Introduction: During studies on chemotherapy-induced apoptosis in lymphoid cells, we noted that aggregation of spectrin occurred early in apoptosis, i.e. before activation of initiator caspase(s) and prior to exposure of phosphatidylserine (PS). We also found that protein kinase C theta (PKC-θ) co-localized with spectrin in these aggregates. Our previously published studies indicated that in formation of early apoptotic spectrin aggregates, either PKC-θ or other apoptosis-related proteins are involved. Taking into consideration above data, we decided to test the effect of PKC-θ and Fas-associated death domain protein (FADD) on spectrin aggregation in these cells during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis.
Material and methods: For PKC-θ gene (PRKCQ) or FADD gene expression silencing in Jurkat T cells we used lentiviral particles containing shRNA and scrambled shRNA, respectively. Spectrin aggregates were detected by Western blotting after Triton-X 100 extraction in pellet and soluble fractions or by confocal imaging.
Results: TRAIL-induced apoptosis results in spectrin aggregation and leads to translocation and aggregation of PKC-θ. We found that phorbol-myristate acetate, a PKC activator and translocation inducer, has only a small effect on spectrin aggregation. To further confirm this, we have also shown that knock down ofPRKCQin Jurkat T cells accelerates the formation of TRAIL-induced spectrin aggregates. Transient overexpression of theβ-spectrin C-terminal fragment, containing multiple S/T phosphorylation sites, potential substrate sites for PKC-θ, accelerated the formation of spectrin aggregates. Silencing of downstream TRAIL receptor effector gene,FADD, delayed aggregation of spectrin, but did not reduce PKC-θ localization to the plasma membrane.
Conclusions: In summary, our results show for the first time involvement of spectrin aggregation in TRAIL receptor-FADD apoptotic pathway and indicate that TRAIL-induced spectrin aggregate formation is mediated by FADD and negatively regulated by PKC-θ.
Keywords: FADD; Jurkat T cell; PKC theta; TRAIL; apoptosis; shRNA; spectrin aggregation.