Protein kinase C (PKC) isoforms regulate a number of processes crucial for the fate of a cell. In this study we identify previously unrecognized interaction partners of PKCα and a novel role for PKCα in the regulation of stress granule formation during cellular stress. Three RNA-binding proteins, cytoplasmic poly(A)+ binding protein (PABPC1), IGF-II mRNA binding protein 3 (IGF2BP3), and RasGAP binding protein 2 (G3BP2) all co-precipitate with PKCα. RNase treatment abolished the association with IGF2BP3 and PABPC1 whereas the PKCα-G3BP2 interaction was largely resistant to this. Furthermore, interactions between recombinant PKCα and G3BP2 indicated that the interaction is direct and PKCα can phosphorylate G3BP2 in vitro. The binding is mediated via the regulatory domain of PKCα and the C-terminal RNA-binding domain of G3BP2. Both proteins relocate to and co-localize in stress granules, but not to P-bodies, when cells are subjected to stress. Heat shock-induced stress granule assembly and phosphorylation of eIF2α are suppressed following downregulation of PKCα by siRNA. In conclusion this study identifies novel interaction partners of PKCα and a novel role for PKCα in regulation of stress granules.

A hallmark of the afflicted nervous tissue in amyotrophic lateral sclerosis is the presence of protein aggregates, which to a large extent contain the intermediate filament protein peripherin. Here we show that activation of protein kinase C (PKC) or overexpression of PKC⑀ induces the aggregation of peripherin in cultured neuroblastoma cells with elevated amounts of peripherin. The formation of aggregates was coupled to an increased apoptosis, suggesting a functional link between these events. Both induction of aggregates and apoptosis were suppressed in cells that had been transfected with small interfering RNAs targeting PKC⑀. PKC⑀ and peripherin associate as shown by co-immunoprecipitation, and the interaction is dependent on and mediated by the C1b domain of PKC⑀. The interaction was specific for PKC⑀ since corresponding structures from other isoforms did not co-precipitate peripherin, with the exception for PKC and -, which pulled down minute amounts. PKC⑀ interacts with vimentin through the same structures but does not induce its aggregation. When the PKC⑀ C1b domain is expressed in neuroblastoma cells together with peripherin, both phorbol ester-induced peripherin aggregation and apoptosis are abolished, supporting a model in which PKC⑀ through its interaction with peripherin facilitates its aggregation and subsequent cell death. These events may be prevented by expressing molecules that bind peripherin at the same site as PKC⑀.

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