Mutant Protein Kinase Cγ Found in Spinocerebellar Ataxia Type 14 Is Susceptible to Aggregation and Causes Cell Death

Abstract

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease characterized by various 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, we examined 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 the cytoplasm of CHO cells, whereas mutant ␥PKC-GFP tended to aggregate in the cytoplasm. The insolubility of mutant ␥PKC-GFP to Triton X-100 was increased and correlated with the extent of aggregation. ␥PKC-GFP in the Triton-insoluble fraction was rarely phosphorylated at Thr 514 , whereas ␥PKC-GFP in the Triton-soluble fraction was phosphorylated. Furthermore, the stimulation of the P2Y receptor triggered the rapid aggregation of mutant ␥PKC-GFP within 10 min after transient translocation to the plasma membrane. Overexpression of the mutant ␥PKC-GFP caused cell death that was more prominent than wild type. The cytotoxicity was exacerbated in parallel with the expression level of the mutant. These results indicate that SCA14 mutations make ␥PKC form cytoplasmic aggregates, suggesting the involvement of this property in the etiology of SCA14.

Effect of Trehalose on the Properties of Mutant γPKC, Which Causes Spinocerebellar Ataxia Type 14, in Neuronal Cell Lines and Cultured Purkinje Cells*

“…Although we have shown that most of the SCA14 mutant ␥PKCs have increased basal kinase activities, several mutations actually have lower or unchanged activities (48). In contrast to controversial results concerning the kinase activity of mutant ␥PKC, we confirmed that most of the mutant ␥PKCs found in SCA14 tend to form aggregates (19). Furthermore, we and another group have recently demonstrated that the aggregation of mutant ␥PKC triggers apoptosis by impairing the ubiquitin proteasome system and inducing endoplasmic reticulum stress (21,49).…”

“…Immunoblotting and Chase Assay-Cells on 6-cm-diameter dishes were analyzed by immunoblotting using an anti-GFP antibody as described previously (19). Briefly, cells were lysed in radioimmune precipitation assay buffer (1% Nonidet P40, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 20 g/ml leupeptin, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM sodium orthovanadate, 1 mM NaF, 100 nM Calyculin A and 10 mM Tris/HCl, pH 7.4) by sonication (UR-20P, TOMY SEIKO, Tokyo; output, 4; duty, 50%).…”

“…mutations that have been identified in SCA14 families, two mutant versions of ␥PKCs (S119P and G128D) were selected for this study; these mutants have been demonstrated to form aggregates at a high frequency (19). Wild-type and mutant ␥PKC-GFPs (WT-GFP, S119P-GFP, and G128D-GFP) were expressed in SH-SY5Y cells, a neuronal cell line, using two adenoviral vectors: Ad-CMV-tTA, which carries the tetracycline transactivator (tTA) gene downstream of the CMV promoter, and Ad-TetOp-␥PKC-GFP, which uses the tTA-operated promoter (TetOp promoter) to transactivate ␥PKC-GFP expression (supplemental Fig.…”

“…We have previously demonstrated that mutant versions of ␥PKC tend to form aggregates in cultured cells (19) and mouse primary cultured PCs (20). This aggregation causes apoptotic cell death by inhibiting the ubiquitin proteasome system and inducing endoplasmic reticulum stress (21).…”

Protein kinase C γ mutations in the C1B domain cause caspase-3-linked apoptosis in lens epithelial cells through gap junctions

“…PKCγ acts as an oxidative stress sensor to prevent the lens from oxidative damage through proper control of gap junctions. It has been reported recently that missense mutations in PKCγ cause the dominant spinocerebellar ataxia type 14 (SCA14), a neurodegenerative disorder with onset age as early as three years (Chen et al, 2003, 005;van de Warrenburg et al, 2003;Stevanin et al, 2004;Yabe et al, 2003, Verbeek et al, 2005Seki et al, 2005;Alonso et al, 2005;Klebe et al, 2005;Vlak et al, 2006;Fahey et al, 2005). Most of the PKCγ SCA14 mutations occur in the C1B subdomain.…”