Wnt-signaling in Development and Disease

Personnel

Marek Mlodzik, Ph.D.
Program Director, PI Project 1

Frizzled (Fz) receptors act as signal transducers of Wnt growth factors via several effector pathways, notably Wnt/β-catenin (β-cat) and Fz/planar cell polarity (PCP) signaling. Fz/PCP and Wnt-Fz/β-cat signaling diverge downstream of the cytoplasmic factor Dishevelled (Dsh). Often both pathways act in the same tissues, and a tight regulation of signaling specificity is essential for normal development and cellular homeostasis. In deregulated scenarios, selection of the wrong Fz-pathway often leads to disease (i.e. cancer). Although a molecular framework for both pathways is established, very little is known about the regulation of Wnt-Fz signaling specificity. This application investigates the molecular aspects of Fz signaling specificity distinguishing between Wnt-Fz/β-Cat and Wnt-Fz/PCP signaling. We will address these issues in Drosophila (the model organism where both pathways were discovered) and in mammalian cell culture. The specific focus is the regulation of Dsh, a cytoplasmic Fz-associated factor shared between both pathways. Based on preliminary data we hypothesize that Dsh is phosphorylated in a pathway specific manner. A combination of cell culture, in vivo, and biochemical studies will dissect the phosphorylation requirements of Dsh that switch its function between the Wnt-Fz pathways. The Specific Aims are: 1. To identify and functionally define kinases acting in Dsh regulation and pathway specific responses; 2. to determine specific phosphorylation event(s) on Dsh and their role in the selection of the signaling outcome; and 3. to define the role of PKC family kinases with common or pathway-specific Dsh regulation. This will be complemented with a small compound screening assay selecting for pathway specific inhibitors. Our genome wide RNAi screen indicated that differential phosphorylation of Dsh contributes to pathway selection, identifying several kinases/phosphatases. Their specific role will be defined in both pathways. In addition, our mass-spectrometry analyses have identified the phosphorylated Dsh residues. Physiological and medical relevance of the phosphorylation events will be established in vivo.

Performance Sites

Icahn School of Medicine (MSSM), New York, NY

Personnel

Sergei Sokol, Ph.D.
PI Project 2

TCF3 is a vertebrate TCF family member that functions as a transcriptional repressor in several models, but it is unclear how TCF3 is regulated by Wnt signaling. Preliminary studies demonstrate that a Wnt protein stimulates specific TCF3 phosphorylation, which correlates with the function of Wnt signaling during anteroposterior embryonic axis specification. Additionally, initial experiments point to homeodomain-interacting protein kinase 2 (HIPK2) as a specific protein kinase that is responsible for this phosphorylation. Wnt signaling is hypothesized to alleviate TCF3-mediated transcriptional repression during axis development and that HIPK2 mediates this effect of Wnt signaling. To test these hypotheses, new transcriptional targets of TCF3 that are also regulated by Wnt signaling will be identified. Experiments are proposed to investigate the significance of TCF3 phosphorylation for the mechanism of transcriptional derepression of Wnt target genes and study the function of HIPK2 in vertebrate axis specification. Xenopus embryos are chosen as an experimental system, because they allow to combine biochemical and cell biological approaches with rapid functional analysis in vivo. These studies should provide insight into basic regulatory mechanisms, which operate during early vertebrate development and are likely to be misregulated in cancer.

Performance Sites

Icahn School of Medicine (MSSM), New York, NY