title: Protein kinase Cδ negatively regulates Notch1-dependent transcription via a kinase-independent mechanism in vitro.
abstract: Protein kinase Cδ (PKCδ) plays a significant role in the regulation of growth, apoptosis, and differentiation in a diversity of cell types. We investigated the effect of PKCδ on Notch1 intracellular domain (NICD)-mediated transcription with Notch transcription reporter constructs. The results indicate that co-expression of PKCδ down-regulated NICD-dependent transcription. Co-expression of a dominant negative PKCδ (K376R) variant lacking kinase activity was also able to downregulate NICD-dependent transcription, suggesting that PKCδ exerts its inhibitory effect via a kinase-independent mechanism(s). Interestingly, expression of PKCδ 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 PKCδ and K376R effectively impaired proper nuclear localization of NICD, possibly via a physical association between NICD and PKCδ, which was confirmed by co-immunoprecipitation experiments. Chromatin immunoprecipitation assays revealed that both PKCδ and K376R inhibit the association of NICD with the promoter region of its target gene, Hes1. Furthermore, silencing of PKCδ resulted in increased NICD nuclear localization and NICD transcriptional activity in MCF-7 cells. PKCδ silencing-induced increase in anti-apoptotic survivin could not rescue apoptosis induced by doxorubicin. The data herein indicate that PKCδ 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.
authors.affiliation: Department of Life Science, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: Minsoon
authors.initials: M
authors.lastname: Kim
authors.forename: Ji-hyun
authors.initials: JH
authors.lastname: Ju
authors.forename: Kibeom
authors.initials: K
authors.lastname: Jang
authors.forename: Sunhwa
authors.initials: S
authors.lastname: Oh
authors.forename: Jieun
authors.initials: J
authors.lastname: Song
authors.forename: Chul Geun
authors.initials: CG
authors.lastname: Kim
authors.forename: Incheol
authors.initials: I
authors.lastname: Shin
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D015536:Down-Regulation; D005786:Gene Expression Regulation; D020868:Gene Silencing; D017930:Genes, Reporter; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: C497762:Notch1 protein, mouse; D051881:Receptor, Notch1; D051057:Proto-Oncogene Proteins c-akt; D051745:Protein Kinase C-delta; D046988:Proteasome Endopeptidase Complex; D006655:Histone Deacetylases