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 th e pathways and nuclear targets of NGF signaling in regulating the NR1 promo ter. PD98059 and wortmannin, but not rapamycin, significantly attenuated NG F-induced transcriptional activity from an NR1 promoter-luciferase construc t. Coexpressing constitutively active forms of Ras, Raf, or MAPK/ERK kinase 1 (MEK1) increased promoter activity dramatically. The MEK1-induced increa se was largely prevented by mutations of the tandem GC boxes in the promote r. Promoter activity was also increased significantly by coexpressed GC box -binding proteins (Sp1, 3, or 4) in nonstimulated PC12 cells. Either an ext racellular signal-regulated kinase-1 (ERK1)- or Sp1-specific antibody copre cipitated Spl with ERKs, and the coprecipitation was enhanced significantly by NGF treatment of PC12 cells. ERK2 also incorporated radioactivity of [g amma P-32]ATP into recombinant Sp1. However, ERK2-treated Sp1 and PC12 nucl ear extracts or nuclear extracts from NGF-treated cells exhibited reduced b inding 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-act ivated ERKs. NGF-increased NR1 promoter activity may involve a complicated mechanism of Sp1 phosphorylation and possible transcription factor exchange .