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. Preliminary experiments suggest that PKCiota may be regulated by CK1d and/or Dvl. 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 preliminary 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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Greer, Yoshimi Endo; Westlake, Christopher J; Gao, Bo et al. (2014) Casein kinase 1? functions at the centrosome and Golgi to promote ciliogenesis. Mol Biol Cell 25:1629-40
González-Sancho, José M; Greer, Yoshimi Endo; Abrahams, Cristina L et al. (2013) Functional consequences of Wnt-induced dishevelled 2 phosphorylation in canonical and noncanonical Wnt signaling. J Biol Chem 288:9428-37
Greer, Yoshimi Endo; Fields, Alan P; Brown, Anthony M C et al. (2013) Atypical protein kinase C? is required for Wnt3a-dependent neurite outgrowth and binds to phosphorylated dishevelled 2. J Biol Chem 288:9438-46
Greer, Yoshimi Endo; Rubin, Jeffrey S (2011) The role of centrosomal casein kinase 1 delta in neurite outgrowth and beyond. Cell Cycle 10:2605-6
Greer, Yoshimi Endo; Rubin, Jeffrey S (2011) Casein kinase 1 delta functions at the centrosome to mediate Wnt-3a-dependent neurite outgrowth. J Cell Biol 192:993-1004
Beauchamp, Elspeth; Bulut, Gulay; Abaan, Ogan et al. (2009) GLI1 is a direct transcriptional target of EWS-FLI1 oncoprotein. J Biol Chem 284:9074-82