Bili Inhibits Wnt Signaling Figure 5. Bili associates with LRP6 and attenuates Axin recruitment to LRP6 in HEK293T cells. (A) hBili interacts with LRP6. HA-LRP6 was cotransfected with flag-GFP or flag-hBili in HEK293T cells. Cell lysates were immunoprecipitated with anti-flag antibody and immunoblotted with anti-HA antibody. (B) dBili interacts with LRP6. dBili-HA and DNLrp6-myc were cotransfected in S2R+ cells. Cell lysates were immunoprecipitated with anti-myc antibody and immunoblotted with anti-HA antibody. (C) Overexpression of Bili attenuates GSK3 induced AXIN/LRP6 association. HEK293T cells stably expressing LRP6-eGFP were cotransfected with AXIN-myc, GSK3B and Bili-flag or vector control. Cell lysates were immunoprecipitated with anti-eGFP antibody and immunoblotted with anti-myc. Data shown are representative of three independent experiments. (D) Bili knockdown enhances GSK3B induced AXIN/LRP6 association. HEK293T cells stably expressing LRP6-eGFP were transfected with Bili siRNA or control siRNA. 48 hours later BilisiRNA or control siRNA treated cells were transfected with AXIN-myc and GSKB. Cell lysates were immunoprecipitated with anti- eGFP antibody and immunoblotted with anti-myc. Data shown are representative of three independent experiments. doi:10.1371/journal.pone.0006129.g005 events occurring at the plasma membrane following Wnt activation of the Frizzled/LRP6 coreceptor complex. Studies over the last several years have shed light on these events by uncovering several kinases that phosphorylate LRP6 resulting in its recruit- ment of AXIN [4,5]. As AXIN recruitment to LRP6 appears to be a key step in the propagation of a WNT signal, it is likely a hotspot for pathway regulation. Our identification of Bili as a negative regulator of Wnt/b-catenin signaling and inhibitor of AXIN recruitment to LRP6 is the first report of negative regulation at this step in pathway transduction. The specific details of how Bili affects AXIN recruitment will be the focus of future studies. One proposed mechanism is that Bili sterically hinders the recruitment of the kinases required for LRP6 phosphorylation and subsequent AXIN recruitment. A second possibility is that Bili inhibits the formation of LRP6 aggregates or ‘signalosomes’ which have been shown to be the LRP6 population associated with AXIN (Bilic et al. 2007). We propose that inhibitors of the Wnt/b-catenin pathway fall into two groups: ‘constitutive inhibitors’ that keep basal levels of b- catenin low, such as members of the degradation complex; and ‘inducible inhibitors’ that act outside of the degradation complex and may function only when the pathway has been activated. The ‘constitutive inhibitors’ would include APC and AXIN, while ‘inducible inhibitors’ would include NKD and Bili. Consistent with this categorization, NKD and Bili both antagonize a WNT signal between receptor activation and the degradation machinery as well as having similar phenotypes in both flies [22] and fish [23]. In concert, constitutive and inducible inhibitors allow for rapid pathway activation in response to WNT ligand and tight regulation of the strength and duration of the WNT signal. Continued characterization and identification of both constitutive and inducible inhibitors is key to our understanding of the regulation of Wnt/b-catenin signaling in development and disease. Materials and Methods Fly screen The screen was performed as previously described [10]. Reporter assays b-catenin reporter assays were carried out in the 24-well or 48- well plate format in HEK293T cells transiently expressing SuperTOPFLASH (1–5 ng) or BAR (b-catenin Activated Report- er) and Renilla luciferase (10–40 ng) for normalization. FOPFlash and fuBAR, which contain mutated response elements and do not respond to WNT/b-catenin signaling, were used as control PLoS ONE | www.plosone.org 7 July 2009 | Volume 4 | Issue 7 | e6129