title: Purified Wnt-5a increases differentiation of midbrain dopaminergic cells and dishevelled phosphorylation.
abstract: The Wnt family of lipoproteins regulates several aspects of the development of the nervous system. Recently, we reported that Wnt-3a enhances the proliferation of midbrain dopaminergic precursors and that Wnt-5a promotes their differentiation into dopaminergic neurones. Here we report the purification of hemagglutinin-tagged Wnt-5a using a three-step purification method similar to that previously described for Wnt-3a. Haemagglutinin-tagged Wnt-5a was biologically active and induced the differentiation of immature primary midbrain precursors into tyrosine hydroxylase-positive dopaminergic neurones. Using a substantia nigra-derived dopaminergic cell line (SN4741), we found that Wnt-5a, unlike Wnt-3a, did not promote beta-catenin phosphorylation or stabilization. However, both Wnt-5a and Wnt-3a activated dishevelled, as assessed by a phosphorylation-dependent mobility shift. Moreover, the activity of Wnt-5a on dishevelled was blocked by pre-treatment with acyl protein thioesterase-1, indicating that palmitoylation of Wnt-5a is necessary for its function. Thus, our results suggest that Wnt-3a and Wnt-5a, respectively, activate canonical and non-canonical Wnt signalling pathways in ventral midbrain dopaminergic cells. Furthermore, we identify dishevelled as a key player in transducing both Wnt canonical and non-canonical signals in dopaminergic cells.
authors.affiliation: Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Schulte
authors.forename: Vítezslav
authors.initials: V
authors.lastname: Bryja
authors.forename: Nina
authors.initials: N
authors.lastname: Rawal
authors.forename: Goncalo
authors.initials: G
authors.lastname: Castelo-Branco
authors.forename: Kyle M
authors.initials: KM
authors.lastname: Sousa
authors.forename: Ernest
authors.initials: E
authors.lastname: Arenas
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D000072261:Dishevelled Proteins; D004298:Dopamine / Q000378:metabolism*; D006388:Hemagglutinins / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D019308:Palmitic Acid / Q000378:metabolism; D010750:Phosphoproteins; D010766:Phosphorylation / Q000187:drug effects; D011506:Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D013378:Substantia Nigra / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D013869:Thiolester Hydrolases / Q000494:pharmacology; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D060508:Wnt3 Protein; D060509:Wnt3A Protein
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000072261:Dishevelled Proteins; D006388:Hemagglutinins; D010750:Phosphoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C555510:WNT3A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D060508:Wnt3 Protein; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; C491837:Wnt5a protein, rat; D019308:Palmitic Acid; C402226:LYPLA1 protein, human; D013869:Thiolester Hydrolases; D004298:Dopamine