Enzymological Analysis of Mutant Protein Kinase C Causing Spinocerebellar Ataxia Type 14 and Dysfunction in Ca²⁺ Homeostasis^*

Naoko Adachi, Takeshi Kobayashi, Hideyuki Takahashi, Takumi Kawasaki, Yasuhito Shirai, Takehiko Ueyama, Toshio Matsuda^¶, Takahiro Seki^||, Norio Sakai^||, , and Naoaki Saito¹

Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, the Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, the ^¶Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, and the ^||Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan

Abstract: Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase C (PKC). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKC kinase activity. To determine the effect of these mutations on PKC function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKCs. We found that wild-type PKC, 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 PKC. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca²⁺ influx regulated by PKC. 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 PKCs 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.

Received for publication February 25, 2008. Revision received April 16, 2008.

^* This work was supported in part by a Grant-in-aid for Scientific Research from the Global Center of Excellence (COE) Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan, and grants from the Astellas Foundation for Research on Metabolic Disorders and Takeda Science Foundation. 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.

¹ To whom correspondence should be addressed. Tel.: 81-78-803-5962; Fax: 81-78-803-5971; E-mail: naosaito{at}kobe-u.ac.jp.

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES: