Schulte Gunnar, Bryja Vítezslav, Rawal Nina, Castelo-Branco Goncalo, Sousa Kyle M, Arenas Ernest
Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
J Neurochem. 2005 Mar;92(6):1550-3. doi: 10.1111/j.1471-4159.2004.03022.x.
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.
Wnt脂蛋白家族调节神经系统发育的多个方面。最近，我们报道Wnt-3a可增强中脑多巴胺能前体细胞的增殖，而Wnt-5a则促进其分化为多巴胺能神经元。在此，我们报告了使用一种类似于先前用于Wnt-3a的三步纯化方法对血凝素标记的Wnt-5a进行纯化。血凝素标记的Wnt-5a具有生物活性，并可诱导未成熟的原代中脑前体细胞分化为酪氨酸羟化酶阳性的多巴胺能神经元。利用源自黑质的多巴胺能细胞系（SN4741），我们发现，与Wnt-3a不同，Wnt-5a不会促进β-连环蛋白的磷酸化或稳定。然而，通过磷酸化依赖性迁移率变化评估，Wnt-5a和Wnt-3a均能激活散乱蛋白。此外，用酰基蛋白硫酯酶-1预处理可阻断Wnt-5a对散乱蛋白的活性，这表明Wnt-5a的棕榈酰化对其功能是必需的。因此，我们的结果表明，Wnt-3a和Wnt-5a分别在腹侧中脑多巴胺能细胞中激活经典和非经典Wnt信号通路。此外，我们确定散乱蛋白是在多巴胺能细胞中转导Wnt经典和非经典信号的关键因子。
