Identification and characterization of PKCγ, a kinase associated with SCA14, as an amyloidogenic protein

Abstract

Amyloid assemblies are associated with a wide range of human disorders, including Alzheimer's and Parkinson's diseases. Here, we identify protein kinase C (PKC) γ, a serine/threonine kinase mutated in the neurodegenerative disease spinocerebellar ataxia type 14 (SCA14), as a novel amyloidogenic protein with no previously characterized amyloid-prone domains. We found that overexpression of PKCγ in cultured cells, as well as in vitro incubation of PKCγ without heat or chemical denaturants, causes amyloid-like fibril formation of this protein. We also observed that SCA14-associated mutations in PKCγ accelerate the amyloid-like fibril formation both in cultured cells and in vitro. We show that the C1A and kinase domains of PKCγ are involved in its soluble dimer and aggregate formation and that SCA14-associated mutations in the C1 domain cause its misfolding and aggregation. Furthermore, long-term time-lapse imaging indicates that aggregates of mutant PKCγ are highly toxic to neuronal cells. Based on these findings, we propose that PKCγ could form amyloid-like fibrils in physiological and/or pathophysiological conditions such as SCA14. More generally, our results provide novel insights into the mechanism of amyloid-like fibril formation by multi-domain proteins.

IP3 receptor mutations and brain diseases in human and rodents

“…(Takahashi et al . )). Importantly, although it is not confirmed in vivo (Nelson and Glitsch ), PKC phosphorylates TRPC3 at a threonine residue in the S4‐S5 linker segment, thereby inactivating it (Venkatachalam et al .…”

“…; Takahashi et al . ). Although apoptotic cell death of PCs expressing mutant PKCγ has not been observed in vivo (Shuvaev et al .…”

“…Since PKCc-KO mice exhibit only mild cerebellar ataxia and have no gross morphological abnormalities in the cerebellum Chen et al 1995), it has been thought that a gain-of-toxic function, rather than a loss-of-function mutant form of PKCc, underlies the prominent cerebellar atrophy and degeneration of PCs in SCA14. Recent findings have suggested that mutant PKCc is prone to form aggregates, and amyloid-like fibrils in cells, causing an impairment in the ubiquitinproteasome system in addition to ER stress, eventually leading to cell death (Seki et al 2005(Seki et al , 2007Seki et al 2009;Takahashi et al 2015). Although apoptotic cell death of PCs expressing mutant PKCc has not been observed in vivo (Shuvaev et al 2011), dark cell degeneration in PCs are present in mice expressing the mutated PKCc (see discussion in a recent paper by Takahashi et al (Takahashi et al 2015)).…”

Protein kinase C: perfectly balanced

“…Over 30 germline mutations have been identified in spinocerebellar ataxia type 14 (SCA14) in PKCγ (Adachi et al 2008; Verbeek et al 2008; Takahashi et al 2015), an isozyme whose expression is restricted to the brain in normal physiology (Ding et al 2005). Curiously, almost all mutations occur in the C1B domain, but not to a specific position, suggesting a general role in perturbing the structure of the domain.…”

Genetic landscape remodelling in spinocerebellar ataxias: the influence of next-generation sequencing

“…More strikingly, PKCc was recently established as an amyloidogenic protein. SCA14-linked mutations seem to accelerate the amyloid-like fibril formation [97].…”