title: Mutant protein kinase Cgamma 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 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.
authors.affiliation: Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Seki
authors.forename: Naoko
authors.initials: N
authors.lastname: Adachi
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Ono
authors.forename: Hideki
authors.initials: H
authors.lastname: Mochizuki
authors.forename: Keiko
authors.initials: K
authors.lastname: Hiramoto
authors.forename: Taku
authors.initials: T
authors.lastname: Amano
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Matsubayashi
authors.forename: Masayasu
authors.initials: M
authors.lastname: Matsumoto
authors.forename: Hideshi
authors.initials: H
authors.lastname: Kawakami
authors.forename: Naoaki
authors.initials: N
authors.lastname: Saito
authors.forename: Norio
authors.initials: N
authors.lastname: Sakai
mesh_terms: D000818:Animals; D016466:CHO Cells; D002449:Cell Aggregation; D016923:Cell Death; D002462:Cell Membrane / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D005192:Family Health; D005434:Flow Cytometry; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D009154:Mutation; D020125:Mutation, Missense; D017830:Octoxynol / Q000494:pharmacology; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics*; D021381:Protein Transport; D020754:Spinocerebellar Ataxias / Q000201:enzymology* / Q000235:genetics*; D013997:Time Factors; D014162:Transfection
chemical_list: D004396:Coloring Agents; D003902:Detergents; D049452:Green Fluorescent Proteins; D017830:Octoxynol; C087857:protein kinase C gamma; D011493:Protein Kinase C