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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011224-04
Application #
8553055
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2012
Total Cost
$498,109
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Greer, Yoshimi Endo; Westlake, Christopher J; Gao, Bo et al. (2014) Casein kinase 1δ functions at the centrosome and Golgi to promote ciliogenesis. Mol Biol Cell 25:1629-40
Greer, Yoshimi Endo; Fields, Alan P; Brown, Anthony M C et al. (2013) Atypical protein kinase Cι is required for Wnt3a-dependent neurite outgrowth and binds to phosphorylated dishevelled 2. J Biol Chem 288:9438-46
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; 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
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
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