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

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 PKC␥s. We found that wild-type PKC␥, but not C1 domain mutants, inhibits Ca 2؉ influx in response to muscarinic receptor stimulation. The sustained Ca 2؉ 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 Ca 2؉ 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 PKC␥s 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 Ca 2؉ entry. Such an alteration in Ca 2؉ homeostasis and Ca 2؉ -mediated signaling in Purkinje cells may contribute to the neurodegeneration characteristic of SCA14.Autosomal dominant SCA14 is a genetically heterogenous group of neurodegenerative disorders characterized by progressive motor incoordination affecting the gait and limbs, cerebellar dysarthria, and nystagmus due to degeneration of cerebellar Purkinje cells. SCA14 is caused by missense or in-frame deletion mutations in the PRKCG gene encoding protein kinase C␥ (PKC␥) 2 (1). PKC␥ is a member of the PKC family that plays critical roles in many cellular functions, affecting diverse signal transduction pathways (2). PKC␥ is selectively expressed in neurons throughout the brain and is most abundant in cerebellar Purkinje cells (3), which specifically degenerate in SCA14 patients.One of the characteristic features of PKC␥ is its translocation from the cytoplasm to the plasma membrane (4). Translocation is a hallmark of enzyme activation and is triggered by the stimulation of G protein-coupled receptors. It is well known that activation of such receptors causes elevations of DAG and intracellular Ca 2ϩ (5). PKC␥ contains C1 and C2 domains in its regulatory domain (6). The C1 domain has two zinc-finger motifs, C1A and C1B, that contain highly conserved Cys residues that bind to diacylglycerol (DAG) and tumor promoting phorbol esters. The C2 domain is a Ca 2ϩ sensor that binds phosphatidylserine (PS) in the presence of elevated Ca 2ϩ . The C1 and C2 domains play crucial roles in PKC␥ transloca...

Elucidation of the Molecular Mechanism and Exploration of Novel Therapeutics for Spinocerebellar Ataxia Caused by Mutant Protein Kinase Cγ

“…Therefore, it is possible that these missense mutations influence the kinase activity and targeting of γPKC. In vitro kinase assays have revealed that most mutant γPKCs have higher basal kinase activities (i.e., without a PKC activator), which are not increased in the presence of PKC activators (22). Similar elevations of basal kinase activity have also been reported from other groups (23,24).…”

“…Verbeek et al have demonstrated that mutant γPKC rapidly translocates to the plasma membrane in response to calcium ionophore and phorbol ester (23,26). We have revealed previously that mutant γPKC, especially the C1 domain mutant, shows prolonged membrane localization after translocation in response to receptor activation (22). This effect was due to sustained Ca 2+ influx from TRPC3, which is phosphorylated and inactivated by WT γPKC but not by mutant γPKC.…”

Effect of Trehalose on the Properties of Mutant γPKC, Which Causes Spinocerebellar Ataxia Type 14, in Neuronal Cell Lines and Cultured Purkinje Cells*

“…More recently, Asai et al has revealed that mutant ␥PKC with higher activity induces aberrant phosphorylation and localization of aprataxin, a DNA repair protein (47). Although we have shown that most of the SCA14 mutant ␥PKCs have increased basal kinase activities, several mutations actually have lower or unchanged activities (48). In contrast to controversial results concerning the kinase activity of mutant ␥PKC, we confirmed that most of the mutant ␥PKCs found in SCA14 tend to form aggregates (19).…”

Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

“…Most disease-causing mutations in PKC␥ occur in the C1 domain, which binds DAG and is necessary for translocation and regulation of PKC␥. Although the C1 domain mutants display kinase activity, they are unable to phosphorylate and thereby inactivate TRPC channels in vivo (Adachi et al, 2008). This failure to phosphorylate TRPC3 channels alters Ca 2ϩ homeostasis, which likely contributes to neurodegeneration (Matilla-Dueñas et al, 2010).…”