Activation of Protein Kinase C Induces Nuclear Translocation of RFX1 and Down-regulates c-myc via an Intron 1 X Box in Undifferentiated Leukemia HL-60 Cells

• Published in: The Journal of biological chemistry 2000-10, Vol.275 (41), p.32227-32233
• Main Authors: Chen, Lei, Smith, Lucinda, Johnson, Martin R., Wang, Kangsheng, Diasio, Robert B., Smith, Jeffrey Bingham
• Format: Article
• Language: English
• Subjects: Active Transport, Cell Nucleus - drug effects; Binding Sites; Bryostatins; Cell Differentiation; Cell Nucleus - drug effects; Cell Nucleus - metabolism; Cytosol - drug effects; Cytosol - metabolism; DNA-Binding Proteins - metabolism; Down-Regulation - drug effects; Enzyme Activation - drug effects; Genes, myc - genetics; Genes, Reporter; HL-60 Cells; Humans; Indoles - pharmacology; Introns - genetics; Lactones - pharmacology; Macrolides; Maleimides - pharmacology; myc gene; Nuclear Proteins - metabolism; Protein Binding - drug effects; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - metabolism; Proto-Oncogene Proteins c-myc - biosynthesis; Proto-Oncogene Proteins c-myc - genetics; Proto-Oncogene Proteins c-myc - metabolism; Regulatory Factor X Transcription Factors; Regulatory Factor X1; Response Elements; RFX1 protein; RNA, Messenger - genetics; RNA, Messenger - metabolism; Tetradecanoylphorbol Acetate - pharmacology; Transcription Factors - metabolism; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M002645200

Treatment of human promyelocytic leukemia cells (HL-60) with phorbol 12-myristate 13-acetate (PMA) is known to decrease c-myc mRNA by blocking transcription elongation at sites near the first exon/intron border. Treatment of HL-60 cells with either PMA or bryostatin 1, which acutely activates protein kinase C (PKC), decreased the levels of myc mRNA and Myc protein. The inhibition of Myc synthesis accounted for the drop in Myc protein, because PMA treatment had no effect on Myc turnover. Treatment with PMA or bryostatin 1 increased nuclear protein binding to MIE1, a c-myc intron 1 element that defines an RFX1-binding X box. RFX1 antiserum supershifted MIE1-protein complexes. Increased MIE1 binding was independent of protein synthesis and abolished by a selective PKC inhibitor, which also prevented the effect of PMA onmyc mRNA and protein levels and Myc synthesis. PMA treatment increased RFX1 in the nuclear fraction and decreased it in the cytosol without affecting total RFX1. Transfection of HL-60 cells with myc reporter gene constructs showed that the RFX1-binding X box was required for the down-regulation of reporter gene expression by PMA. These findings suggest that nuclear translocation and binding of RFX1 to the X box cause the down-regulation of myc expression, which follows acute PKC activation in undifferentiated HL-60 cells.