Protein kinase C delta negatively regulates Notch1-dependent transcription via a kinase-independent mechanism in vitro

Abstract

Protein kinase C delta (PKC delta) plays a significant role in the regulation of growth, apoptosis, and differentiation in a diversity of cell types. We investigated the effect of PKC delta on Notch1 intracellular domain (NICD)-mediated transcription with Notch transcription reporter constructs. The results indicate that co-expression of PKC delta down-regulated NICD-dependent transcription. Co-expression of a dominant negative PKC delta (K376R) variant lacking kinase activity was also able to downregulate NICD-dependent transcription, suggesting that PKC delta exerts its inhibitory effect via a kinase-independent mechanism(s). Interestingly, expression of PKC delta as well as K376R induced NICD up-regulation by inhibiting proteasome-mediated degradation of NICD, indicating that NICD protein quantity is not proportional to its transcriptional activity. When the subcellular distribution of NICD was investigated by both subcellular fractionation and immunocytochemistry, it was found that PKC delta and K376R effectively impaired proper nuclear localization of NICD, possibly via a physical association between NICD and PKC delta, which was confirmed by co-immunoprecipitation experiments. Chromatin immunoprecipitation assays revealed that both PKC delta and K376R inhibit the association of NICD with the promoter region of its target gene, Hes1. Furthermore, silencing of PKC delta resulted in increased NICD nuclear localization and NICD transcriptional activity in MCF-7 cells. PKC delta silencing-induced increase in anti-apoptotic survivin could not rescue apoptosis induced by doxorubicin. The data herein indicate that PKC delta can induce down-regulation of NICD transcriptional activity via a kinase-independent inhibition of NICD nuclear targeting and dissociation of NICD from target gene promoters.