Atypical protein kinase Ci is required for Wnt3a-dependent neurite outgrowth and binds to phosphorylated Dishevelled 2

Yoshimi Endo Greer, Alan P Fields, Anthony M C Brown, Jeffrey S. Rubin

Research output: Contribution to journalArticle

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Abstract

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 1δ, 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 PKCζ, but the role of Dvl phosphorylation was not evaluated. Here we report that Ewing sarcoma family tumor cells express PKCi but not PKCζ. Wnt3a stimulated PKCi activation and caused a punctate distribution of pPKCi in the neurites and cytoplasm, with a particularly intense signal at the centrosome. Knockdown of PKCi expression with siRNA reagents blocked neurite formation in response to Wnt3a. Aurothiomalate, a specific inhibitor of PKCi/Par6 binding, also suppressed neurite extension. Wnt3a enhanced the coimmunoprecipitation of endogenous PKCi and Dvl2. Although FLAG-tagged wild-type Dvl2 immunoprecipitated with PKCi, a phosphorylation-deficient Dvl2 derivative did not. This derivative also was unable to rescue neurite outgrowth when endogenous Dvl2/3 was suppressed by siRNA (González-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 PKCi. This interaction is likely to be one of the mechanisms essential for Wnt3a-dependent neurite outgrowth.

Original languageEnglish (US)
Pages (from-to)9438-9446
Number of pages9
JournalJournal of Biological Chemistry
Volume288
Issue number13
DOIs
StatePublished - Mar 29 2013

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Phosphorylation
Protein Kinases
Neurites
Ewing's Sarcoma
Small Interfering RNA
Tumors
Casein Kinase I
Cells
Gold Sodium Thiomalate
Derivatives
Centrosome
JNK Mitogen-Activated Protein Kinases
Tumor Cell Line
Neurons
Axons
Neuronal Outgrowth
Cytoplasm
Chemical activation
Association reactions
Specifications

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Atypical protein kinase Ci is required for Wnt3a-dependent neurite outgrowth and binds to phosphorylated Dishevelled 2. / Greer, Yoshimi Endo; Fields, Alan P; Brown, Anthony M C; Rubin, Jeffrey S.

In: Journal of Biological Chemistry, Vol. 288, No. 13, 29.03.2013, p. 9438-9446.

Research output: Contribution to journalArticle

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abstract = "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 1δ, 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 PKCζ, but the role of Dvl phosphorylation was not evaluated. Here we report that Ewing sarcoma family tumor cells express PKCi but not PKCζ. Wnt3a stimulated PKCi activation and caused a punctate distribution of pPKCi in the neurites and cytoplasm, with a particularly intense signal at the centrosome. Knockdown of PKCi expression with siRNA reagents blocked neurite formation in response to Wnt3a. Aurothiomalate, a specific inhibitor of PKCi/Par6 binding, also suppressed neurite extension. Wnt3a enhanced the coimmunoprecipitation of endogenous PKCi and Dvl2. Although FLAG-tagged wild-type Dvl2 immunoprecipitated with PKCi, a phosphorylation-deficient Dvl2 derivative did not. This derivative also was unable to rescue neurite outgrowth when endogenous Dvl2/3 was suppressed by siRNA (Gonz{\'a}lez-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 PKCi. This interaction is likely to be one of the mechanisms essential for Wnt3a-dependent neurite outgrowth.",
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