The potent lipid mitogen sphingosylphosphocholine activates the DNA binding activity of upstream stimulating factor (USF), a basic helix-loop-helix-zipper protein.
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.
Berger
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Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20007, USA.
Cultaro
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CM
Segal
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Spiegel
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D016475:3T3 Cells; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs / Q000502:physiology; D016350:Leucine Zippers / Q000502:physiology; D051379:Mice; D008934:Mitogens / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013696:Temperature; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism*; D051720:Upstream Stimulatory Factors
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D008934:Mitogens; C067850:Myc associated factor X; D009687:Nuclear Proteins; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; C497186:Usf2 protein, mouse; C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; C068422:Max protein, mouse; D013110:Sphingosine