Casein Kinase 1 Delta in Wnt Signaling and Beyond

我们确定了尤文瘤细胞对Wnt-3a的反应是形成神经突，并开始确定这种作用的机制。 Frizzled 3（Fzd 3）被鉴定为介导该过程的主要Wnt受体，该过程还需要Dishevelled-2（Dvl-2）、Dishevelled-3（Dvl-3）和氨基末端c-Jun激酶（JNK）。 我们发现Dickkopf-1也促进这些细胞中的神经突生长，显然是通过促进内源性Wnt激活Fzd 3/JNK。 Wnt-3a诱导的神经突生长与Dvl-2/3磷酸化相关;神经突形成和Dvl磷酸化均被酪蛋白激酶1 δ/β（CK 1d/e）抑制剂IC 261阻断。 用小干扰RNA敲低CK 1d抑制Wnt-3a依赖的神经突形成，而敲低CK 1 e在没有外源性Wnt-3a的情况下刺激神经突形成。 CK 1d，但没有CK 1 e检测中心体，与神经突起形成的细胞器。 缺失分析将CK 1d的中心体定位信号（CLS）定位到其羧基端结构域。 含有CLS和EGFP的融合蛋白从中心体置换全长CK 1d并抑制Wnt-3a依赖的神经突生长。与野生型CK 1 e相反，由CK 1 e的激酶结构域和CK 1d的CLS组成的嵌合体定位于中心体，并通过CK 1d敲低抑制了Wnt-3a依赖的轴突生长。 这些结果提供了强有力的证据，CK 1d的中心体定位是Wnt-3a依赖的轴突发生所必需的。 敲除非典型PKCiota也阻断Wnt-3a依赖性神经突生长。 Wnt-3a刺激PKCi的磷酸化。Dvl 2与PKCi共免疫沉淀，当Dvl 2中的CK 1磷酸化位点被丙氨酸残基取代时，这种相互作用不会发生。 这些结果表明，Dvl 2磷酸化CK 1所需的Dvl 2-PKCi结合，这反过来可能是必要的神经突生长。这项工作意义重大，不仅因为它提供了有关神经突形成机制的见解。 参与神经突生长的许多因素在其他情况下有助于细胞极性，例如形成对细胞迁移至关重要的细胞延伸。 此外，我们有证据表明，CK 1d也参与初级纤毛的形成。 有缺陷的初级纤毛是负责几种疾病，包括神经管缺陷，多囊肾病和内脏逆位。 异常的Wnt信号传导也可以引起这些异常。 因此，我们对CK 1d和Dvl的研究可能为Wnt信号控制胚胎发育及其失调导致发病机制的方式提供新的见解。

We established that cells from Ewing tumors form neurites in response to Wnt-3a and have begun to define the mechanisms that account for this effect. Frizzled3 (Fzd3) was identified as the primary Wnt receptor that mediates the process, which also requires Dishevelled-2 (Dvl-2), Dishevelled-3 (Dvl-3), and amino-terminal c-Jun kinase (JNK). We showed that Dickkopf-1 also promotes neurite outgrowth in these cells, apparently by facilitating Fzd3/JNK activation by endogenous Wnts. Neurite outgrowth induced by Wnt-3a was associated with Dvl-2/3 phosphorylation; both neurite formation and Dvl phosphorylation were blocked by the casein kinase 1 delta/epsilon (CK1d/e) inhibitor, IC261. Knockdown of CK1d with small interfering RNA suppressed Wnt-3a-dependent neuritogenesis, whereas knockdown of CK1e stimulated neurite formation in the absence of exogenous Wnt-3a. CK1d but not CK1e was detected at the centrosome, an organelle associated with neurite formation. Deletion analysis mapped the centrosomal localization signal (CLS) of CK1d to its carboxyl-terminal domain. A fusion protein containing the CLS and EGFP displaced full-length CK1d from the centrosome and inhibited Wnt-3a-dependent neurite outgrowth. In contrast to wild-type CK1e, a chimera comprised of the kinase domain of CK1e and the CLS of CK1d localized to the centrosome and rescued Wnt-3a-dependent neurite outgrowth suppressed by CK1d knockdown. These results provide strong evidence that the centrosomal localization of CK1d is required for Wnt-3a-dependent neuritogenesis. Knockdown of the atypical PKCiota also blocked Wnt-3a-dependent neurite outgrowth. Wnt-3a stimulated the phosphorylation of PKCi. Dvl2 co-immunoprecipitated with PKCi, and this interaction did not occur when CK1 phosphorylation sites in Dvl2 were replaced with alanine residues. These results suggested that Dvl2 phosphorylation by CK1 was required for Dvl2-PKCi binding, which in turn might be necessary for neurite outgrowth. This work is significant not only because it provides insights about mechanisms involved in the formation of neurites. Many of the factors that participate in neurite outgrowth contribute to cell polarity in other contexts such as the formation of cellular extensions critical for cell migration. Moreover, we have evidence that CK1d also participates in the formation of primary cilia. Defective primary cilia are responsible for several disorders including neural tube defects, polycystic kidney disease and situs inversus. Aberrant Wnt signaling also can elicit these abnormalities. Thus, our studies of CK1d and Dvl may provide new insight about the ways in which Wnt signaling controls embryonic development and its dysregulation contributes to pathogenesis.