The potent lipid mitogen sphingosylphosphocholine activates the DNA binding activity of upstream stimulating factor (USF), a basic helix-loop-helix-zipper protein

• Published in: Biochimica et biophysica acta 1998-02, Vol.1390 (2), p.225-236
• Main Authors: Berger, Alvin, Cultaro, Constance M., Segal, Shoshana, Spiegel, Sarah
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Basic-Leucine Zipper Transcription Factors; DNA-Binding Proteins - metabolism; E-box; Helix-Loop-Helix Motifs - physiology; Leucine Zippers - physiology; Max; Mice; Mitogens - pharmacology; Myc; Nuclear Proteins - metabolism; Phosphorylation; Phosphorylcholine - analogs & derivatives; Phosphorylcholine - pharmacology; Protein Binding - drug effects; Proto-Oncogene Proteins c-myc - genetics; RNA, Messenger - metabolism; Sphingolipid; Sphingosine - analogs & derivatives; Sphingosine - pharmacology; Sphingosylphosphocholine; Temperature; Transcription Factors - analysis; Transcription Factors - metabolism; Upstream Stimulatory Factors; USF
• ISSN: 0005-2760; 0006-3002; 1879-145X
• DOI: 10.1016/S0005-2760(97)00180-X

We previously demonstrated that the sphingolipid, sphingosylphosphocholine (SPC) increased DNA binding activity of AP-1 proteins accompanying cellular proliferation. Herein, the effects of SPC on DNA binding activity and transcription of the basic, helix-loop-helix, leucine zipper (bHLH-ZIP) proteins Myc, Max, and USF were investigated because they regulate genes involved in mitogenesis. E-box (CACGTG) DNA binding proteins were detected by electrophoretic mobility shift assays in nuclear extracts from Swiss 3T3 fibroblasts. The slowest migrating complex (complex I) increased within 1–3 min after treatment with SPC, remained elevated for 10 min, and increased again after 12 h. Complexes I and II contained USF-1 and USF-2 proteins, and complex I migrated similarly to recombinant USF-1 protein/DNA complex. Treatment of nuclear extracts with alkaline phosphatase decreased these complexes suggesting USF might be a phosphoprotein, post-translationally modified by SPC. max and usf-1 mRNA levels were unaffected by SPC treatment. In contrast, c-myc mRNA was rapidly elevated, reached maximum levels at 0.5–1 h, and showed an additional increase after 12 h, just preceding S phase. Thus, certain bHLH-ZIP transcription factors may be involved in cell growth regulation by SPC.