title: GSK3β/axin-1/β-catenin complex is involved in semaphorin3A signaling.
abstract: Semaphorin3A (Sema3A) exerts a wide variety of biological functions by regulating reorganization of actin and tubulin cytoskeletal proteins through signaling pathways including sequential phosphorylation of collapsin response mediator protein 1 (CRMP1) and CRMP2 by cyclin-dependent kinase-5 and glycogen synthase kinase-3β (GSK3β). To delineate how GSK3β mediates Sema3A signaling, we here determined the substrates of GSK3β involved. Introduction of either GSK3β mutants, GSK3β-R96A, L128A, or K85M into chick dorsal root ganglion (DRG) neurons suppressed Sema3A-induced growth cone collapse, thereby suggesting that unprimed as well as primed substrates are involved in Sema3A signaling. Axin-1, a key player in Wnt signaling, is an unprimed substrate of GSK3β. The phosphorylation of Axin-1 by GSK3β accelerates the association of Axin-1 with β-catenin. Immunocytochemical studies revealed that Sema3A induced an increase in the intensity levels of β-catenin in the DRG growth cones. Axin-1 siRNA knockdown suppressed Sema3A-induced growth cone collapse. The reintroduction of RNAi-resistant Axin-1 (rAxin-1)-wt rescued the responsiveness to Sema3A, while that of nonphosphorylated mutants, rAxin S322A/S326A/S330A and T485A/S490A/S497A, did not. Sema3A also enhanced the colocalization of GSK3β, Axin-1, and β-catenin in the growth cones. The increase of β-catenin in the growth cones was suppressed by the siRNA knockdown of Axin-1. Furthermore, either Axin-1 or β-catenin RNAi knockdown suppressed the internalization of Sema3A. These results suggest that Sema3A induces the formation of GSK3β/Axin-1/β-catenin complex, which regulates signaling cascade of Sema3A via an endocytotic mechanism. This finding should provide clue for understanding of mechanisms of a wide variety of biological functions of Sema3A.
authors.affiliation: Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.
authors.forename: Tomonobu
authors.initials: T
authors.lastname: Hida
authors.forename: Naoya
authors.initials: N
authors.lastname: Yamashita
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Usui
authors.forename: Fumio
authors.initials: F
authors.lastname: Nakamura
authors.forename: Yukio
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Akira
authors.initials: A
authors.lastname: Kikuchi
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Goshima
mesh_terms: D000818:Animals; D060466:Axin Protein / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D002642:Chick Embryo; D004705:Endocytosis / Q000235:genetics; D005260:Female; D005727:Ganglia, Spinal / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000502:physiology*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009924:Organ Culture Techniques; D010766:Phosphorylation / Q000235:genetics; D051381:Rats; D040121:Semaphorin-3A / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D013379:Substrate Specificity / Q000235:genetics; D051176:beta Catenin / Q000235:genetics / Q000502:physiology*
chemical_list: C555451:AXIN1 protein, human; D060466:Axin Protein; C495270:CTNNB1 protein, human; C108639:SEMA3A protein, human; D040121:Semaphorin-3A; D051176:beta Catenin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3