PKC
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 for publication 22 April 2008.
Abstract:
Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disorder caused by mutations in the neuronal-specific protein kinase C gamma (PKC
) gene. Since most mutations causing SCA14 are located in the PKC C1B regulatory subdomain, we investigated the impact of three C1B mutations on the intracellular kinetics, protein conformation and kinase activity of PKC in living cells. SCA14 mutant PKC proteins showed enhanced phorbol-ester-induced kinetics when compared with wild-type PKC. The mutations led to a decrease in intramolecular FRET of PKC, suggesting that they `open' PKC protein conformation leading to unmasking of the phorbol ester binding site in the C1 domain. Surprisingly, SCA14 mutant PKC 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 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
