylation by PLKs
at CHINA MEDICAL UNIVERSITY LIBRARY, on March 30, 2013www.jbc Downloaded from
results suggest that PLK1to -3phosphorylate ␣-syn at a single site.To determine if PLK1to -3-mediated phosphorylation occurs at Ser-129,the phosphorylation reactions were analyzed by Western blotting using an antibody against Ser(P)-129␣-syn and subjected to trypsin digestion and peptide mapping by MALDI-TOF and liquid chromatography-electrospray ioniza-tion MS/MS.␣-Syn samples incubated with the PLK1to -3showed a band at 14kDa that was detectable with both anti-␣-syn and anti-Ser(P)-129antibodies (Fig.2B ).Comparative anal-ysis of the tryptic fragments of the bands revealed that phos-phorylation of ␣-syn occurs only within peptide fragments that encompass Ser-129.Trypsin digestion resulted in a peptide with a molecular mass of 4353.0Da (M ϩH)ϩcorresponding to the monophosphorylated C-terminal fragment 103–140
containing Ser-129(calculated mass 4272.4Da (M)(NEEG-APQ EGILEDMPVDPDNEAYEMPpSEEGYQDYEPEA,where pS reprents phosphorine)(data not shown).This peptide fragment does not contain other rine or threonine residues,suggesting that phosphorylation indeed occurs at Ser-129.To further confirm the results,in vitro phosphorylation was also performed with a mutant that cannot be phosphorylated at position 129,S129A.As predicted,none of the PLKs could phosphorylate S129A mutant ␣-syn (data not shown).
Real-time Spectroscopy of the Phosphorylation Reaction —Hetereonuclear NMR spectroscopy on 1
5N-labeled protein modified by PLK allows identification of all phosphorylation sites,measures the level of integration,and yields kinetic data for the enzymatic modification of the individual sites.
Although
FIGURE 2.In vitro phosphorylation of synucleins by PLK1to -4.A ,MALDI-TOF analysis of the WT ␣-syn after phosphorylation by PLK1to -4.For PLK1to -3,there is an 80-Da increa in the molecular mass of WT ␣-syn (14,461ϩ80ϭ14,541),corresponding to one phosphorylation.B ,Western blot analysis of the same samples in A .The anti-Ser(P)-129antibody detected a band,suggesting that the phosphorylation detected by mass spectrometry is at position 129.C ,comparison of two-dimensional 1H,15N HSQC spectra of unphosphorylated WT (green )and ␣-syn phosphorylated by PLK3(red ).A dashed rectangle marks glutamine (Q )and asparagine (N )side chain resonances.D ,kinetics of in vitro phosphorylation of Ser-129in ␣-syn by PLK3(black ),PLK2(red ),and PLK1(blue )as monitored by real-time NMR spectroscopy.NMR samples contained ϳ0.1m M 15N-labeled ␣-syn in 200m M HEPES,10m M MgCl 2,2m M dithiothreitol,and 1.09m M ATP,pH 6.9.The real-time assay was started by the addition of kina into the NMR sample using a protein/kina ratio of 100:0.5mg.The error bars were determined bad on the signal/noi ratio obrved in the NMR spectra.
Synucleins Phosphorylation by PLKs
at CHINA MEDICAL UNIVERSITY LIBRARY, on March 30, 2013
www.jbc Downloaded from
