Protein kinase C activation potently down-regulates the expression of its major substrate, 80K, in Swiss 3T3 cells.
The amino acid sequence of 80K, the major acidic protein kinase C (PKC) substrate of Swiss 3T3 fibroblasts, was deduced from a cDNA nucleotide sequence. Overall, 25% of the predicted amino acid sequence is supported by direct protein sequence data. Southern blot analysis suggests that the mouse genome contains a single copy of this gene. Two 80K mRNA species, a major band of 2.25 kb and a minor band of 3.9 kb, were detected by Northern blot analysis. Stimulation of PKC by biologically active phorbol esters, including phorbol-12, 13-dibutyrate (PDB), reduced the steady state level of 80K mRNA to 8.8% of control within 5-7 h. This effect was dose-dependent, and was abolished by prior depletion of PKC. The PDB-induced down-regulation of 80K mRNA levels was transient, and recovery coincided with the disappearance of PKC activity. A similar transient decrease in 80K mRNA levels was also demonstrated in tertiary cultures of mouse embryo fibroblasts. The down-regulation of 80K mRNA levels was completely abolished by actinomycin D, cycloheximide or anisomycin if added up to 30 min after PDB addition. Since the rate of transcription of the 80K gene was unaltered by PDB treatment, we concluded that the PKC-induced down-regulation of 80K mRNA is mediated by a post-transcriptional mechanism. In addition, PDB transiently decreased the level of 80K protein within 14-18 h, thus reflecting the effects of this phorbol ester on mRNA expression.
Brooks
S F
SF
Imperial Cancer Research Fund, Lincoln's Inn Fields, London, UK.
Herget
T
T
Erusalimsky
J D
JD
Rozengurt
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015536:Down-Regulation; D004789:Enzyme Activation; D005959:Glucosidases; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010750:Phosphoproteins; D011493:Protein Kinase C / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; C512949:Prkcsh protein, mouse; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D012333:RNA, Messenger; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015240:Phorbol 12,13-Dibutyrate; D004247:DNA; D011493:Protein Kinase C; D005959:Glucosidases