SCA14 mutation V138E leads to partly unfolded PKCγ associated with an exposed C‐terminus,altered kinetics,phosphorylation and enhanced insolubilization

The protein kinase C γ (PKCγ) undergoes multistep activation and participates in various cellular processes in Purkinje cells. Perturbations in its phosphorylation state, conformation or localization can disrupt kinase signalling, such as in spinocerebellar ataxia type 14 (SCA14) that is caused by missense mutations in PRKCG encoding for PKCγ. We previously showed that SCA14 mutations enhance PKCγ membrane translocation upon stimulation owing to an altered protein conformation. As the faster translocation did not result in an increased function, we examined how SCA14 mutations induce this altered conformation of PKCγ and what the consequences of this conformational change are on PKCγ life cycle. Here, we show that SCA14‐related PKCγ‐V138E exhibits an exposed C‐terminus as shown by fluorescence resonance energy transfer‐fluorescence lifetime imaging microscopy in living cells, indicative of its partial unfolding. This conformational change was associated with faster phorbol 12‐myristate 13‐acetate‐induced translocation and accumulation of fully phosphorylated PKCγ in the insoluble fraction, which could be rescued by coexpressing PDK1 kinase that normally triggers PKCγ autophosphorylation. We propose that the SCA14 mutation V138E causes unfolding of the C1B domain and exposure of the C‐terminus of the PKCγ‐V138E molecule, resulting in a decrease of functional kinase in the soluble fraction.