Bili Inhibits Wnt/b-Catenin Signaling by Regulating the Recruitment of Axin to LRP6 Lorna S. Kategaya1., Binita Changkakoty2., Travis Biechele1., William H. Conrad 1 , Ajamete Kaykas1, Ramanuj DasGupta2, Randall T. Moon 1 * 1 Department of Pharmacology, Howard Hughes Medical Institute (HHMI), University of Washington School of Medicine and Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington, United States of America, 2 Department of Pharmacology and Cancer Institute, New York University School of Medicine, New York, New York, United States of America Abstract Background: Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ß-catenin pathway. Methodology/Principal Findings: In pursuit of such insights, we performed a genome-wide RNAi screen in Drosophila cells expressing an activated form of LRP6 and a b-catenin-responsive reporter. This screen resulted in the identification of Bili, a Band4.1-domain containing protein, as a negative regulator of Wnt/b-catenin signaling. We found that the expression of Bili in Drosophila embryos and larval imaginal discs significantly overlaps with the expression of Wingless (Wg), the Drosophila Wnt ortholog, which is consistent with a potential function for Bili in the Wg pathway. We then tested the functions of Bili in both invertebrate and vertebrate animal model systems. Loss-of-function studies in Drosophila and zebrafish embryos, as well as human cultured cells, demonstrate that Bili is an evolutionarily conserved antagonist of Wnt/b-catenin signaling. Mechanistically, we found that Bili exerts its antagonistic effects by inhibiting the recruitment of AXIN to LRP6 required during pathway activation. Conclusions: These studies identify Bili as an evolutionarily conserved negative regulator of the Wnt/b-catenin pathway. Citation: Kategaya LS, Changkakoty B, Biechele T, Conrad WH, Kaykas A, et al. (2009) Bili Inhibits Wnt/b-Catenin Signaling by Regulating the Recruitment of Axin to LRP6. PLoS ONE 4(7): e6129. doi:10.1371/journal.pone.0006129 Editor: Carl-Philipp Heisenberg, Max Planck Institute of Molecular Cell Biology and Genetics, Germany Received September 29, 2008; Accepted June 1, 2009; Published July 2, 2009 Copyright: 2009 Kategaya et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rtmoon@u.washington.edu . These authors contributed equally to this work. Introduction Wnt signaling through b-catenin regulates the expression of genes involved in cell proliferation and cell fate during development, in adult homoeostasis, and in diverse diseases [1]. In the absence of a Wnt ligand, steady-state levels of b-catenin are maintained at a low level by a ‘degradation complex’ that promotes the phosphorylation, ubiquitination, and proteosomal degradation of b-catenin [2]. The binding of Wnt ligands to the transmembrane FZD (frizzled) and LRP5/6 (low-density-lipopro- tein-related protein 5/6) co-receptors triggers an inhibition of the degradation complex resulting in the stabilization and nuclear accumulation of b-catenin, where it binds to transcription factors of the TCF/LEF family and regulates target gene expression. Signaling events immediately following Wnt binding to the receptor complex are poorly understood. Previous studies have shown that in the presence of Wnt ligand, Dishevelled (DVL) facilitates phosphorylation of the cytoplasmic tail of LRP5/6 [3] by Casein Kinase 1c (CSNK1G1) and Glycogen Synthase Kinase- 3b (GSK3B) [4,5]. Phosphorylation of LRP5/6 is followed by the recruitment of AXIN away from the degradation complex. As AXIN is likely a rate-limiting scaffold protein required for the degradation of b-catenin [6], the relocation of AXIN to the membrane away from the degradation complex likely promotes stabilization of b-catenin [7]. We performed a genome-wide RNAi screen in Drosophila cells to identify genes that regulate LRP6 mediated activation of a b- catenin reporter. Here we describe Bili (Band4.1 inhibitor LRP interactor), a previously uncharacterized FERM domain contain- ing protein. Analyses of Bili function in Drosophila, zebrafish, and cultured human cells support the conclusion that Bili is an evolutionarily conserved antagonist of b-catenin signaling. Mech- anistically, we show that Bili regulates the recruitment of AXIN to LRP6. Results RNAi screen in Drosophila identifies Bili as a negative regulator of Wg signaling Drosophila mutagenesis screens have resulted in the identification of several core Wg signaling proteins [8]. The potential for discovering additional contextually relevant molecular players in the pathway is enhanced by robust read-outs such as transcrip- tional reporter assays coupled with genome-wide RNAi libraries PLoS ONE | www.plosone.org 1 July 2009 | Volume 4 | Issue 7 | e6129