(A) 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. 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. (B) Inhibition of casein kinase 1 delta (CK1d), but not CK1e, blocked primary ciliogenesis in hTERT-RPE and mIMCD3 cells. Mouse embryonic fibroblasts (MEFs) and retinal cells from Csnk1d (CK1d) null mice also exhibited ciliogenesis defects. CK1d catalytic activity and centrosomal localization signal (CLS) were required to rescue cilia formation in CK1d null MEFs. Furthermore, expression of a truncated derivative containing the CLS displaced full-length CK1d from the centrosome and decreased ciliary length in control MEFs, suggesting that centrosomal CK1d has a role in ciliogenesis. CK1d inhibition also altered the pericentrosomal or ciliary distribution of several proteins involved in ciliary transport, including Rab11a, Rab8a, CEP290, PCM1 and polycystin-2, as well as the Golgi distribution of its binding partner, AKAP450. As reported for AKAP450, CK1d was required for microtubule nucleation at the Golgi and maintenance of Golgi integrity. Overexpression of an AKAP450 fragment containing the CK1d binding site inhibited Golgi-derived microtubule nucleation, Golgi distribution of IFT20 and ciliogenesis. Our results suggest that CK1d mediates primary ciliogenesis by multiple mechanisms, one involving its centrosomal function and another dependent on its interaction with AKAP450 at the Golgi where it is important for maintaining Golgi organization and polarized trafficking of multiple factors that mediate ciliary transport. (C) Current research is focused on observations that inhibition of CK1d expression or catalytic activity disrupts DNA damage repair and cell cycle regulation.
