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

Minsoon Kim, Ji hyun Ju, Kibeom Jang, Sunhwa Oh, Jieun Song, Chul Geun Kim, Incheol Shin

Research output: Contribution to journalArticle

3 Scopus citations

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.

Original languageEnglish
Pages (from-to)387-397
Number of pages11
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1823
Issue number2
DOIs
StatePublished - 2012 Feb

Keywords

  • Notch
  • Protein Kinase Cδ
  • Transcription

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