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.
Highlights
Gressive motor incoordination affecting the gait and limbs, cerebellar dysarthria, and nystagmus due to degeneration of cerebellar Purkinje cells
Spinocerebellar ataxia type 14 (SCA14) Mutant PKC␥ Exhibits Prolonged Membrane Localization—As 18 of 22 mutations in SCA14 families are located in the C1 domain of PKC␥ (Fig. 1) and the C1 domain is responsible for membrane binding in PKC translocation, we first examined whether these C1 domain mutations affect the membrane targeting of PKC␥
CHOhm1 cells expressing wild-type or mutant PKC␥-DsRed (H101Y, G118D, S119P, plasm within 60 s (Fig. 2, C and D). These results indicate that extracellular Ca2ϩ is responsible for the prolonged membrane localization of C1 domain mutant PKC␥
Summary
PKC␥, protein kinase C␥; SCA14, spinocerebellar ataxia type 14; Cch, carbachol; DAG, diacylglycerol; TRPC, canonical transient receptor potential; PS, phosphatidylserine; TPA, 12-O-tetradecanoylphorbol 13-acetate; DsRed, DsRed monomer; CHOhm, Chinese hamster ovary cells expressing human muscarinic acetylcholine receptor 1; [Ca2ϩ]i, intracellular Ca2ϩ; t1⁄2 [Ca2ϩ]i, the period required to reach the half-maximal level of intracellular Ca2ϩ; TIRF, total internal reflection fluorescence; DiC10, (Ϯ)-1,2-didecanoylglycerol; GFP, green fluorescent protein; GST, glutathione S-transferase. 22 different PKC␥ mutations have been found in SCA14 families, 18 of which map to the C1 domain [11,12,13,14,15,16]. This fact strongly suggests that these mutations disturb a fundamental property of PKC␥ including membrane translocation and activator-dependent regulation of its kinase activity. Our results show that C1 domain mutants have a shorter residence time on the plasma membrane. This results in decreased phosphorylation of the TRPC3 channel and alters Ca2ϩ influx. Our results suggest that alteration in Ca2ϩ homeostasis induced by mutant PKC␥ may contribute to SCA14
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