Enzymological analysis of mutant protein kinase Cgamma causing spinocerebellar ataxia type 14 and dysfunction in Ca2+ homeostasis

J Biol Chem. 2008 Jul 11;283(28):19854-63. doi: 10.1074/jbc.M801492200. Epub 2008 May 22.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Diglycerides
  • Receptors, Muscarinic
  • TRPC Cation Channels
  • TRPC3 cation channel
  • protein kinase C gamma
  • Protein Kinase C
  • Calcium