Mutant Protein Kinase C� Found in Spinocerebellar Ataxia Type 14Is Susceptible to Aggregation and Causes Cell Death*□S

Received for publication, February 15, 2005, and in revised form, June 15, 2005Published, JBC Papers in Press, June 17, 2005, DOI 10.1074/jbc.M501716200

Takahiro Seki‡, Naoko Adachi§, Yoshitaka Ono¶, Hideki Mochizuki‡�, Keiko Hiramoto‡**,Taku Amano‡, Hiroaki Matsubayashi‡, Masayasu Matsumoto‡‡, Hideshi Kawakami‡‡,Naoaki Saito§, and Norio Sakai‡§§

From the ‡Department of Molecular and Pharmacological Neuroscience, the �Department of Ophthalmology and VisualSciences, the **Department of Neurosurgery, and the ‡‡Department of Clinical Neuroscience and Therapeutics, GraduateSchool of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan and the §Laboratory of MolecularPharmacology and ¶Biosignal Research Center, Kobe University, Kobe 657-8501, Japan

Spinocerebellar ataxia type 14 (SCA14) is an autosomaldominant neurodegenerative disease characterized byvarious symptoms including cerebellar ataxia. Recently,several missense mutations in the protein kinase C�(�PKC) gene have been found in different SCA14 families.To elucidate how the mutant �PKC causes SCA14, weexamined the molecular properties of seven mutant(H101Y, G118D, S119P, S119F, Q127R, G128D, and F643L)�PKCs fused with green fluorescent protein (�PKC-GFP).Wild-type �PKC-GFP was expressed ubiquitously in thecytoplasm of CHO cells, whereas mutant �PKC-GFPtended to aggregate in the cytoplasm. The insolubility ofmutant �PKC-GFP to Triton X-100 was increased and cor-related with the extent of aggregation. �PKC-GFP in theTriton-insoluble fraction was rarely phosphorylated atThr514, whereas �PKC-GFP in the Triton-soluble fractionwas phosphorylated. Furthermore, the stimulation of theP2Y receptor triggered the rapid aggregation of mutant�PKC-GFP within 10 min after transient translocation tothe plasma membrane. Overexpression of the mutant�PKC-GFP caused cell death that was more prominentthan wild type. The cytotoxicity was exacerbated in par-allel with the expression level of the mutant. These resultsindicate that SCA14 mutations make �PKC form cytoplas-mic aggregates, suggesting the involvement of this prop-erty in the etiology of SCA14.

The autosomal dominant spinocerebellar ataxias (SCAs)1 area heterogeneous group of neurological disorders clinically char-

acterized by progressive ataxia of gait and limbs, cerebellardysarthria, abnormal eye movements, and so on. SCAs areclassified at least into 25 types (SCA1–8, 10–19, 21–23, 25, 26DRPLA, FGF14) by genetic patterns, clinical features, andpathological findings (1, 2). The genes involved and the respon-sible mutations have been identified in several types of SCAs.Among these, CAG trinucleotide repeat expansions are com-monly found in seven types (SCA1, 2, 3, 6, 7, 17 and DRPLA) (1,2). Diseases caused by such expansions, including Huntingtondisease and spinal and bulbar muscular atrophy (SBMA), arecalled polyglutamine diseases (3, 4). The aggregation of mutantproteins having an abnormally elongated polyglutamine tractis considered to be the molecular basis of neuronal degenera-tion in polyglutamine diseases (5).

Recently, six different missense mutations in protein kinaseC� (�PKC) gene (PRKCG) have been found in SCA14 families(6–9). Five mutations are located in exon 4, encoding the C1Bregion in the regulatory domain of �PKC, and one mutation isin exon 18, encoding the C terminus of the catalytic domain of�PKC (Fig. 1). Furthermore, we found a novel mutation in aJapanese SCA14 family (Fig. 1, bold lined box).2 Because mu-tations associated with SCA14 affect highly conserved aminoacids among the PKC family members, it is possible that thesemutations disturb the fundamental function or conformation of�PKC. However, how these mutations cause cerebellar degen-eration remains controversial.

PKC is a family of serine/threonine kinases which playsimportant roles in signal transduction and the regulation ofvarious cellular functions. Among PKC subtypes, �PKC is spe-cifically present in the central nervous system and is especiallyabundant in cerebellar Purkinje cells and hippocampal pyram-idal cells (10). Therefore, �PKC is thought to be involved invarious neuronal functions including synaptic plasticity andmemory via modulating long term potentiation and long termdepression (11). �PKC knock-out mice showed mildly impairedmotor coordination and incomplete developmental eliminationof synapses between Purkinje cell and climbing fibers (12, 13).Furthermore, in model mice of SCA1 overexpressing mutantataxin-1 with elongated polyglutamine, �PKC was down-regu-lated and abnormally localized to the cytoplasmic vacuoles inPurkinje cells (14). These findings suggest that �PKC may beinvolved in SCA.

Previous live imaging studies using green fluorescent protein(GFP)-tagged PKC (PKC-GFP) demonstrated that PKCs aretranslocated to several cellular organelles in an isoform- and

* This work was supported by a grant-in-aid for Scientific Researchfrom the Ministry of Education, Sports and Culture and by a grant fromTakeda Science Foundation and the Japanese Smoking Research Asso-ciation. The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org)contains a Supplemental Video.

§§ To whom correspondence should be addressed: Dept. of Molecularand Pharmacological Neuroscience, Graduate School of Biomedical Sci-ences, Hiroshima University, Minami-ku, 1-2-3 Kasumi, Hiroshima734-8551, Japan. Tel.: 81-82-257-5142; Fax: 81-82-257-5144; E-mail:nsakai@hiroshima-u.ac.jp.

1 The abbreviations used are: SCAs, spinocerebellar ataxias; PKC,protein kinase C; CHO, Chinese hamster ovary; GFP, green fluorescentprotein; RIPA, radioimmunoprecipitation assay; PVDF, polyvinylidinedifluoride; NGS, normal goat serum; 7-AAD, 7-amino-actinomycin D;UPS, ubiquitin-proteasome system; PDK1, 3-phosphoinositide-depend-ent protein kinase-1; WGA, wheat germ agglutinin; FI/A, fluorescenceintensity per area; FRAP, fluorescent recovery after photobleaching;WT, wild type; PBS, phosphate-buffered saline.

2 K. Hiramoto, H. Kawakami, K. Inoue, T. Seki, H. Maruyama, H.Morino, M. Matsumoto, K. Kurisu, and N. Sakai, submitted data.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 32, Issue of August 12, pp. 29096–29106, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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stimulation-specific manner when PKCs are activated by dif-ferent stimulations. Thereafter, PKCs recognize and phospho-rylate their substrates at the targeted subcellular regions andcause the subsequent cellular responses (PKC targeting). ThisPKC targeting is considered to be the molecular basis under-lining the multiplicity of PKC-mediated functions. Using trans-genic mice overexpressing �PKC-GFP, we have recently re-ported that the translocation of �PKC-GFP, which was inducedby the electrical stimulation of parallel fibers, propagated alongthe dendritic shaft of the cerebellar Purkinje cells (15), indicat-ing that PKC targeting is prerequisite for various PKC-in-volved neuronal functions in Purkinje cells.

�PKC is a member of the classical PKCs (cPKCs), which areactivated by diacylglycerol (DG) and Ca2� in the presence ofphosphatidylserine (16). �PKC has C1 and C2 domains, whichbind DG and Ca2�, respectively (17), in its regulatory domain(Fig. 1). The C1 domain of �PKC is subdivided into two cys-teine-rich repeats (C1A and C1B), both of which bind with DGand phorbol ester with high affinity (18, 19). The C1 and C2domains have crucial roles in PKC targeting through binding tothese PKC activators (20). As described above, 6 of 7 missensemutations found in SCA14 families are located in the C1Bdomain of �PKC (Fig. 1). Therefore, it is possible that thesemissense mutations influence the targeting of �PKC. In thepresent study, to elucidate how mutant �PKCs induced theneuronal degeneration and the pathology of SCA14, we focusedon PKC targeting. We expressed mutant �PKC-GFP in culturecells and compared its localization and receptor-mediatedtranslocation with those of wild type.

EXPERIMENTAL PROCEDURES

Materials—ATP was purchased from Research Biochemical Interna-tional (Natick, MA). Anti-GFP rabbit polyclonal antibody, Alexa546-conjugated anti-rabbit IgG goat antibody and Alexa633-conjugatedwheat germ agglutinin (WGA) were from Molecular Probes (Leiden,Netherlands). Anti-phospho-�PKC (Thr514), anti-phospho-�PKC(Thr655)and anti-phospho-�PKC(Thr674) polyclonal antibodies were from BIO-SOURCE International (Camarillo, CA). Horseradish peroxidase-conju-gated goat anti-rabbit IgG antibody was from Jackson ImmunoResearchLaboratories (West Grove, PA). Anti-�PKC polyclonal antibody was fromSanta Cruz Biotechnology (Santa Cruz, CA).

Plasmid Construction—Human �PKC cDNA was cloned from a hu-man cDNA library by PCR and subcloned into pBluescript II KS(�) vector(Stratagene, La Jolla, CA). Mutant human �PKC cDNAs were con-structed by using QuickChange multisite-directed mutagenesis kit (Strat-agene). To construct the plasmids encoding wild-type or mutant �PKC-GFP, �PKC and GFP cDNAs were together subcloned into the expressionvector, pcDNA3 (Invitrogen). The GFP cDNA followed the �PKC cDNA sothat GFP protein was fused with the C terminus of �PKC. All wild-typeand mutant �PKC cDNAs were verified by sequencing.

Cell Culture—The CHO-K1 cell strain was a gift from Dr. Nishijima(National Institute of Health, Tokyo, Japan). CHO cells were culturedin Ham’s F12 medium (Sigma), supplemented with 10% fetal bovineserum, 100 units/ml of penicillin, and 100 �g/ml of streptomycin in ahumidified atmosphere containing 5% CO2 at 37 °C.

Immunoblotting—Plasmids (5 �g) were transfected into CHO cells(2 � 105 cells) by lipofection using the FugeneTM6 transfection reagent(Roche Applied Science) according to the manufacturer’s directions.Transfected CHO cells were spread onto 6-cm diameter dishes andcultured for 2 days. Cells were harvested by 500 � g centrifugation,followed by washing with 1 ml of homogenate buffer (250 mM sucrose,10 mM EGTA, 2 mM EDTA, and 50 mM Tris-HCl, pH 7.4). For preparingtotal cell fractions, cells were resuspended in 100 �l of RIPA buffer (1%Nonidet P40, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM

EDTA, 20 �g/ml of leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1mM sodium orthovanadate, 1 mM NaF, and 100 nM calyculin A, and 10mM Tris-HCl, pH 7.4) and sonicated (UR-20P, TOMY SEIKO, Tokyo,Japan) (output, 4; duty, 50%) for 15 times at 4 °C. For immunoblotting,the same amounts (10–20 �g) of samples were subjected to 7.5% SDS-PAGE, and the separated proteins were electrophoretically transferredonto polyvinylidine difluoride (PVDF) filters (Millipore, Bedford, MA).Nonspecific binding sites on PVDF filters were blocked by incubationwith 5% skim milk in PBS-T (0.01 M phosphate-buffered saline contain-

ing 0.03% Triton X-100) for �1 h at room temperature. After washingwith PBS-T, the PVDF filters were incubated with anti-GFP polyclonalantibody (diluted 1:2000) or anti-phospho-PKC� (Thr514) polyclonal an-tibody (diluted 1:1000) for �1 h at room temperature. After furtherwashing, the filters were incubated with horseradish peroxidase-conju-gated anti-rabbit IgG antibody (diluted 1:10,000) for �30 min at roomtemperature. After three more washes, the immunoreactive bands werevisualized with a chemiluminescence detection kit (ECLTM Westernblotting detection reagents, Amersham Biosciences) The band densitieswere quantified with Fluor-S MultiImager (Bio-Rad).

For preparing Triton-soluble (S) and -insoluble (I) fractions, cellswere suspended in lysis buffer (homogenate buffer containing 1% TritonX-100, 20 �g/ml of leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM

sodium orthovanadate, 1 mM NaF, and 100 nM calyculin A) and soni-cated. Samples were centrifuged at 15,000 � g for 15 min at 4 °C, andthe supernatants were collected as the S fraction. The pellets wereresuspended with 50 �l of RIPA buffer, sonicated, and used as the Ifraction. One-twentieth volume of each fraction was subjected to 7.5%SDS-PAGE and immunoblotted by the same method as described above.

Observation of �PKC-GFP Localization—CHO cells (1�105 cells)were spread onto poly-D-lysine-coated glass bottom culture dishes (Mat-Tek Corp., Ashland, MA) and were transfected with 2.5 �g of plasmid bylipofection. Transfected cells were cultured for 2 days until the obser-vation. After the culture medium was replaced with 1 ml of HEPESbuffer (135 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM

HEPES, and 10 mM glucose, pH 7.3), the fluorescence of GFP wasmonitored with a confocal laser scanning fluorescent microscope(LSM510META, Carl Zeiss, Esslingen, Germany) at 488-nm argon la-ser excitation using a 505–530-nm band pass barrier filter.

To analyze the expression level of wild-type or mutant �PKC-GFP inindividual cells, fluorescence images of randomly selected CHO cellsexpressing �PKC-GFPs were obtained. For this purpose, parameters ofconfocal laser scanning fluorescent microscope (e.g. pinhole, laser in-tensity, and sensitivity of fluorescence) were adjusted to the same level.The fluorescence intensity and the area of the whole cell were measuredusing LSM510META software. The fluorescence intensity per area(FI/A) was used as an index for estimating the expression level of�PKC-GFPs in each cell.

Fluorescent Recovery after Photobleaching (FRAP) Analysis—Circu-lar regions in the cytoplasm of CHO cells expressing mutant �PKC-GFPwere photobleached by scanning for 15 s with an argon laser of thehighest power. Before and after photobleaching, the bleached cells weremonitored for 30 min.

Observation of �PKC-GFP Translocation—Wild-type or mutant�PKC-GFP-transfected CHO cells were cultured in glass bottom dishesfor 2 days until observation. After the culture medium was replacedwith 0.9 ml of HEPES buffer, the GFP fluorescence was monitored witha confocal laser scanning fluorescent microscope. Translocations ofGFP-fused proteins were triggered by a direct application of 0.1 ml ofATP solution at 10� higher concentration into HEPES buffer to obtainthe appropriate final concentration. Images were recorded every 5 s for5–10 min before and after the stimulation. All experiments were per-formed at room temperature.

Immunostaining and Staining with Golgi Complex Marker—Twodays after transfection, CHO cells were fixed with 4% paraformalde-hyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4, for morethan 30 min. After washing twice with PBS-T, the cells were treatedwith PBS containing 0.3% Triton X-100 and 5% normal goat serum(NGS) for 5 min at room temperature. For immunostaining, the cellswere then incubated with the anti-�PKC polyclonal antibody (1:1000)and 5% NGS in PBS-T for 1 h at room temperature. After three timeswashing with PBS-T, the cells were incubated with Alexa546-conju-gated goat anti-rabbit IgG antibody (1:500) and 5% NGS in PBS-T for1 h at room temperature, followed by three washes with PBS-T. Forstaining with Golgi complex marker, the cells were incubated with 1�g/ml Alexa633-conjugated WGA and 5% NGS in PBS-T for 40 min atroom temperature, followed by three washes with PBS-T. The fluo-rescence of Alexa546 and Alexa633 was observed with a confocalscanning fluorescent microscope at 543-nm and 633-nm HeNe laserexcitation using a 560-nm and 650 nm-long pass barrier filter,respectively.

Evaluating and Counting Cells with Aggregation—CHO cells trans-fected with �PKC-GFP were cultured for 2 days and fixed as describedabove. After two washes with PBS, cells were observed using fluores-cent microscopy. We classified cells expressing �PKC-GFP into threetypes: cells without aggregation, with massive aggregations, and withdot-like aggregations (Fig. 2, A–C). We evaluated the cell type andcounted the number of each cell type in 50–60 GFP-positive cells

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Analyzing Cell Death Using Flow Cytometry—Flow cytometric anal-yses were conducted using FACSCalibur (BD Biosciences). We used7-amino-actinomycin D (BD Biosciences) as a marker for dead cells.Transfected CHO cells were cultured on 6-cm diameter dishes for 3days. Cells were isolated with 0.125% trypsin and 0.5 mM EDTA,washed three times, and suspended in 10 mM HEPES buffer (pH 7.4)containing 140 mM NaCl and 2.5 mM CaCl2. We added 5 �l of 50 �g/ml7-AAD into 1 � 105 cells in 100 �l of HEPES buffer and incubated cellsfor 10 min at room temperature. Stained cells were immediately ana-lyzed by flow cytometry at a 488-nm argon laser excitation using a515–545-nm band pass barrier filter for GFP and 650-nm long passfilter for 7-AAD. For each sample, the fluorescence of 2 � 104 cells wasrecorded and analyzed by the Cell QuestTM software (BD Biosciences).

RESULTS

Mutant �PKC-GFPs Tended to Aggregate in CHO Cells—Sixdifferent missense mutations (5 around the C1 domain and 1 inthe catalytic domain) have been reported in the �PKC gene indifferent SCA14 families (Fig. 1). Recently, we found a novelmutation around the C1 domain (Ser119 replaced with Phe) of�PKC in a Japanese SCA family (Fig, 1, bold lined box).2 Toclarify how these mutations affect the molecular properties of�PKC and trigger neurodegeneration, we introduced these mis-sense mutations into �PKC-GFP and investigated molecularproperties of 7 mutant �PKC-GFPs (H101Y, G118D, S119P,S119F, Q127R, G128D, and F643L) expressed in CHO cells. Mostof the CHO cells expressing wild-type �PKC-GFP had a ubiqui-tous GFP fluorescence in the cytoplasm, but not in the nucleus(Fig. 2A). On the other hand, cells expressing mutant �PKC-GFPfrequently had aggregated GFP fluorescence in the cytoplasm(Fig. 2, B and C). Precise observation revealed that there weretwo patterns of mutant �PKC-GFP aggregation, massive aggre-gation and dot-like aggregation as described in Fig. 2, B and C,respectively. Massive aggregation was seen in the vicinity of thenucleus (Fig. 2B), whereas dot-like ones were seen in cytoplasm(Fig. 2C). Therefore, we classified aggregation patterns into thesetwo types. We evaluated the extent of �PKC-GFP aggregations in50–60 GFP-positive CHO cells expressing wild-type or mutant�PKC-GFP. Results are shown in Fig. 2D. Although wild-type�PKC-GFP aggregations were observed in 20.9 � 2.7% of ex-pressed cells, all seven mutants were preferably aggregated, andthe percentage of cells having aggregation was over 30%. In sixmutants (G118D, S119P, Q119F, Q127R, G128D, and F643L),the percentages were significantly greater than that in wild-type�PKC-GFP. In cells expressing H101Y, S119F, and F643L mu-tant �PKC-GFPs, massive aggregations were more often ob-served than in cells expressing wild type, whereas G118D,S119P, Q127R, G128D, and F643L mutants more frequentlyformed dot-like aggregations than wild type (Table I). Similarmassive and dot-like aggregations were observed when �PKC-GFPs were expressed in other cell lines such as COS-7 andSH-SY5Y cells (data not shown).

To exclude the possibility that the aggregate formation of

�PKC-GFPs depends on their excessive expression, but noton the properties of mutated proteins, we investigated therelationship between the expression level and the aggregateformation of �PKC-GFP. In CHO cells expressing wild typeand S119P and G128D �PKC-GFPs, fluorescent images ofrandomly selected 30–40 cells were obtained using the sameparameters of confocal laser scanning microscope. The fluo-rescence intensity per area (FI/A), which represents themean of the fluorescence intensity throughout the cell, wasused as an index exhibiting the expression level of �PKC-GFPs in each cell. In Fig. 3A, histograms show the distribu-tion of cells with or without aggregation classified by �PKC-GFP expression level. In wild-type �PKC-GFP, all cells withFI/A �100 did not have any aggregation (0/23 cells). Aggre-gations were observed only in cells with FI/A �100 (7/12cells). In contrast, in S119P and G128D mutant �PKC-GFPs,aggregations were frequently observed in cells whose FI/Awere 20–100 (22/25 cells in S119P and 19/26 cells in G128D).Fig. 3B shows the representative images of cells expressingwild-type and mutant (S119P and G128D) �PKC-GFPs withFI/A 40–60, indicating that the expression levels of �PKC-GFPs were similar among these three cell types. Althoughwild-type �PKC-GFP was uniformly expressed in the cyto-plasm, mutant �PKC-GFPs formed dot-like aggregation inthe cytoplasm. These results indicate that the susceptibilityto aggregate formation of mutant �PKC-GFPs is determinedby their properties, but not by their expression level.

Next, we examined whether massive and dot-like aggrega-tion of �PKC-GFP were colocalized with cellular organelles.The massive aggregation of wild-type �PKC-GFP was stronglycolocalized with the wheat germ agglutinin (WGA), a Golgi

FIG. 1. Schematic illustrations of �PKC protein and the mu-tated amino acid found in SCA14 families. Seven mutations andsubstituted amino acids are listed in the box below the diagram. Thebold lined box represents a mutation found by our group. Three phos-phorylation sites are shown in italic above the diagram. The Thr514 inthe activation loop is phosphorylated by PDK1. The other two sites,Thr655 and Thr674, located near the C terminus, are autophosphorylatedafter PDK1 initially phosphorylates Thr514.

FIG. 2. Mutant �PKC-GFPs aggregates in the cytoplasm ofCHO cells in two different manners. A–C, representative images ofWT or mutant �PKC-GFP expression in CHO cells. A, WT �PKC-GFPis uniformly expressed in the cytoplasm. B, massive aggregation ofmutant �PKC-GFP (G118D) is seen near the nucleus. C, dot-like ag-gregations of mutant �PKC-GFP (Q127R) is seen in the cytoplasm.Cells were observed using confocal laser microscope 2 days after trans-fection. Bar, 10 �m. D, extent of aggregation of wild-type and mutant�PKC-GFP. We evaluated 50–60 GFP-positive cells expressing wild-type or mutant �PKC-GFP. Each bar indicates the percentage of cellsshowing aggregation of wild-type or mutant �PKC-GFP. Black andhatched parts of each bar represent the percentage of cells showingmassive and dot-like aggregations, respectively. *, p � 0.05; **, p � 0.01versus WT (unpaired Student’s t test, n � 6 in WT and S119P, n � 5 inS119F, n � 4 in other mutants).

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complex marker (Fig. 4A). However, the massive aggregation ofmutant �PKC-GFP was partially colocalized with WGA (Fig.4B). This result indicates that massive aggregation of mutant�PKC-GFP was qualitatively different from that of wild type.In the case of dot-like aggregations, we could not establishcolocalization of these aggregations with markers for Golgicomplex, lysosome, or early or late endosomes (data not shown).

To elucidate whether �PKC-GFP irreversibly formed dot-likeaggregates, we performed a FRAP study. A �PKC-GFP aggre-gation was photobleached with an argon laser at 488 nm,followed by observing fluorescence recovery. As shown in Fig. 5,an application of photobleaching into a circular area arounddot-like aggregations (a and b) abolished the fluorescence of�PKC-GFP aggregation, and the GFP fluorescence was notrecovered, at least within 30 min. In contrast, the fluorescenceof non-aggregated �PKC-GFP (Fig, 5c) was recovered to a levelsimilar to that in the unbleached cytoplasm within 1 min afterphotobleaching (Fig. 5d). This result indicates that mutant�PKC-GFP of dot-like aggregation tightly associates each otherand that the aggregates were not exchangeable with free�PKC-GFP in the cytoplasm.

To exclude the possibility that the addition of GFP to the�PKC was critical for aggregate formation, we expressed mu-tant �PKC alone in CHO cells. We attempted to immunostainmutant �PKCs with anti-�PKC antibody. To confirm whetherthis antibody could properly detect the aggregation of �PKCs,CHO cells expressing mutant �PKC-GFP were stained withthis antibody. As shown in Fig. 6A, the �PKC immunofluores-cence detected with this antibody is consistent with the S119P�PKC-GFP fluorescence, although the antibody only recog-nized edges, not centers, of massive aggregations of S119P�PKC-GFP (upper panels, arrows). In contrast, the antibodyrecognized the whole of dot-like aggregations (lower panels,arrowheads). These results suggest that the anti-�PKC anti-body properly recognizes �PKC-GFP aggregation, although itmight be inaccessible to the centers of these aggregations. Asshown in Fig. 6B, both massive (arrow) and dot-like (arrow-

heads) aggregations were also observed in CHO cells express-ing S119P �PKC. In contrast, wild-type �PKC rarely aggre-gated in CHO cells (data not shown). This result indicates thatthe addition of GFP to �PKC is not critical for the aggregateformation of mutant �PKC.

Phosphorylation Level and Solubility to Triton X-100 of Mu-tant �PKC-GFP Was Decreased—�PKC has three phosphoryl-ation sites in its kinase domain: activation loop (Thr514), turnmotif (Thr655), and hydrophobic motif (Thr674) site (Fig. 1).Phosphorylation of these three sites is necessary for the fullactivation of PKC in response to various stimulations (21–23).We examined whether the aggregate formation affected thephosphorylation level of these three sites by immunoblottingwith each phosphospecific antibody. As shown in Fig. 7 andTable I, the phosphorylation levels of mutant �PKC-GFPs weresignificantly decreased or tended to be decreased at three phos-phorylation sites, compared with those of wild type. Specifi-cally, phosphorylation levels of three mutants (S119P, G128D,and F643L) were significantly lower at all three sites thanthose of wild type.

Various neurodegenerative diseases are accompanied by theformation of disease-specific inclusion bodies, for examples Lewybodies in Parkinson disease, neurofibrillary tangles and senileplaques in Alzheimer disease, and nuclear inclusion bodies inHuntington disease (3, 24). These inclusion bodies are generatedby aggregated, unfolded, or misfolded protein such as�-synuclein, tau, amyloid �-protein, and expanded polyglu-tamine, respectively. It has been reported that these unfolded ormisfolded proteins became detergent-insoluble in cellular andanimal models of various neurodegenerative diseases (25–27).Therefore, we examined whether mutated �PKC became insolu-ble to the detergent. Transfected CHO cells were separated intothe 1% Triton-soluble (S) and -insoluble (I) fractions, and theamounts of �PKC-GFP in both fractions were quantified by im-munoblotting with anti-GFP antibody. As shown in the upperpanel of Fig. 8A, wild-type �PKC-GFP was detected with almostequal amounts in both S and I fractions. On the other hand,

TABLE IProperties of WT and mutant �PKC-GFPs found in the present study

Results are represented as mean � S.E. except aggregation after translocation, which is indicated as observed cells/examined cells.

WT H101Y G118D S119P S119F Q127R G128D F643L

Cells havingAggregates 20.9 � 2.7 32.7 � 6.8 41.4 � 5.6a 60.2 � 6.4b 35.0 � 5.2c 49.8 � 2.7b 56.9 � 11.9a 45.6 � 4.3b

Massive aggregates 14.4 � 1.7 24.0 � 4.3c 27.0 � 6.4 20.5 � 4.9 28.6 � 5.2c 16.3 � 2.0 27.9 � 7.4 28.5 � 2.2b

Dot-like aggregates(% of total �PKC-GFP expressing

cells)

6.5 � 1.4 8.7 � 2.9 14.4 � 1.5a 39.7 � 3.6b 6.4 � 0.7 33.5 � 2.7b 29.0 � 5.8a 17.1 � 2.7a

Phosphorylation level atThr514 100 � 4.4 68.5 � 10.7 55.1 � 5.4a 26.2 � 2.9b 55.1 � 2.4b 88.0 � 22.1 43.2 � 3.4b 29.6 �8.0a

Thr655 100 � 8.7 74.4 � 4.5 78.2 � 13.9 53.5 � 10.9c 76.3 � 4.9 84.0 � 12.0 46.8 � 7.6a 65.5 � 8.0c

Thr674

(% of WT)100 � 0.8 79.4 � 3.2a 70.3 � 13.9 45.7 � 6.8a 59.1 � 8.5a 59.3 � 12.5c 37.7 � 2.9b 32.8 � 8.2a

Insolubility toTriton X.100

(% of expressed �PKC-GFP)60.6 � 8.9 74.3 � 6.7 82.7 � 5.7 91.2 � 2.6c 52.1 � 17.1 93.3 � 1.7c 94.3 � 1.0c 92.6 � 1.7c

Translocation induced by ATP (1 mM)Retained period at the plasma

membrane(seconds)

129.4 � 23.2 114.4 � 25.0 137.7 � 9.3 95.0 � 13.1b 91.5 � 8.1 123.3 � 7.7 123.3 � 9.1 117.5 � 12.4

Aggregation after the translocation(observed cells/examined cells)

0/9 0/9 5/11 0/7 0/10 6/9d 7/9d 0/8

Dead cells inTotal GFP(�) cells 20.0 � 0.8 20.3 � 1.9 22.3 � 1.3 31.5 � 2.5c 23.5 � 1.5 25.5 � 1.8c 26.5 � 2.5 25.5 � 2.2Low GFP cells 14.6 � 0.2 11.7 � 2.0 12.8 � 1.3 17.1 � 3.1 13.1 � 1.9 14.6 � 2.0 15.6 � 2.5 15.9 � 1.0High GFP cells

(% of GFP(�) cells)32.2 � 1.0 37.1 � 1.6 40.9 � 2.3c 56.9 � 0.9b 52.7 � 1.6c 43.1 � 4.2 47.1 � 3.1c 45.0 � 3.2c

a p � 0.01.b p � 0.001.c p � 0.05 versus WT (unpaired Student’s t test).d p � 0.01 versus WT (Fisher’s exact test).

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mutant �PKC-GFPs were mostly detected in the I fraction. Es-pecially, in S119P, Q127R, G128D, and F643L mutants, onlyfaint bands were detected in the S fraction and very intensebands in I fraction. The amount of �PKC-GFP in the I fractionwas quantified and indicated as percentage of the total fraction(Fig. 8B and Table I), which was used as an index of the insolu-bility to Triton X-100. Except in S119F, this value tended toincrease, and significant differences were detected in S119P,Q127R, G128D, and F643L mutants compared with wild-type�PKC-GFP. This result suggests that mutant �PKC-GFPs be-came insoluble to 1% Triton X-100. The insolubilities of �PKC-GFPs to Triton X-100 were positively correlated (r � 0.817) withthe extent of aggregated cells (Fig. 2D). For example, both valueswere high in S119P, Q127R, G128D, and F643L mutants andwere relatively low in H101Y and S119F mutants. This correla-tion would imply that the insolubility of mutant �PKC-GFP todetergent was caused by its aggregated form.

Next, we compared the phosphorylation state of Thr514 inthe activation loop of �PKC, which is essential for PKC ki-nase activity (28, 29), between wild-type and mutant �PKC-GFP. The phosphorylation state was also compared between

the S and I fractions. Phospho-Thr514-specific antibody wasused for assessment of the phosphorylation level (Fig. 8A,lower panel). �PKC-GFP in the S fraction was intensely phos-phorylated. However, very few mutant �PKC-GFPs werephosphorylated in the I fraction although sufficient levels ofmutant �PKC-GFP existed (Fig. 8A, upper panel). The rela-tive phosphorylation level of Thr514 per �PKC-GFP amountwas shown in Fig. 8C. As for the S fraction, the phosphoryl-ation level did not significantly differ between the wild-typeand mutant �PKC-GFPs, although the phosphorylation levelof S119F tended to be increased, compared with wild type. Asfor the I fraction, the phosphorylation level was obviouslydecreased in all wild-type and mutant �PKC-GFPs. Theseresults suggest that mutant �PKC-GFPs in the S fractionwere normally phosphorylated at Thr514 to the same extentas the wild type. It is possible that �PKC-GFPs in the Ifraction were mostly unphosphorylated at The514 and inac-tive. As shown in Fig. 7, the phosphorylation levels of mutant�PKC-GFPs in RIPA-soluble total fraction were mostly de-creased. This may be manifested because Triton-insolubleaggregated mutants were unphosphorylated.

FIG. 3. Relationship between ex-pression level and aggregate forma-tion of wild-type and mutant �PKC-GFPs (S119P and G128D). A,histograms indicate the distribution ofCHO cells expressing WT and S119P andG128D mutant �PKC-GFPs, which wereclassified into six groups according totheir expression levels. The expressionlevel was evaluated by the fluorescenceintensity per area (FI/A) of each cell.Closed and open bars indicate the numberof cells with and without aggregation, re-spectively. Total observed cells were 35,31, and 33 cells in WT and S119P andG128D �PKC-GFPs, respectively. Cellswere observed using confocal laser micro-scope 2 days after transfection. B, repre-sentative images of CHO cells expressingWT and S119P and G128D mutant �PKC-GFPs with FI/A 40–60. Bar, 10 �m.

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Mutant �PKC-GFP Aggregated after Receptor-mediatedTransient Translocation—To examine whether receptor-medi-ated translocation of mutant �PKC-GFPs differed from that of

wild type, we observed the translocation of these �PKC-GFPstriggered by the stimulation of P2Y receptors, which are endo-genously expressed in CHO cells (30). As reported previously(31), the application of ATP (1 mM) induced a rapid transloca-tion of �PKC-GFP from the cytoplasm to the plasma membranewithin 10 s after the stimulation, followed by a re-translocationfrom the membrane to cytoplasm around 2 min (Fig. 9A). Like-wise, all mutant �PKC-GFPs were transiently translocatedfrom cytoplasm to the plasma membrane in CHO cells withoutany aggregations (Fig. 9). The translocation of mutant �PKC-GFP occurred within 10 s after the stimulation, and there wereno differences in the retention period at the plasma membranebetween wild-type and mutants (Table I).

However, in many cells expressing G118D, Q127R, andG128D, �PKC-GFP formed punctuate aggregates in the cyto-plasm after the reversion from plasma membrane to cytoplasm(Fig. 9C and Supplemental Video). In these cells, mutant �PKC-GFP behaved in a similar manner to wild type until 2 min afterthe stimulation. Thereafter, �PKC-GFP started to aggregate inthe cytoplasm and almost all �PKC-GFP in the cytoplasm aggre-gated within 10 min (Fig. 9C and Supplemental Video). Thesephenomena were seen in 45, 67, and 78% of cells expressingG118D, Q127R, and G128D, respectively, to which translocationstudies were applied (Table I). These results suggest that theaggregate formation of mutant �PKC-GFP was frequently trig-gered by its receptor-mediated translocation.

As is shown in Fig. 2, in some cells, aggregation had alreadyobserved before the stimulation. In these cells, diffusely ex-pressed cytoplasmic �PKC-GFP was translocated, but aggre-gated �PKC-GFP was not (data not shown).

Mutant �PKC-GFP Induced Cell Death in Parallel with ItsExpression Level—Overexpression of etiological gene productshas been reported to cause cell death in cellular and animalmodels of various neurodegenerative diseases (5, 32, 33). Weinvestigated whether these mutant �PKC-GFPs caused celldeath. Three days after transfection of wild-type or mutant�PKC-GFPs, CHO cells were stained with 7-AAD, a fluorescentDNA dye that selectively enters dead cells (34). Stained cellswere applied to flow cytometry and were classified by twoparameters, GFP and 7-AAD fluorescence. Representative re-sults using wild type and S119P are shown in Fig. 10, A and B,respectively. The GFP and 7-AAD fluorescence of each cell was

FIG. 4. Alexa633-conjugated WGA, a marker of Golgi complex,was strongly colocalized with the massive aggregation of WT�PKC-GFP (A), but partially with that of G128D �PKC-GFP (B).CHO cells expressing WT (A) and G128D (B) �PKC-GFP were fixed 2days after transfection, followed by the staining with Alexa633-conju-gated WGA to make the Golgi complex visible. The fluorescence of GFPand Alexa633 are shown in green (left) and red (center), respectively. Inthe merged image, the overlapping of GFP and Alexa633 signals ap-pears yellow (right). Bar, 10 �m.

FIG. 5. The fluorescence of �PKC-GFP was not recovered afterphotobleaching in a dot-like aggregation. A, FRAP study wasperformed on cytoplasmic dot-like aggregation of �PKC-GFP. The im-ages were obtained from CHO cells expressing S119P �PKC-GFP inevery 10 s before and after the photobleaching. The images before(upper left) and 10 s (upper center) and 1, 5, 10, and 30 min (upper right,lower left, lower center, and lower right, respectively) after photobleach-ing are shown. Circles of dotted lines indicates bleached areas arounddot-like aggregations (a and b), bleached areas in the cytoplasm withoutaggregation (c), and the unbleached area in the cytoplasm (d). Bar, 10�m. B, time-dependent recoveries of fluorescence in the bleached areas(a and b, dot-like aggregations; c, cytoplasm without aggregation) andfading of fluorescence in the unbleached area (d) are shown as percent-ages of the fluorescence before photobleaching.

FIG. 6. Fused GFP was not critical for aggregation of S119P�PKC-GFP. Two days after transfection, CHO cells expressing S119P�PKC-GFP (A) and S119P �PKC (B) were fixed with 4% paraformalde-hyde plus 0.2% picric acid and then immunostained with anti-�PKCantibody. Immunoreactivity was visualized with Alexa543-conjugatedsecondary antibody. The fluorescence of GFP and Alexa543 are shownin green (left) and red (center, right), respectively. A, anti-�PKC anti-body recognizes both massive (arrows in upper panels) and dot-like(arrowheads in lower panels) aggregations of S119P �PKC-GFP al-though it only immunoreacts with the margin of massive aggregation.B, S119P �PKC alone forms both massive (arrow) and dot-like (arrow-heads) aggregations, similar to S119P �PKC-GFP. Bar, 10 �m.

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positioned as a dot in the graph. Cells in the right lower part(GFP(�)/7-AAD(�)) represented viable cells expressing �PKC-GFPs, and cells in the right upper part (GFP(�)/7-AAD(�))represented dead cells expressing �PKC-GFPs. The GFP-posi-tive cells expressing S119P �PKC-GFP were better stainedwith 7-AAD than cells expressing wild-type �PKC-GFP (Fig.10, A and B). The percentages of dead cells in total GFP-positive cells were shown in Fig. 10C. In CHO cells expressingS119P and Q127R mutant �PKC-GFPs, the percentages ofdead cells were significantly higher (31.5 � 2.5 and 25.5 �1.8%, respectively) than that in cells expressing wild type(20.0 � 0.8%). To evaluate whether the expression level of�PKC-GFP affects the cell viability, GFP-positive cells weredivided into two groups: groups with low and high GFP fluo-rescence (low and high GFP group, Fig. 10B), and the percent-ages of dead cells were calculated in each group. In the low GFPgroup, there were no significant differences in cell viabilitiesbetween wild-type and mutant �PKC-GFP-expressing cells(Fig. 10D). However, in the high GFP group, cytotoxic effects ofmutant �PKC-GFPs were more prominently manifested thanin total GFP-positive cells (Fig. 10E). G118D, S119P, S119F,and G128D significantly exacerbated cell death, compared withwild type. In addition, H101Y and Q127R mutants tended tocause cell death more prominently than wild type. These re-sults suggest that mutant �PKC-GFP induced cell death inparallel with its expression level.

DISCUSSION

Several autosomal dominant SCAs have been revealed to becaused by abnormal expansions of CAG trinucleotide repeats(1, 2). In these polyglutamine diseases, expanded polyglu-tamine chains can easily form �-sheet structures and insolubleaggregates in the neuronal cells (3, 4). Similar insoluble aggre-gations of mutant or misfolded proteins are frequently ob-served in various inherited neurodegenerative disorders in-cluding Alzheimer disease and Parkinson disease (3, 24, 35).The gain of toxic function derived from aggregated mutantproteins is considered to be the etiology of these late-onsetneurodegenerative disorders. In the present study, we demon-strated that missense mutations of the �PKC gene, found inSCA14, induced the formation of insoluble �PKC aggregatesand cell death in CHO cells.

Two types of aggregation, massive and dot-like aggregationswere frequently observed in CHO cells expressing mutant�PKC-GFP (Fig. 2). Dot-like aggregations, frequently shown inCHO cells expressing S119P, Q127R, and G128D mutants,might be caused by the accumulation of �PKC-GFP to cellularorganelles such as lysosome or endosomes. However, we couldnot clearly find the colocalization of this aggregation with anyorganelles (data not shown). Moreover, FRAP analysis con-firmed that �PKC-GFP in these aggregations was tightly asso-ciated and not exchangeable with free �PKC-GFP in the cyto-

FIG. 7. Mutant �PKC-GFPs had lessphosphorylation at their three phos-phorylation sites. A, immunoblottinganalysis of WT and mutant �PKC-GFPswith anti-GFP and anti-phosphorylatedthreonine (pThr514, pThr655, and pThr673)-specific antibodies. Protein samples wereextracted from CHO cells 2 days aftertransfection. Total fraction of each sample(10 �g of protein) was subjected to 7.5%SDS-PAGE, followed by immunoblotting.Data shown are representative of threeexperiments. B, normalized phosphoryla-tion level at each threonine residue.Chemiluminescence of immunoreactivebands were quantified using Fluor-S Mul-tiImager. In each sample, the intensity ofband detected with a phosphospecific an-tibody was normalized by that detectedwith anti-GFP antibody. Data are pre-sented as percentages of normalizedvalue in WT. *, p � 0.05; **, p � 0.01; ***,p � 0.001 versus WT (unpaired Student’st test, n � 3).

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plasm (Fig. 5). This result is consistent with previous reportsthat the GFP fluorescence of polyglutamine protein aggregatesis not recovered after photobleaching (36, 37). These findingsindicate that dot-like accumulations of mutant �PKC-GFP areindeed aggregates, but are not the simple result of targeting toparticular cell organelles.

Massive aggregations, frequently shown in cells expressingH101Y, G118D, S119F, and F643L mutants, were located near

the nucleus, but partially localized to Golgi complex, whereasmassive aggregations of wild type showed strong colocalizationwith the Golgi complex (Fig. 4). This result might imply thatthe mechanism of massive-aggregate formation of mutant�PKC-GFPs was different from that of wild type. The long termtime-lapse imaging of mutant �PKC-GFP aggregation is nec-essary to elucidate how massive aggregations are formed.

In seven missense mutations examined in the present study,six mutations were located around the C1B domain (Fig. 1),which is involved in the binding to various lipid messengerslike DG (17–19). These mutations might affect the lipid bind-ing, resulting in the aggregate formation.

Both wild-type and mutant �PKC-GFPs were transientlytranslocated from cytoplasm to plasma membrane in CHO cellsby P2Y receptor stimulation with ATP (Fig. 9). No significantdifferences were found in parameters of PKC translocation,such as translocation or re-translocation speed and PKC-re-taining periods at plasma membrane, between wild type andmutants (Table I). It suggests that the six missense mutationsaround C1B domain did not robustly affect the properties ofreceptor-mediated PKC translocation. This is inconsistent withresults reported by Verbeek et al. (38), showing that calciumionophore-induced translocation was hastened in two mutant�PKC-GFPs found in SCA14 families; one is G118D and theother is C150F, a newly found missense mutation. This discrep-ancy may be explained by the difference in stimulation and celltypes; however, it is still controversial whether mutations af-fect the PKC regulation mechanism and subsequently alter itstranslocation.

Although the translocation process of mutant PKC is likelysimilar to that of wild type, three mutant (G118D, Q127R, andG128D) �PKC-GFPs rapidly and irreversibly aggregated in thecytoplasm after the receptor-mediated translocation (Fig. 9C

FIG. 8. Mutant �PKC-GFPs were in-soluble to Triton X-100 and weremarkedly less phosphorylated atThr514 in the Triton-insoluble fraction.A, immunoblotting analysis of Triton-solu-ble (S) and Triton-insoluble (I) fractions,which were prepared from CHO cells ex-pressing WT and mutant �PKC-GFPs, wasperformed using anti-GFP and anti-phos-phorylated Thr514 (pThr514) -specific anti-bodies. Two days after transfection, CHOcells were harvested and separated into Sand I fractions as described in the text.Five percent of each fraction was subjectedto 7.5% SDS-PAGE, followed by immuno-blotting. Data shown are representative offour experiments. B, insolubility of WT andmutant �PKC-GFP to Triton X-100. Theamount of �PKC-GFPs in each fractionwas quantified by the band intensity de-tected with anti-GFP antibody. Theamount of �PKC-GFP in the I fraction ispresented as the percentage of that in thetotal fraction (S plus I fraction). This valuewas used as an index showing insolubilityto Triton X-100. *, p � 0.05 versus WT(unpaired Student’s t test, WT; n � 8, mu-tant; n � 4). C, normalized phosphoryla-tion level at Thr514 in S and I fractions. Theintensity of immunoreactive band detectedwith anti-phosphorylated Thr514 antibodywas normalized by that detected with anti-GFP antibody. This value was used as thephosphorylation level at Thr514 in eachsample. Data are presented as percentagesof phosphorylation levels at Thr514 in the Sfraction of WT. Black and hatched bars rep-resent the phosphorylation level at Thr514

in the S and I fraction, respectively.

FIG. 9. Several mutant �PKC-GFPs formed cytoplasmic dot-like aggregates after its receptor-mediated transient transloca-tion. CHO cells expressing wild type (A), H101Y (B), and Q127R (C)mutant �PKC-GFPs were stimulated with ATP (1 mM), an agonist ofP2Y receptor. The sequential changes in the fluorescence of GFP fusionprotein were monitored. Images before (pre) and 0.5, 2, 3, 5, and 10 minafter ATP stimulation are shown. ATP induced transient �PKC-GFPtranslocation from the cytoplasm to the plasma membrane in CHO cellsexpressing both wild-type and mutant �PKC-GFPs. Q127R �PKC-GFPgradually aggregated in a dot-like manner after re-translocation tocytoplasm (3, 5, 10 min in C). Data shown are representative of at leastfive experiments. Bar � 10 �m. Live imaging of Q127R�PKC-GFPtranslocation can be seen in a Supplemental Video.

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and Supplemental Video). The aggregation pattern resembledthe dot-like aggregation (Fig. 2C) although we did not confirmwhether these two types of aggregation were identical. Whenthe stimulation of Gq-protein-coupled receptors translocatePKC to the plasma membrane, PKC interacts with its activa-tor, diacylglycerol and Ca2�, which are elevated by the stimu-lation It is well accepted that this interaction triggers theconformational change of the PKC molecule and allows PKC tobe active (21, 39). This conformational change of mutant �PKC-GFP might cause the rapid aggregation of �PKC-GFP. In un-stimulated CHO cells, the endogenous receptor activationmight trigger the translocation, activation, conformationalchange, and aggregation of mutant �PKC-GFP during prolifer-ation of CHO cells. The dot-like aggregation might be formedby repetitive endogenous receptor activation.

In the present study, Triton-insoluble �PKC-GFP seemed tobe inactive because it was marginally phosphorylated at Thr514

in the activation loop (Fig. 8, C and D), which is phosphorylatedby PDK1 and is most essential for kinase activity in the threemain phosphorylation sites of �PKC (Thr514, Thr655, andThr674) (21–23, 28, 29, 40, 41) (Fig. 1). Because the insolubilityof �PKC-GFPs to Triton X-100 were positively correlated (r �

0.817) with the extent of its aggregation, this result wouldimply that aggregated �PKC-GFP was unphosphorylated andinactive. This raises the possibility that the reduction of �PKCfunction, caused by the aggregate formation, is the etiology ofSCA14. Indeed, �PKC knock-out mice showed mildly impairedmotor coordination and mild deficits in spatial and contextuallearning (12, 42), similar pathologies observed in SCA14. How-ever, these symptoms seemed to be more severe in SCA14patients than in knock-out mice although mutations found inSCA 14 are heterozygous, leading to the idea that the gain oftoxic function, not the loss of �PKC function derived frommutant �PKC, would trigger various cerebellar symptoms.

In the recent study reported by Verbeek et al. (38) the in-crease of basal kinase activity was observed in G118D andC150F mutant �PKC-GFPs. They also demonstrated that thesemutant �PKC-GFPs had similar phosphorylation levels atthree sites to wild-type �PKC-GFP. These findings appear toconflict with our findings. This discrepancy is interpreted bythe difference in the timing of observation. In the presentstudy, our experiments were conducted 2 or 3 days after trans-fection, whereas Verbeek et al. observed the phosphorylationand kinase activity 24 h after transfection. We confirmed that

FIG. 10. Flow cytometric analysis of cell death in CHO cells expressing wild-type and mutant �PKC-GFPs. Transfected cells werecultured for 3 days and then stained with 7-AAD, a dye targeting dead cells. Stained cells were separated by two dimensions, GFP and 7-AADfluorescence using flow cytometry. A and B, representative GFP/7-AAD dot plots of CHO cells expressing wild type (A) and S119P (B) mutant�PKC-GFPs. Cells in right upper and lower quadrants (GFP(�) in A) were considered to express �PKC-GFP. Cells in upper right and left quadrants(7-AAD(�) in A) were considered to be dead. C–E, percentages of dead cells in GFP-positive CHO cells expressing WT and mutant �PKC-GFPsanalyzed by flow cytometry. The results of total GFP(�) cells were shown in C. Total GFP(�) cells were divided into two groups, cells with low andhigh GFP fluorescence (Low GFP and High GFP, respectively). The percentages of dead cells in low and high GFP groups are shown in D and E,respectively. *, p � 0.05; **, p � 0.01 versus WT (unpaired Student’s t test, n � 3).

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lower aggregation and lower insolubility to Triton X-100 ofmutant �PKC-GFPs were observed 1 day after transfection inour experiments (data not shown). Furthermore, our prelimi-nary study demonstrated that the basal kinase activities werenot increased in all of the SCA14 �PKC mutants, although weconfirmed the increased basal activity of G118D.2 This resultsuggests that the elevated basal activity found in G118D couldnot simply account for the whole aspect of SCA14 pathogenesis.

Our present study demonstrated that the susceptibility toaggregate formation and solubility to Triton X-100 variedamong mutant �PKC-GFPs found in several SCA14 families(Figs. 2 and 7). To explore whether these variations are corre-lated with the clinical features of each SCA14 family, we in-vestigated the relationship between the Triton solubility ofmutant �PKC-GFPs and average age at onset of ataxia in fiveSCA families (H101Y, G118D, S119F, Q127R, and F643L) inwhich more than five persons were reported to be affected. Asshown in Fig. 11, the average onset age of each family isnegatively correlated with the insolubility of the corresponding�PKC mutant (r � �0.8115). This strongly suggests that insol-uble �PKC formed by the missense mutation is implicated inthe pathogenesis of SCA14.

Although the formation of insoluble aggregation is thoughtto exert neurotoxic effects in several inherited neurodegenera-tive diseases (3, 24), the precise molecular mechanism is un-certain. The ubiquitin-proteasome system (UPS) is one of themajor proteolytic pathways in mammalian cells that is in-volved in the degradation of cytosolic short-lived proteins (43).The UPS is also involved in the elimination system of inappro-priate folded proteins, which prevents them from aggregating.Age-related decline of UPS function results in the increasedaccumulation and aggregation of misfolded proteins. Proteinaggregation further exacerbates UPS dysfunction by seques-trating the 26 S proteasome complex into the aggregation (44).This is the commonly accepted mechanism of neuronal celldeath in neurodegenerative diseases accompanying protein ag-

gregation (44–46). In our preliminary experiments, increasedubiquitination was found in several mutant �PKC-GFPs. Al-though further studies are necessary to elucidate the involve-ment of UPS in SCA14 pathogenesis, it is plausible that mu-tant �PKC aggregation exerts its cytotoxic effects on neuronsby disturbing the UPS.

In various neurodegenerative diseases, the disease-specificinclusion bodies, for example Lewy bodies in Parkinson dis-ease, neurofibrillary tangles and senile plaques in Alzheimerdisease, and nuclear inclusion bodies in Huntington disease (3,24) were observed. However, the autopsy report from theSCA14 patient having the H101Y mutation revealed no aggre-gation of �PKC in the cerebellar neurons (6). At present, we donot have adequate answers to explain why the mutant �PKCdoes not form aggregates in the Purkinje cells of actual SCA14patients. Further autopsy studies from specimens with muta-tions other than H101Y are necessary to resolve the discrep-ancy because the H101Y mutant had the lowest tendency toaggregate among the mutant PKC-GFP examined in the pres-ent study (Fig. 2D and Table I).

The findings in the present study provide the possibility thatSCA14 is caused by a mechanism similar to other neurodegen-erative diseases; that is the accumulation of aggregated pro-tein. Although further studies are necessary to identify theprecise molecular mechanism of mutant �PKC to cause SCA14,the identification may lead to effective therapeutic methods notonly for SCA14 but also for other neurodegenerative diseases.

Acknowledgment—This work was carried out with equipment fromthe Analysis Center of Life Science, Hiroshima University.

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FIG. 11. The insolubility of mutant �PKC-GFP is negativelycorrelated with age at onset of affected patients in SCA14 fam-ilies. The dot plot shows the relationship between the insolubility ofeach mutant �PKC-GFP, shown in Fig. 6B, and the distribution of agesat onset in the corresponding SCA14 family. A missense mutation of�PKC in each family is shown in the box. We analyzed five families(H101Y, G118D, S119F, Q127R, and F643L) where more than fivepatients were reported to be affected. The bar in each family representsthe average age at onset. The dotted line indicates the negative corre-lation between the insolubility to Triton X-100 and the average age atonset (r � �0.811).

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and Norio SakaiAmano, Hiroaki Matsubayashi, Masayasu Matsumoto, Hideshi Kawakami, Naoaki Saito Takahiro Seki, Naoko Adachi, Yoshitaka Ono, Hideki Mochizuki, Keiko Hiramoto, Taku

Aggregation and Causes Cell Death Found in Spinocerebellar Ataxia Type 14 Is Susceptible toγMutant Protein Kinase C

doi: 10.1074/jbc.M501716200 originally published online June 17, 20052005, 280:29096-29106.J. Biol. Chem. 

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