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First published online 24 June 2008
doi: 10.1242/jcs.027698

Journal of Cell Science 121, 2339-2349 (2008)
Published by The Company of Biologists 2008
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Research Article

PKC{gamma} mutations in spinocerebellar ataxia type 14 affect C1 domain accessibility and kinase activity leading to aberrant MAPK signaling

Dineke S. Verbeek1,*, Joachim Goedhart2, Laurie Bruinsma1, Richard J. Sinke3 and Eric A. Reits1

1 Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
2 Section Molecular Cytology, Swammerdam Institute for Life Sciences, Centre for Advanced Microscopy, University of Amsterdam, The Netherlands
3 Department of Biomedical Genetics, University Medical Center Utrecht, University of Utrecht, The Netherlands

* Author for correspondence (e-mail: D.S.Verbeek{at}medgen.umcg.nl)

Accepted 22 April 2008

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disorder caused by mutations in the neuronal-specific protein kinase C gamma (PKC{gamma}) gene. Since most mutations causing SCA14 are located in the PKC{gamma} C1B regulatory subdomain, we investigated the impact of three C1B mutations on the intracellular kinetics, protein conformation and kinase activity of PKC{gamma} in living cells. SCA14 mutant PKC{gamma} proteins showed enhanced phorbol-ester-induced kinetics when compared with wild-type PKC{gamma}. The mutations led to a decrease in intramolecular FRET of PKC{gamma}, suggesting that they `open' PKC{gamma} protein conformation leading to unmasking of the phorbol ester binding site in the C1 domain. Surprisingly, SCA14 mutant PKC{gamma} showed reduced kinase activity as measured by phosphorylation of PKC reporter MyrPalm-CKAR, as well as downstream components of the MAPK signaling pathway. Together, these results show that SCA14 mutations located in the C1B subdomain `open' PKC{gamma} protein conformation leading to increased C1 domain accessibility, but inefficient activation of downstream signaling pathways.

Key words: Spinocerebellar ataxia, Protein kinase C gamma, GFP, FLIM, FRET


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PKC{gamma} opens up in ataxia

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