The Wnt signaling pathway is of critical importance in a wide variety of biological processes from the regulation of early embryonic development to the control of stem cell growth. Misregulation of this pathway leads to a variety of cancers, including colon cancer, the second major cause of cancer-related death in the United States. Central to the regulation of this pathway is the """"""""Destruction Complex"""""""", an assembly of proteins including the kinase GSK3, which controls the levels of the transcriptional activator beta-catenin, a principal effector of Wnt signaling. Whereas in many cell types intercellular Wnt signals regulate the activity of the Destruction Complex through an unknown mechanism, in early Xenopus development, the complex is regulated intracellularly by translocating particles containing a GSK3 binding protein, GBP. One major goal of this proposal will build on our result from the last funding period that GBP interacts with kinesin light chain (KLC), which we have proposed provides a critical bridge between a motor protein required for translocation and other regulators of the Wnt pathway such as Dishevelled. Using a variety of approaches to disrupt kinesin function, we will test the proposed role of kinesin in particle movement and axis formation. Furthermore, in order to understand how the particles are assembled in the Xenopus oocyte, we will test the hypothesis that casein kinase 1epsilon (CK1epsilon), potentially activated by the Frizzled 7 receptor, has an essential role in particle formation. Finally, using zebrafish as a model system, we will test our proposition that these dorsalizing particles are used in other lower vertebrates. The second major area of research will continue our structural studies of the Wnt intracellular pathway, focusing on CK1epsilon, a kinase that is essential for transducing Wnt signals. We will examine how CK1epsilon inhibits its own activity, since recent studies have shown that Wnt signaling regulates the autoinhibition of CK1epsilon. We will solve the structure of CK1epsilon bound to Dishevelled, one of the principle targets of CK1epsilon activity. Finally, using a structure-function approach, we will determine if Dishevelled is the essential target of CK1epsilon activity in regulating the Wnt pathway. Since the Wnt pathway plays key roles in vertebrate development and oncogenesis, these studies will be important in advancing knowledge of the molecular basis of birth defects and cancer.
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