Nerve growth factor uses Ras/ERK and phosphatidylinositol 3-kinase cascades to up-regulate the N-methyl-D-aspartate receptor 1 promoter.
We reported previously that nerve growth factor (NGF) up-regulates activity of the N-methyl-D-aspartate receptor 1 (NR1) promoter. We have explored the pathways and nuclear targets of NGF signaling in regulating the NR1 promoter. PD98059 and wortmannin, but not rapamycin, significantly attenuated NGF-induced transcriptional activity from an NR1 promoter-luciferase construct. Coexpressing constitutively active forms of Ras, Raf, or MAPK/ERK kinase 1 (MEK1) increased promoter activity dramatically. The MEK1-induced increase was largely prevented by mutations of the tandem GC boxes in the promoter. Promoter activity was also increased significantly by coexpressed GC box-binding proteins (Sp1, 3, or 4) in nonstimulated PC12 cells. Either an extracellular signal-regulated kinase-1 (ERK1)- or Sp1-specific antibody coprecipitated Sp1 with ERKs, and the coprecipitation was enhanced significantly by NGF treatment of PC12 cells. ERK2 also incorporated radioactivity of [gamma(32)P]ATP into recombinant Sp1. However, ERK2-treated Sp1 and PC12 nuclear extracts or nuclear extracts from NGF-treated cells exhibited reduced binding to the promoter or a consensus GC box. Our results suggest that NGF utilizes both the Ras/ERK and phosphatidylinositol 3-kinase pathways to up-regulate NR1 promoter activity and that Sp1 is a novel substrate of NGF-activated ERKs. NGF-increased NR1 promoter activity may involve a complicated mechanism of Sp1 phosphorylation and possible transcription factor exchange.
Liu
A
A
Department of Oral and Craniofacial Biological Sciences, University of Maryland Dental School, Baltimore, Maryland 21201, USA.
Prenger
M S
MS
Norton
D D
DD
Mei
L
L
Kusiak
J W
JW
Bai
G
G
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation; D020932:Nerve Growth Factor / Q000378:metabolism* / Q000502:physiology*; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D005419:Flavonoids; D016194:Receptors, N-Methyl-D-Aspartate; D016329:Sp1 Transcription Factor; D004247:DNA; D020932:Nerve Growth Factor; D008156:Luciferases; D019869:Phosphatidylinositol 3-Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D020123:Sirolimus; D000077191:Wortmannin