RÉSUMÉ
The Wnt-induced planar cell polarity (PCP) signaling pathway is essential for polarized cell migration and morphogenesis. Dishevelled (Dvl) and its binding protein Daam1 mediate RhoA activation in this pathway. WGEF, a member of the Rho-guanine nucleotide exchange factor (Rho-GEF) family, was shown to play a role in Wnt-induced RhoA activation in Xenopus embryos. However, it has remained unknown which member(s) of a Rho-GEF family are involved in Wnt/Dvl-induced RhoA activation in mammalian cells. Here we identified p114-RhoGEF and Lfc (also called GEF-H1) as the Rho-GEFs responsible for Wnt-3a-induced RhoA activation in N1E-115 mouse neuroblastoma cells. We screened for Rho-GEF-silencing short-hairpin RNAs (shRNAs) that are capable of suppressing Dvl-induced neurite retraction in N1E-115 cells and found that p114-RhoGEF and Lfc shRNAs, but not WGEF shRNA, suppressed Dvl- and Wnt-3a-induced neurite retraction. p114-RhoGEF and Lfc shRNAs also inhibited Dvl- and Wnt-3a-induced RhoA activation, and p114-RhoGEF and Lfc proteins were capable of binding to Dvl and Daam1. Additionally, the Dvl-binding domains of p114-RhoGEF and Lfc inhibited Dvl-induced neurite retraction. Our results suggest that p114-RhoGEF and Lfc are critically involved in Wnt-3a- and Dvl-induced RhoA activation and neurite retraction in N1E-115 cells.
Sujet(s)
Protéines adaptatrices de la transduction du signal/métabolisme , Facteurs d'échange de nucléotides guanyliques/métabolisme , Neurites/enzymologie , Neuroblastome/enzymologie , Phosphoprotéines/métabolisme , Protéines proto-oncogènes/métabolisme , Protéines de type Wingless/métabolisme , Protéine G RhoA/métabolisme , Animaux , Lignée cellulaire tumorale , Protéines Dishevelled , Activation enzymatique/effets des médicaments et des substances chimiques , Analyse de profil d'expression de gènes , Régulation de l'expression des gènes tumoraux/effets des médicaments et des substances chimiques , Techniques de knock-down de gènes , Humains , Lysophospholipides/pharmacologie , Souris , Protéines des microfilaments/métabolisme , Neuroblastome/génétique , Neuroblastome/anatomopathologie , Liaison aux protéines/effets des médicaments et des substances chimiques , Cartographie d'interactions entre protéines , Petit ARN interférent/métabolisme , Rho guanine nucleotide exchange factors , Protéine Wnt3 , Protéine Wnt3A , Protéines de Xénope , Protéines G rho/métabolismeRÉSUMÉ
The presence of the geranylgeranyl diphosphate synthase (GGS) gene is a common feature of gene clusters for diterpene biosynthesis. We demonstrated identification of a diterpene gene cluster using homology-based PCR of GGS genes and the subsequent genome walking in the fungus Phomopsis amygdali N2. Structure determination of a novel diterpene hydrocarbon phomopsene provided by enzymatic synthesis with the recombinant terpene synthase PaPS and screening of fungal broth extracts with reference to characteristic NMR signals of phomopsene allowed us to isolate a new diterpene, methyl phomopsenonate. The versatility of the gene-based screening of unidentified diterpenes is discussed in regard to fungal genomic data.
