Abstract
Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline mutations in the diacylglycerol (DG)/Ca2+‐regulated protein kinase C gamma (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Curiously, the majority of the approximately 50 missense mutations identified in PKCγ cluster to the DG‐sensing C1A and C1B domains. Here, we use a genetically‐encoded FRET‐based C Kinase Activity Reporter (CKAR) to show that ataxia‐associated PKCγ mutants have higher basal activity in cells, and thus are less autoinhibited, than wild‐type enzyme. However, whereas reduced autoinhibition generally renders PKC sensitive to degradation, we show that mutations in the C1B domain allow translocation to membranes but protect PKCγ from phorbol ester‐induced down‐regulation. Indeed, deletion of the C1B domain prevents PKCγ down‐regulation with phorbol esters, potent ligands for the C1 domains. Strikingly, the degree of impaired autoinhibition correlates inversely with age of disease onset. Patients with the most severe mutation we examined (V138E) present with symptoms as young children, whereas symptoms in patients with the least severe mutation examined (D115Y) manifested in their 40s. To understand the structural basis of mutations outside the C1 domains, we generated a model of PKCγ using homology modeling and molecular docking. Mutations outside the C1 domains occur in regions also predicted to disrupt autoinhibition, including the pseudosubstrate, a predicted interface between the kinase and C1B domains, and the C‐terminal tail of PKCγ. Taken together, our data support a model in which SCA14 mutations enhance PKCγ activity without compromising stability. Furthermore, because many of the genetic causes of the 40+ types of SCA alter Ca2+ homeostasis, deregulated PKCγ activity may be a common cause for the disease. This raises the possibility that inhibition of PKCγ will be a potentially viable therapeutic target for SCA.Support or Funding InformationNIH T32 GM007752
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