Protein Kinase C-δ mRNA Is Down-regulated Transcriptionally and Post-transcriptionally by 12-O-Tetradecanoylphorbol-13-acetate

Neng-Yao Shih,Georgia Floyd-Smith

doi:10.1074/jbc.271.27.16040

Neng-Yao Shih, Georgia Floyd-Smith

Open Access

https://doi.org/10.1074/jbc.271.27.16040

Copy DOI

Abstract

Activation of protein kinase C-delta (PKC-delta) by 12-O-tetradecanoylphorbol-13-acetate (TPA) is followed by a gradual decrease in detectable protein 12-24 h later in the mouse B lymphoma cell line A20. Down-regulation is associated with TPA-induced proteolysis and a 50-86% decrease in PKC-delta mRNA 0.5-24 h post-treatment which is due to both a 50% decrease in transcription and accelerated degradation of PKC-delta mRNA as determined using the pulse-chase method. Destabilization of PKC-delta mRNA is also observed when actinomycin D is added to cells pretreated with TPA for 2 h; however, addition of actinomycin D or cycloheximide prior to TPA treatment blocks destabilization. Addition of PKC inhibitors to TPA-treated cells also blocks destabilization of PKC-delta mRNA. Cells treated with TPA for 4 h contain an activity not found in control cells which destabilizes PKC-delta mRNA but not glyceraldehyde-3-phosphate dehydrogenase mRNA in vitro. Addition of TPA to control extracts fails to increase degradation of PKC-delta mRNA in vitro, suggesting that treatment of intact cells is required to induce the synthesis of a factor(s) that destabilizes PKC-delta mRNA. This factor(s) then acts along with transcriptional and post-translational regulatory mechanisms to down-regulate PKC-delta.

Highlights

The biological functions of Protein kinase C (PKC)-␦ are largely unknown; this enzyme has been implicated as a regulator of cell growth
Following TPA treatment, about 50% of the total PKC-␦ translocated to the membrane fraction within 10 min and was retained there for at least 18 h (Fig. 1A)
In A20 cells, some PKC-␦ is translocated to the membrane fraction; most remains in the cytosol following activation by TPA

Summary

Introduction

The biological functions of PKC-␦ are largely unknown; this enzyme has been implicated as a regulator of cell growth. Since several PKC isozymes were found previously to be down-regulated rapidly by TPAinduced proteolysis (14, 44 – 46), the amount of PKC-␦ in control and TPA-treated cells was determined in the presence or absence of the translational inhibitor, cycloheximide (Fig. 1B). To determine if PKC-␦ mRNA levels were down-regulated, Northern blots of RNA from control and TPA-treated cells were analyzed (Fig. 2).

Results

Conclusion