The objective of this application is to determine how p-catenin signaling is regulated by EGF-induced tyrosine phosphorylation of the p-catenin protein. Aberrant activation of Wnt/p-catenin signaling plays an important role in tumorigenesis. We have found that p-catenin signaling is frequently activated in human osteosarcoma. Many receptor tyrosine kinases (e.g., EGFR) and non-receptor tyrosine kinases (e.g., Src) are up-regulated in cancer cells. We previously reported that p-catenin signaling can be inhibited by tyrosine kinase inhibitor STI-571/Gleevec and EGFR inhibitor AG-1478. We have recently demonstrated that EGF activates p-catenin signaling and induces tyrosine phosphorylation of the C-terminal region of P-catenin. The importance of tyrosine phosphorylation in Wnt/p-catenin signaling has further been highlighted by a recent discovery, in which an atypical receptor tyrosine kinase Ryk has been shown to be a Wnt coreceptor and is required for WntSa-induced neurite outgrowth. However, the mechanisms behind the regulatory role of tyrosine phosphorylation in p-catenin signaling are unknown. We hypothesize that tyrosine phosphorylation of p-catenin can induce conformational changes which affect the interactions of p-catenin with its binding partners, resulting in an increased association with factorsthat enhance p-catenin-mediated transactivation. In this application, we propose to use EGF-induced phosphorylation as an exemplary systemto dissect the regulatory mechanisms behind tvrosine phosphorylation in B-catenin signaling by focusing on the following three aims. The first aim is to determine the EGF-induced tyrosine phosphorylation site(s) ofP- catenin by using immunoprecipitation/phosphotyrosine Western blotting, EGFR in vitro phosphorylation assays, and mass spectral analysis. The second aim is to determine whether EGF regulates the interactions of P-catenin with its C-terminal binding partners ICAT and p300, by using immunoprecipitation and FRET protein interaction analysis. The third aim is to determine whether tyrosine mutants of p-catenin affect canonical Wnt signal-induced axis duplication in Xenopus embryos and tumor growth in mousexenograft tumor models. We believe that results from the proposed studies should expand our knowledge about the functional role of tyrosine phosphorylation in regulating the p-catenin signaling activity. Because both deregulation of p-catenin signaling and elevation of tyrosine kinase activities are common in human cancer, successful execution of this application should increase our knowledge about p-catenin signaling in cancer development, and may lead to the development of innovative strategiesfor cancer treatment and prevention by targeting both tyrosine kinases and p-catenin signaling pathways.

R01CA106569

2006-01-26

2011-12-31