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

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