Enzymological Analysis of Mutant Protein Kinase Cγ Causing Spinocerebellar Ataxia Type 14 and Dysfunction in Ca2+ Homeostasis*

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase Cγ (PKCγ). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKCγ kinase activity. To determine the effect of these mutations on PKCγ function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKCγs. We found that wild-type PKCγ, but not C1 domain mutants, inhibits Ca2+ influx in response to muscarinic receptor stimulation. The sustained Ca2+ influx induced by muscarinic receptor ligation caused prolonged membrane localization of mutant PKCγ. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca2+ influx regulated by PKCγ. Although in vitro kinase assays revealed that most C1 domain mutants are constitutively active, they could not phosphorylate TRPC3 channels in vivo. Single molecule observation by the total internal reflection fluorescence microscopy revealed that the membrane residence time of mutant PKCγs was significantly shorter than that of the wild-type. This fact indicated that, although membrane association of the C1 domain mutants was apparently prolonged, these mutants have a reduced ability to bind diacylglycerol and be retained on the plasma membrane. As a result, they fail to phosphorylate TRPC channels, resulting in sustained Ca2+ entry. Such an alteration in Ca2+ homeostasis and Ca2+-mediated signaling in Purkinje cells may contribute to the neurodegeneration characteristic of SCA14.

gressive motor incoordination affecting the gait and limbs, cerebellar dysarthria, and nystagmus due to degeneration of cerebellar Purkinje cells. SCA14 is caused by missense or in-frame deletion mutations in the PRKCG gene encoding protein kinase C␥ (PKC␥) 2 (1). PKC␥ is a member of the PKC family that plays critical roles in many cellular functions, affecting diverse signal transduction pathways (2). PKC␥ is selectively expressed in neurons throughout the brain and is most abundant in cerebellar Purkinje cells (3), which specifically degenerate in SCA14 patients.
One of the characteristic features of PKC␥ is its translocation from the cytoplasm to the plasma membrane (4). Translocation is a hallmark of enzyme activation and is triggered by the stimulation of G protein-coupled receptors. It is well known that activation of such receptors causes elevations of DAG and intracellular Ca 2ϩ (5). PKC␥ contains C1 and C2 domains in its regulatory domain (6). The C1 domain has two zinc-finger motifs, C1A and C1B, that contain highly conserved Cys residues that bind to diacylglycerol (DAG) and tumor promoting phorbol esters. The C2 domain is a Ca 2ϩ sensor that binds phosphatidylserine (PS) in the presence of elevated Ca 2ϩ . The C1 and C2 domains play crucial roles in PKC␥ translocation through binding to DAG and Ca 2ϩ , respectively.
The pathogenesis of SCA14 is not well understood. PKC␥ knock-out mice exhibit persistent cerebellar multiple climbing fiber innervation and a slight ataxia. However, they do not have cerebellar degeneration (7,8). Thus, gain-of-function, rather than loss-of-function, of PKC␥ may be responsible for the cerebellar neurodegeneration in SCA14. Previously, we have reported that mutant PKC␥s aggregate and cause cell death of several cultured cell systems (9,10). However, the aggregation was not observed in the Purkinje cells of a SCA14 patient who carried the H101Y mutation (1). This suggests that not only the * This work was supported in part by a Grant-in-aid for Scientific Research from the Global Center of Excellence (COE) Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan, and grants from the Astellas Foundation for Research on Metabolic Disorders and Takeda Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4 and Videos 1-4. 1 To whom correspondence should be addressed. Tel.: 81-78-803-5962; Fax: 81-78-803-5971; E-mail: naosaito@kobe-u.ac.jp.
aggregated, but also the soluble, fraction of mutant PKC␥ may contribute to the pathogenesis of SCA14. To date, 22 different PKC␥ mutations have been found in SCA14 families, 18 of which map to the C1 domain (11)(12)(13)(14)(15)(16). This fact strongly suggests that these mutations disturb a fundamental property of PKC␥ including membrane translocation and activator-dependent regulation of its kinase activity. In the present study, we tested whether these C1 domain mutations influence membrane translocation and activation of PKC␥. Our results show that C1 domain mutants have a shorter residence time on the plasma membrane. This results in decreased phosphorylation of the TRPC3 channel and alters Ca 2ϩ influx. Our results suggest that alteration in Ca 2ϩ homeostasis induced by mutant PKC␥ may contribute to SCA14.
Plasmid Constructions-Wild-type and mutant PKC␥s were cloned into pcDNA3 with GFP or DsRed monomer (DsRed) as described previously (9). All mutant PKC␥ cDNAs were verified by sequencing. For cDNA cloning of human TRPC3, total RNA was extracted from HEK-293 cells using the SV Total RNA Isolation System (Promega Corporation, Madison, WI). After DNase I treatment, total RNA was reverse-transcribed into first-strand cDNA using random primers and the Thermo-Script TM RT-PCR System (Invitrogen). PCR primers were designed based on a published nucleotide sequence of human TRPC3 (GenBank TM accession number NM_003305). Amplified TRPC3 gene was cloned into p3XFLAG-CMV-10 (Sigma). TRPC3 cDNA was verified by sequencing.
Observation of PKC␥ Translocation-The culture medium of CHOhm1 cells expressing wild-type or mutant PKC␥-GFPs (or PKC␥-DsReds) was replaced with Ringer's solution (135 mM NaCl, 5.4 mM KCl, 1 mM MgCl 2 , 5 mM HEPES pH 7.35, 10 mM glucose). CaCl 2 was added to the appropriate Ca 2ϩ concentration depending on the experimental conditions. The fluorescence of the GFP was monitored with a confocal laser scanning fluorescent microscope (LSM510, Carl Zeiss, Jena, Germany) at 488-nm argon excitation with a 515-535-nm band pass barrier filter, DsRed was excited with a 543-nm HeNe laser and detected using a 560-nm long pass filter. All experiments were done at 37°C. Image analysis was performed using the Zeiss LSM 510 software, and the membrane fluorescence ratio was calculated as described previously (19).
Intracellular Ca 2ϩ Measurement-CHOhm1 cells expressing wild-type or mutant PKC␥-DsRed were incubated with 4 M Fluo-4 (Dojindo, Kumamoto, Japan) and 0.02% pluronic F-127 (Dojindo) in Ringer's solution containing 2 mM EGTA for 25 min at room temperature. Fluo-4 was loaded into SH-SY5Y and HEK-293 cells in Ringer's solution containing 2 mM Ca 2ϩ (20 min, 37°C). The cells were then washed twice with Ca 2ϩfree Ringer's solution. The fluorescence of Fluo-4 was monitored by confocal microscopy. The time course of Ca 2ϩ measurement was recorded using the Zeiss LSM 510 software. Fluorescence changes (⌬F/F 0 ) within the cytoplasm of the cells were quantified as the fraction of the evoked change in Fluo-4 Ca 2ϩ signal divided by the resting fluorescence level in Ca 2ϩ free buffer recorded for 30 s prior to drug application. All cells within the plane of focus that had a change in Ca 2ϩ signal were analyzed. All experiments were done at room temperature.
In Vitro PKC Kinase Assay-COS-7 cells expressing wildtype or mutant PKC␥-GFPs were harvested and only the supernatant fraction of the recombinant PKC was used for the PKC kinase assay. Briefly, the cells were centrifuged, and resus- pended in 400 l of homogenization buffer (250 mM sucrose, 10 mM EGTA, 2 mM EDTA, 20 mM Tris-HCl, 200 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, pH 7.4). After sonication, samples were centrifuged (200,000 ϫ g for 15 min at 4°C), and the supernatant was collected. To equalize the amount of PKC␥-GFP or its mutant proteins, GFP fluorescence intensity of the supernatants were measured by Mithras LB 940 (Berthold Technologies, Tokyo, Japan). For immunoprecipitation of PKC␥-GFP, an equivalent amount of GFP from each sample was rotated with anti-GFP antibody (1 h at 4°C) and then precipitated with protein A-Sepharose for an additional 1 h. The beads were collected and washed five times with phosphatebuffered saline (Ϫ). Finally, 10 l of suspended pellet was used for kinase assay as described previously (4). Briefly, the kinase activity was assayed by measuring the incorporation of 32 P into myelin basic protein from [␥-32 P]ATP in the absence of activators (20 mM Tris-HCl and 0.25 mM EGTA only) or in the presence of 8 g/ml PS (Sigma), 0.8 g/ml (Ϯ)-1,2-didecanoylglycerol (DiC10, BIOMOL, Plymouth Meeting, PA), and 50 M Ca 2ϩ .
Single-molecule Imaging of PKC␥-GFP in Living CHOhm1 Cells-The apparatus, data acquisition, and data analysis will be described in detail. 3 Briefly, individual PKC␥-GFP molecules in the plasma membrane of CHOhm1 cells were visualized using an objective-type total internal reflection microscope constructed on an inverted fluorescence microscope (IX81, Olympus, Japan) (20,21). Cells were illuminated with 488 nm light from a blue laser (Sapphire 488; Coherent, Japan) using a ϫ100 objective (PlanApo, NA ϭ 1.45; Olympus, Japan). Fluorescence images were intensified with image intensifier (C8600; Hamamatsu Photonics, Japan) and obtained at video rate with an electron bombardment CCD camera (C7190-23; Hamamatsu Photonics). The fluorescent spots were tracked and analyzed to determine the residence time of PKC␥ molecules on the plasma membrane.

SCA14 Mutant PKC␥ Exhibits Prolonged Membrane
Localization-As 18 of 22 mutations in SCA14 families are located in the C1 domain of PKC␥ (Fig. 1) and the C1 domain is responsible for membrane binding in PKC translocation, we first examined whether these C1 domain mutations affect the membrane targeting of PKC␥. CHOhm1 cells expressing wild-type or mutant PKC␥-DsRed (H101Y, G118D, S119P, Q127R, and G128D) were stimulated with Cch and membrane translocation assessed by confocal microscopy. Both wild-type and mutant PKC␥-DsRed translocated in response to Cch (supplemental Fig. S1). However, whereas wildtype PKC␥ rapidly returned ("retranslocated") to the cytoplasm, retranslocation was delayed in the C1 domain mutants. To assess the temporal differences in re-translocation between wild-type and mutant PKC␥, wild-type PKC␥-DsRed and GFP-conjugated PKC␥ carrying the G128D mutation were independently expressed in CHOhm1 cells and simultaneously observed (Fig. 2,  A and B). Both wild-type and mutant PKC␥ rapidly translocated to the plasma membrane in response to Cch. However, whereas re-translocation of wild-type PKC␥ was complete by 80 s, membrane levels of the G128D mutant were still maximal. In fact, this prolonged membrane localization continued for at least 90 s; in some cases, it remained more than 3 min after stimulation (data not shown). Extracellular Ca 2ϩ Is Responsible for Prolonged Membrane Localization of Mutant PKC␥-We have evidence that C1B peptides carrying SCA14 mutations (H101Y, S119P, Q127R, and V138E) have a decreased affinity for phorbol esters (PDBu). 4 Not surprisingly, the G128D mutation also exhibited lower phorbol 12,13-dibutyrate binding (data not shown). Thus, the affinity of the G128D mutant for endogenous DAG is also likely to be attenuated by mutations in the C1B domain. How then, are the mutants retained at the membrane? We tested the hypothesis that one of the other translocation factors, namely Ca 2ϩ , is involved in retaining the C1 domain mutants at the plasma membrane. Indeed, addition of the Ca 2ϩ chelator, EGTA, to the extracellular solution resulted in immediate retranslocation of G128D mutant PKC␥-GFP (Fig. 2, A and B). In addition, in Ca 2ϩ -free Ringer's solution, although G128D mutant exhibited a slight delay (about 10 s) in re-translocation compared with wild-type, both enzymes returned to the cytoplasm within 60 s (Fig. 2, C and D). These results indicate that extracellular Ca 2ϩ is responsible for the prolonged membrane localization of C1 domain mutant PKC␥.
The Kinase Activity of PKC␥ Is Required for Negative Regulation of Ca 2ϩ Entry-We then investigated the effect of overexpression of PKC␥ on Cch-induced Ca 2ϩ entry in CHOhm1 cells. Ca 2ϩ mobilization was assessed in Fluo4-loaded cells expressing wild-type PKC␥-DsRed or unconjugated DsRed as a control. As shown in Fig. 3A, Cch induced a rapid elevation of intracellular Ca 2ϩ ([Ca 2ϩ ] i ) in both cells. However, the kinetics of the Ca 2ϩ decrease in the cells expressing PKC␥-DsRed was distinct from DsRed. Specifically, the t1 ⁄ 2 [Ca 2ϩ ] i of PKC␥-DsRed was 28.34 Ϯ 5.44 s compared with 70.30 Ϯ 8.69 s in DsRed-expressing cells. In addition, in Ca 2ϩ -free solution, the kinetics of Ca 2ϩ decrease for PKC␥-DsRed and DsRed were identical (Fig. 3B). This data suggests that PKC␥ modulates [Ca 2ϩ ] i through plasma membrane calcium channels.
To investigate whether catalytic activity is required for the negative regulation of Cch-induced Ca 2ϩ entry, we examined the effect of PKC inhibitor, GF 109203X, on cells expressing wild-type PKC␥-DsRed or DsRed. The t1 ⁄ 2 [Ca 2ϩ ] i of PKC␥-DsRed was prolonged in a dose-dependent manner in the presence of GF109203X (Fig. 3C). Moreover, the t1 ⁄ 2 [Ca 2ϩ ] i for cells expressing a kinase-negative mutant (K380M) was significantly longer than that of wild-type in Ringer's solution containing Ca 2ϩ (Fig. 3D). These results suggest that kinase activity of PKC␥ is necessary for a rapid Ca 2ϩ decay in response to Cch.
C1 Domain Mutants Cannot Inhibit Ca 2ϩ Entry-We then examined the effect of 17 missense mutations and one deletion mutation on the regulation of [Ca 2ϩ ] i in CHOhm1 cells (Fig.  4A). Interestingly, the t1 ⁄ 2 [Ca 2ϩ ] i of all C1 domain mutants was significantly longer in the presence of extracellular Ca 2ϩ com-pared with its absence; t1 ⁄ 2 [Ca 2ϩ ] i of wild-type was not significantly altered by addition or depletion of the extracellular Ca 2ϩ (Fig. 4A). Thus, we concluded that the sustained high levels of [Ca 2ϩ ] i are likely responsible for the prolonged membrane localization of these C1 domain mutant PKC␥s.
The kinase activity of PKC␥ is required for a rapid Ca 2ϩ decay in the cells (Fig. 3, C-E). However, the C1 domain mutants could not decrease [Ca 2ϩ ] i after Cch stimulation. Therefore, we hypothesized that the C1 domain mutants were catalytically inactive. It is known that PKC␥ is activated by PS and the DAG analog, DiC10. In addition, its kinase activity can be further elevated by the addition of Ca 2ϩ (supplemental Fig. S3). In this system, the kinase activity of wild-type PKC␥ was enhanced 2.5-3-fold in the presence of PS, DiC10, and Ca 2ϩ compared with their absence (i.e. Tris-HCl buffer with EGTA only). In contrast, all C1 domain mutants had high kinase activity, even in the absence of activators (Fig. 4B). Despite their high intrinsic kinase activity, the C1 domain mutants could not regulate [Ca 2ϩ ] i levels.
In regard to other four kinase domain mutants, we also observed translocation (supplemental Fig. S1), regulation of [Ca 2ϩ ] i (Fig. 4A), and kinase activity (Fig.  4B). The V692G mutant was indistinguishable from wild-type. On the other hand, the inhibitory effect of Ca 2ϩ entry in cells expressing constitutively active S361G and F643L mutants was exaggerated compare with wild-type (supplemental Fig. S2). Interestingly, the kinase negative G360S mutant exhibited prolonged membrane localization and sustained [Ca 2ϩ ] similar to the C1 domain mutants. These data suggest that in addition to disruption of the C1 domain, a decrease or increase in its kinase activity ablates Cch-induced Ca 2ϩ regulation in CHOhm1 cells.
PKC␥ Negatively Regulates TRPC Channels in Response to Cch-Cch induces Ca 2ϩ influx through receptor-mediated Ca 2ϩ channels such as TRPC channels (22). Therefore, to test the hypothesis that PKC␥ modulates TRPC channel activity to inhibit the Cch-induced Ca 2ϩ entry, we examined the effect of various Ca 2ϩ channel inhibitors on Ca 2ϩ levels in cells expressing wild-type or G128D mutant PKC␥-DsRed. As shown in Fig.  5A, the t1 ⁄ 2 [Ca 2ϩ ] i of G128D mutant was significantly longer than that of wild-type. Treatment with the TRPC channel inhibitors 2-aminoethoxydiphenyl borate and SKF-96395 completely abolished the excessive Ca 2ϩ entry in cells expressing the G128D mutant. Conversely, blocking the Na ϩ /Ca 2ϩ exchanger or the L-type Ca 2ϩ channel with nifedipine and SEA0400, respectively, had no effect. These results suggest that the Cch-induced change in [Ca 2ϩ ] i is mediated by TRPC channels. We next examined the effect of PKC␥ on the Cch-induced Ca 2ϩ response in HEK-293 and human neuroblastoma SH-SY5Y cells, cells that endogenously express muscarinic receptors. Similarly to CHOhm1 cells, wild-type, but not the G128D mutant, negatively regulated Ca 2ϩ influx in both cell types (Fig. 5B). This result suggests that the negative regulation of [Ca 2ϩ ] i by PKC␥ likely occurs in neurons that endogenously express PKC␥ and TRPC channels. Interestingly, although much of the data on Ca 2ϩ responses in HEK-293 cells were similar to CHOhm1 and SH-SY5Y cells, Ca 2ϩ oscillations were frequently observed in cells expressing wild-type but not the G128D mutant (data not shown). Taken together, these results suggest that Ca 2ϩ influx regulated by PKC␥ likely depends on plasma membrane Ca 2ϩ channels, especially TRPC channels. However, the G128D mutant lacks the ability to regulate these channels.
A C1 Domain Mutant Fails to Phosphorylate TRPC3 Channel in Vivo-Among the TRPC subfamily, the TRPC3 channel is expressed predominantly in the brain, especially cerebellar Purkinje cells (23,24), which is rich in PKC␥. In addition, both TRPC3 channels and PKC␥ are activated by DAG (25), suggest-ing that PKC␥ could phosphorylate the TRPC3 channel at the plasma membrane of Purkinje cells. In fact, PKC␣ has been reported to phosphorylate TRPC3 channels in vitro and in vivo (26). We first investigated whether the TRPC3 channel can be phosphorylated by PKC␥ in vitro. As shown in Fig. 6A, significant phosphorylation of FLAG-TRPC3 by GST-PKC␥ occurred in the presence of PS, DiC10, and Ca 2ϩ and phosphorylation was abolished by the PKC inhibitor GF 109203X. Thus, PKC␥ can directly phosphorylate TRPC3 in vitro. We next investigated whether wild-type and the G128D PKC␥ could phosphorylate TRPC3 channels in intact cells. For this purpose, we first stimulated the CHOhm1 cells co-expressing PKC␥-GFP and FLAG-TRPC3 with Cch. However, we failed to detect significant phophorylation of TRPC3 by wild-type PKC␥, possibly due to the transient nature of its association with membranes. Indeed, the translocation of wild-type PKC␥ elicited by Cch occurs transiently ( Fig. 2A). Therefore, TPA was used as it induces irreversible translocation of wildtype PKC␥ to the plasma membrane and maximally stimulates PKC␥ (4). In COS-7 cells co-expressing FLAG-TRPC3 and wild-type PKC␥-GFP, 200 nM TPA significantly enhanced the phosphorylation of FLAG-TRPC3, an increase of 1.89 Ϯ 0.14-fold in PKC␥ overexpressing cells (Fig.  6, B and C). On the other hand, in COS-7 cells co-expressing FLAG-TRPC3 and constitutively active G128D mutant PKC␥-GFP, FLAG-TRPC3 was significantly less phosphorylated, exhibiting an increase only of 1.33 Ϯ 0.25 in response to TPA, which was not significantly different from the GFP control. Similar results were obtained when another constitutively active C1 domain mutant (H101Y) was used (data not shown). These results suggest that C1 domain mutants cannot phosphorylate endogenous substrates, despite their high catalytic activity.
C1 Domain Mutant Decreases the Residence Time of PKC␥ on the Plasma Membrane-Inaccessibility of PKC␥ to the substrate may explain why the constitutively active C1 domain mutant PKC␥ do not phosphorylate TRPC3 channels in the cells. As C1B domain mutations have a decreased affinity for membrane lipids, 4 we used total internal reflection fluorescence (TIRF) microscopy to determine how long wild-type and mutant PKC␥-GFP (H101Y, G128D, and G360S) stay on the plasma membrane. When we imaged the ventral surface of the CHOhm1 cells expressing PKC␥-GFP using TIRF microscopy and a highly sensitive imaging system at the video rate (20,27), many fluorescent spots appeared on the plasma membrane (supplementary videos 1-4). We could not observe such spots in the mocktreated control cells. Because the distribution of their fluorescence intensity exhibited a single peak and the peak position of the distribution is slightly lower than that for recombinant GFP attached on the coverslip, or for GFP-tagged Ras imaged at the single molecule level (27) (supplementary Fig.  S4), we concluded that each spot mostly represents a single molecule of PKC␥-GFP.
Cells were stimulated with TPA to analyze membrane targeting through the C1 domain. In response to TPA, a single molecule of wild-type appeared at the plasma membrane and moved around in the plane of the membrane for more than 20 s. The residence time of wild-type on the membrane averaged 22.1 s (Fig. 7, left column, supplemental Videos 1-4). In contrast, the residence time of the H101Y and G128D C1 domain mutants was dramatically short (3.0 and 1.3 s, respectively). On the other hand, the G360S mutant, which has an intact C1 domain, was indistinguishable from wild type. We also measured the residence time of the C1 domain mutant in response to Cch. Compared with wild-type PKC␥, the H101Y and G128D mutants had significantly shorter residence times (ϳ60% of wild type) (Fig. 7, right column), suggesting that C1 domain mutants do not stably associate with their physiological binding partner DAG. The result of this shortened membrane retention time is decreased phosphorylation of PKC␥ substrates, such as TRPC3 channels, on the plasma membrane.

DISCUSSION
SCA14 is inherited as autosomal dominant and PKC␥-deficient mice do not exhibit atrophy of the cerebellum or loss of Purkinje cells (8). Therefore, we considered that gainof-function, rather than loss-offunction, of mutant PKC␥ causes neurodegeneration in SCA14. Previously, we demonstrated that, similar to other neurodegenerative disorders, the mutant PKC␥ found in SCA14 tends to aggregate and cause cell death (9). However, although immunoreactivity of PKC␥ was reduced, the aggregation was not observed in Purkinje cells of an autopsy of a SCA14 patient who carried the H101Y mutation (1). Therefore, we suspected the gainof-toxic function of the soluble protein in addition to the aggregation that mutant PKC␥ may contribute to neurodegeneration in SCA14.
In the present study, we have shown that expression of C1 domain mutants of PKC␥s results in sustained Ca 2ϩ influx into the cells due to a decrease in membrane residence time. In addition, cells expressing the G360S mutant PKC␥ also caused sustained Ca 2ϩ influx resulting from its kinase inactivity. Wildtype PKC␥ negatively regulated Cch-induced Ca 2ϩ entry via phosphorylation of TRPC3 channels. For TRPC3 phosphorylation, both C1 domain-mediated membrane binding and kinase activity of PKC␥ are indispensable.
On the other hand, cells expressing S361G and F643L PKC␥s with an intact C1 domain and a high kinase activity, were able to suppress Ca 2ϩ entry after physiological stimulation. Interestingly, this inhibitory effect was exaggerated compared with wild-type and Ca 2ϩ influx was completely blocked. This finding suggests that a constitutively active PKC␥ with a functional C1 domain may cause hyperphosphorylation of substrates on the plasma membrane. Thus, not only the sustained Ca 2ϩ influx but also inhibition of Ca 2ϩ entry might alter intracellular Ca 2ϩ homeostasis in SCA14 patients (Fig. 8).
By confocal microscopy, TPA and the Ca 2ϩ ionophore induced similar PKC␥ localization at the plasma membrane (4,28). However, electron microscopy revealed that PKC␥ has similar, but significantly different, localization depending on stimulation. Whereas DAG or TPA tightly anchored this enzyme on the plasma membrane, Ca 2ϩ ionophore resulted in the accumulation of PKC␥ to a submembrane region Ͻ500 nm from the plasma membrane (28). Thus, we, in these studies, employed TIRF microscopy, which is generally able to visualize the PKC␥ within 100 -150 nm of the plasma membrane. TIRF provides a readout of events occurring essentially at the membrane. These studies revealed that the C1B mutants are at the plasma membrane for significantly less time that wild-type. This results in decreased signaling as measured by the lower phosphorylation of the TRPC3 channels.
PKC␥ has two DAG binding regions, C1A and C1B, and it is reported that both bind equally to DAG or phorbol ester (29). Although SCA14 mutations affect only one region of the C1 domain, the membrane residence time of mutant PKC␥s induced by Cch or TPA were significantly shorter than that of wild-type. These results suggest that both C1A and C1B con-tribute to the stable association of PKC␥ with membranes; mutations in either decrease membrane residence time.
The data in Fig. 2A suggested that C1 domain mutants stay longer on the plasma membrane than wild-type. Thus, we predicted increased substrate phosphorylation. However, this was not the case. Mutations in the C1 domain decrease PKC␥-DAG binding, membrane retention time, and substrate phosphorylation. Indeed, the G128D mutant could not phosphorylate TRPC3 on the plasma membrane. These results emphasize the importance of the C1 domain for membrane binding and substrate phosphorylation.
It is reported that of the 50 amino acids in the C1B domain in PKC␥, residues 101-143 mediate phorbol ester binding (30). Although C150F mutation is outside this core region, the C150F mutant could not control intracellular Ca 2ϩ concentrations and kinase activity. This indicates that C150F does not have a functional C1B domain and cannot associate with DAG. The substitution of phenylalanine for cysteine is thought to induce a conformational change in the C1B domain that inhibits DAG/phorbol ester binding. Increased kinase activity of the C150F mutant was also reported by Verbeek et al. (31). Moreover, they demonstrated that, in COS-7 cells, Ca 2ϩ ionophore induces more rapid translocation of the C150F mutant than wild-type PKC␥. We also observed that Ca 2ϩ influx-mediated rapid and enhanced translocation of PKC␥s carrying the C1 domain mutation in the CHOhm1 cell (data not shown). PKC is known to enhance Ca 2ϩ efflux through phosphorylation of the plasma membrane Ca 2ϩ -ATPases (32). As is the case with the lower phosphorylation of the TRPC3 channel by the G128D mutant, these C1 domain mutants may also fail to phosphorylate the plasma membrane Ca 2ϩ -ATPases. Therefore, enhancement of the Ca 2ϩ efflux by PKC␥ may not occur in cells expressing C1 domain mutant, thus, an increase of intracellular Ca 2ϩ would induce strong translocation of mutant PKC␥.
In Purkinje cells, TRPC3 is the most abundantly expressed member of the TRPC family (24). However, its specific roles in the neurons are largely unknown. It has been reported that the mGluR-dependent slow excitatory postsynaptic currents require the activation of TRPC channels in Purkinje cells (33). PKC␥ also plays a key role in mGluR signaling. Therefore, the loss of inhibition of TRPC activity due to its decreased phosphorylation by mutant PKC␥ may indirectly activate slow excitatory postsynaptic currents. Interestingly, an ataxic phenotype is observed in various knock-out mice that are deficient in the components of the mGluR cascade (e.g. mGluR1, G q , PLC␤, inositol 1,4,5-trisphosphate receptor, and PKC␥) (34 -37). Moreover, TRPC3 knock-out mice also exhibit abnormal motor coordination. 5 Therefore, abnormal regulation of TRPC channels, as seen in cells expressing C1B mutants of PKC␥, may contribute to cerebellar dysfunction.
Although intracellular Ca 2ϩ signals are critical for the regulation of synaptic functions in the central nervous system, sustained high levels of intracellular Ca 2ϩ can be toxic for neurons. For example, dysregulation of intracellular Ca 2ϩ has been reported in aging brain and been implicated in vulnerability to 5 A. Konnerth, personal communication. neurodegenerative diseases such as Alzheimer disease (38,39). In addition, increases in intracellular Ca 2ϩ through Ca 2ϩ -permeable ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid channels produce reactive oxygen species and, in some cases, nitric oxide (40). These reactive oxygen species and nitric oxide are known to cause neuronal injury in amyotrophic lateral sclerosis. Recent data demonstrates an abundance of S-nitosylated proteins in the brains of Alzheimer and Parkinson victims (41,42). Sustained high levels of intracellular Ca 2ϩ lead to increased nitric oxide, elevated S-nitrosylation of proteins, and protein aggregation. One potential target of nitric oxide is heat shock protein (Hsp) (17), whose function in cells is thought to repair misfolded proteins. However, its activity can be inhibited by S-nitrosylation. Thus, in our model, mutant PKC␥ causes a sustained high level of intracellular Ca 2ϩ that increases nitric oxide and S-nitrosylation of Hsp, and results in the accumulation of aggregated proteins. If this model is correct, we would predict that overexpression of Hsp should bypass the effects of mutant PKC␥, and decrease protein aggregation. In fact, current evidence from our laboratory indicates that protein aggregation and cytotoxicity induced by mutant PKC␥ can be inhibited by overexpression of Hsp70. 4 Although sustained Ca 2ϩ influx was caused by "loss-of-function" of PKC␥, it is possible that aberrant regulation of intracellular Ca 2ϩ enhances the misfolding of mutant PKC␥ in the long term.
In the case of the V692G mutation, which occurs in the variable region of the PRKCG gene, the patterns of kinase activity, membrane targeting, and intracellular Ca 2ϩ regulation were very similar to those of the wild-type enzyme. Interestingly, aggregation of this mutant was not observed in CHOhm1 and SH-SY5Y cells (data not shown). Thus, it is possible that another mutation might be responsible for the SCA phenotype.
To understand the pathogenesis of SCA14, it is necessary to compare Ca 2ϩ -mediated signaling, aggregation, and loss of PKC␥ in Purkinje cells derived from SCA14 patients with normal controls. However, as SCA14 is rare, but not fatal, it is difficult to study the pathogenesis of the disease using human brain tissue. Thus, the generation of an animal model of SCA14 is necessary.
In summary (Fig. 8), we have studied the effects of SCA14 PKC␥ mutations on Cch-induced Ca 2ϩ influx and TRPC channel phosphorylation. We have shown that the phosphorylation of TRPC channels is reduced in cells expressing the C1 domain mutant PKC␥, resulting in sustained high levels of intracellular Ca 2ϩ upon cell stimulation. Surprisingly, the mutant PKC␥ have higher kinase activity in vitro but cannot stably associate with the plasma membrane. These results suggest that mutant PKC␥s fail to phosphorylate TRPC3 channels leading to sustained high levels of intracellular Ca 2ϩ and aberrant intracellular signaling. Such an alteration in Ca 2ϩ homeostasis and Ca 2ϩmediated signaling in Purkinje cells may be responsible for the neurodegeneration characteristic of SCA14.