| RRC ID |
43370
|
| 著者 |
Adachi N, Kobayashi T, Takahashi H, Kawasaki T, Shirai Y, Ueyama T, Matsuda T, Seki T, Sakai N, Saito N.
|
| タイトル |
Enzymological analysis of mutant protein kinase Cgamma causing spinocerebellar ataxia type 14 and dysfunction in Ca2+ homeostasis.
|
| ジャーナル |
J Biol Chem
|
| Abstract |
Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase Cgamma (PKCgamma). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKCgamma kinase activity. To determine the effect of these mutations on PKCgamma function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKCgammas. We found that wild-type PKCgamma, 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 PKCgamma. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca2+ influx regulated by PKCgamma. 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 PKCgammas 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.
|
| 巻・号 |
283(28)
|
| ページ |
19854-63
|
| 公開日 |
2008-7-11
|
| DOI |
10.1074/jbc.M801492200
|
| PII |
S0021-9258(20)81437-9
|
| PMID |
18499672
|
| MeSH |
Animals
CHO Cells
COS Cells
Calcium / metabolism
Calcium Signaling* / genetics
Cell Membrane / enzymology*
Cell Membrane / genetics
Chlorocebus aethiops
Cricetinae
Cricetulus
Diglycerides / genetics
Diglycerides / metabolism
Humans
Mutation*
Phosphorylation
Protein Binding / genetics
Protein Kinase C / genetics
Protein Kinase C / metabolism*
Protein Structure, Tertiary / genetics
Purkinje Cells / enzymology*
Receptors, Muscarinic / metabolism
Spinocerebellar Ataxias / enzymology*
Spinocerebellar Ataxias / genetics
TRPC Cation Channels / genetics
TRPC Cation Channels / metabolism
|
| IF |
4.238
|
| 引用数 |
65
|
|
WOS 分野
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| リソース情報 |
| ヒト・動物細胞 |
COS-7(RCB0539)
293(RCB1637) |
