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

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

doi:10.1074/jbc.m501716200

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 Cgamma (gammaPKC) gene have been found in different SCA14 families. To elucidate how the mutant gammaPKC causes SCA14, we examined the molecular properties of seven mutant (H101Y, G118D, S119P, S119F, Q127R, G128D, and F643L) gammaPKCs fused with green fluorescent protein (gammaPKC-GFP). Wild-type gammaPKC-GFP was expressed ubiquitously in the cytoplasm of CHO cells, whereas mutant gammaPKC-GFP tended to aggregate in the cytoplasm. The insolubility of mutant gammaPKC-GFP to Triton X-100 was increased and correlated with the extent of aggregation. gammaPKC-GFP in the Triton-insoluble fraction was rarely phosphorylated at Thr(514), whereas gammaPKC-GFP in the Triton-soluble fraction was phosphorylated. Furthermore, the stimulation of the P2Y receptor triggered the rapid aggregation of mutant gammaPKC-GFP within 10 min after transient translocation to the plasma membrane. Overexpression of the mutant gammaPKC-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 gammaPKC form cytoplasmic aggregates, suggesting the involvement of this property in the etiology of SCA14.

Highlights

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease characterized by various symptoms including cerebellar ataxia
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)
We found a novel mutation around the C1 domain (Ser119 replaced with Phe) of ␥PKC in a Japanese SCA family (Fig, 1, bold lined box).2. To clarify how these mutations affect the molecular properties of ␥PKC and trigger neurodegeneration, we introduced these missense mutations into ␥PKC-GFP and investigated molecular properties of 7 mutant ␥PKC-GFPs (H101Y, G118D, S119P, S119F, Q127R, G128D, and F643L) expressed in CHO cells

Summary

EXPERIMENTAL PROCEDURES

Materials—ATP was purchased from Research Biochemical International (Natick, MA). Anti-GFP rabbit polyclonal antibody, Alexa546conjugated anti-rabbit IgG goat antibody and Alexa633-conjugated wheat germ agglutinin (WGA) were from Molecular Probes (Leiden, Netherlands). The filters were incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (diluted 1:10,000) for Ͼ30 min at room temperature. The cells were incubated with the anti-␥PKC polyclonal antibody (1:1000) and 5% NGS in PBS-T for 1 h at room temperature. After three times washing with PBS-T, the cells were incubated with Alexa546-conjugated goat anti-rabbit IgG antibody (1:500) and 5% NGS in PBS-T for 1 h at room temperature, followed by three washes with PBS-T. For staining with Golgi complex marker, the cells were incubated with 1 ␮g/ml Alexa633-conjugated WGA and 5% NGS in PBS-T for 40 min at room temperature, followed by three washes with PBS-T. The fluorescence of 2 ϫ 104 cells was recorded and analyzed by the Cell QuestTM software (BD Biosciences)

RESULTS

DISCUSSION

The findings in the present study provide the possibility that