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J. Biol. Chem., Vol. 280, Issue 32, 29096-29106, August 12, 2005

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

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 Visual Sciences, the ^**Department of Neurosurgery, and the Department of Clinical Neuroscience and Therapeutics, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan and the Laboratory of Molecular Pharmacology and ^¶Biosignal Research Center, Kobe University, Kobe 657-8501, Japan

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 Thr⁵¹⁴, 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.

Received for publication, February 15, 2005 , and in revised form, June 15, 2005.

^* This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Sports and Culture and by a grant from Takeda Science Foundation and the Japanese Smoking Research Association. 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.

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 Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, 1-2-3 Kasumi, Hiroshima 734-8551, Japan. Tel.: 81-82-257-5142; Fax: 81-82-257-5144; E-mail: nsakai{at}hiroshima-u.ac.jp.

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