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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
2R01HD027262-15A1
Application #
6926557
Study Section
Special Emphasis Panel (ZRG1-DEV-1 (01))
Program Officer
Klein, Steven
Project Start
1990-08-01
Project End
2010-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
15
Fiscal Year
2005
Total Cost
$323,240
Indirect Cost
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Weiser, Douglas C; Kimelman, David (2012) Analysis of cell shape and polarity during zebrafish gastrulation. Methods Mol Biol 839:53-68
Kimelman, David (2010) On the fast track to organizer gene expression. Dev Cell 19:190-2
Dahlberg, Caroline Lund; Nguyen, Elizabeth Z; Goodlett, David et al. (2009) Interactions between Casein kinase Iepsilon (CKIepsilon) and two substrates from disparate signaling pathways reveal mechanisms for substrate-kinase specificity. PLoS One 4:e4766
Martin, Benjamin L; Kimelman, David (2008) Regulation of canonical Wnt signaling by Brachyury is essential for posterior mesoderm formation. Dev Cell 15:121-33
Weiser, Douglas C; St Julien, Krystal R; Lang, James S et al. (2008) Cell shape regulation by Gravin requires N-terminal membrane effector domains. Biochem Biophys Res Commun 375:512-6
Weiser, Douglas C; Pyati, Ujwal J; Kimelman, David (2007) Gravin regulates mesodermal cell behavior changes required for axis elongation during zebrafish gastrulation. Genes Dev 21:1559-71
Xu, Wenqing; Kimelman, David (2007) Mechanistic insights from structural studies of beta-catenin and its binding partners. J Cell Sci 120:3337-44
Sampietro, James; Dahlberg, Caroline L; Cho, Uhn Soo et al. (2006) Crystal structure of a beta-catenin/BCL9/Tcf4 complex. Mol Cell 24:293-300
Kimelman, D; Xu, W (2006) beta-catenin destruction complex: insights and questions from a structural perspective. Oncogene 25:7482-91
Clements, Wilson K; Kimelman, David (2005) LZIC regulates neuronal survival during zebrafish development. Dev Biol 283:322-34

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