title: SCA14 mutation V138E leads to partly unfolded PKCγ associated with an exposed C-terminus, altered kinetics, phosphorylation and enhanced insolubilization.
abstract: 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. Here, we show that the mutation V138E of the protein kinase C γ (PKCγ) C1B domain (PKCγ-V138E), which is implicated in spinocerebellar ataxia type 14, exhibits a partially unfolded C-terminus. This leads to unusually fast phorbol 12-myristate 13-acetate-induced membrane translocation and accumulation of phosphorylated PKCγ-V138E in the insoluble fraction, causing loss of the functional kinase. In contrast to general chaperones, coexpression of PKCγ's 'natural chaperone', PDK1 kinase, could rescue the PKCγ-V138E phenotype.
authors.affiliation: Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Justyna
authors.initials: J
authors.lastname: Jezierska
authors.forename: Joachim
authors.initials: J
authors.lastname: Goedhart
authors.forename: Harm H
authors.initials: HH
authors.lastname: Kampinga
authors.forename: Eric A
authors.initials: EA
authors.lastname: Reits
authors.forename: Dineke S
authors.initials: DS
authors.lastname: Verbeek
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002273:Carcinogens / Q000494:pharmacology; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D020125:Mutation, Missense / Q000235:genetics / Q000502:physiology; D010766:Phosphorylation; D011092:Polyethylene Glycols / Q000737:chemistry; D017510:Protein Folding; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D012995:Solubility; D012997:Solvents; D020754:Spinocerebellar Ataxias / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002273:Carcinogens; C000631429:PDK1 protein, human; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D012997:Solvents; D011092:Polyethylene Glycols; D004247:DNA; C087857:protein kinase C gamma; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate
keywords: C1B domain; PDK1; conformational change; protein kinase C; spinocerebellar ataxia