Atypical Protein Kinase C iota Is Required for Wnt3a-dependent Neurite Outgrowth and Binds to Phosphorylated Dishevelled 2

作者：Greer Yoshimi Endo; Fields Alan P; Brown Anthony M C; Rubin Jeffrey S^*

来源：Journal of Biological Chemistry, 2013, 288(13): 9438-9446.

DOI：10.1074/jbc.M112.448282

Previously we reported that Wnt3a-dependent neurite outgrowth in Ewing sarcoma family tumor cell lines was mediated by Frizzled3, Dishevelled (Dvl), and c-Jun N-terminal kinase (Endo, Y., Beauchamp, E., Woods, D., Taylor, W. G., Toretsky, J. A., Uren, A., and Rubin, J. S. (2008) Mol. Cell. Biol. 28, 2368-2379). Subsequently, we observed that Dvl2/3 phosphorylation correlated with neurite outgrowth and that casein kinase 1 delta, one of the enzymes that mediate Wnt3a-dependent Dvl phosphorylation, was required for neurite extension (Greer, Y. E., and Rubin, J. S. (2011) J. Cell Biol. 192, 993-1004). However, the functional relevance of Dvl phosphorylation in neurite outgrowth was not established. Dvl1 has been shown by others to be important for axon specification in hippocampal neurons via an interaction with atypical PKC zeta, but the role of Dvl phosphorylation was not evaluated. Here we report that Ewing sarcoma family tumor cells express PKC iota but not PKC zeta. Wnt3a stimulated PKC iota activation and caused a punctate distribution of pPKC iota in the neurites and cytoplasm, with a particularly intense signal at the centrosome. Knockdown of PKC iota expression with siRNA reagents blocked neurite formation in response to Wnt3a. Aurothiomalate, a specific inhibitor of PKC iota/Par6 binding, also suppressed neurite extension. Wnt3a enhanced the co-immunoprecipitation of endogenous PKC iota and Dvl2. Although FLAG-tagged wild-type Dvl2 immunoprecipitated with PKC iota, a phosphorylation-deficient Dvl2 derivative did not. This derivative also was unable to rescue neurite outgrowth when endogenous Dvl2/3 was suppressed by siRNA (Gonzalez-Sancho, J. M., Greer, Y. E., Abrahams, C. L., Takigawa, Y., Baljinnyam, B., Lee, K. H., Lee, K. S., Rubin, J. S., and Brown, A. M. (2013) J. Biol. Chem. 288, 9428-9437). Taken together, these results suggest that site-specific Dvl2 phosphorylation is required for Dvl2 association with PKC iota. This interaction is likely to be one of the mechanisms essential for Wnt3a-dependent neurite outgrowth.

• 出版日期2013-3-29