PKCγ 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 (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.