Bili Inhibits Wnt Signaling Figure 4. Bili inhibits Wnt/b-catenin transcriptional reporters in human cell-lines. (A) Pretreatment with Bili or Axin1 siRNA leads to a 2-fold greater activation of a b-catenin responsive reporter after treatment with WNT3A conditioned media (black bars) in RKO-BAR cells compared to control siRNA. b-catenin (b-cat) siRNA ablated Wnt 3A mediated reporter activation. Error bars represent STDEV. (B) hBili overexpression inhibits Wnt3A mediated b-catenin responsive reporter activation in HEK293T cells. (C) hBili overexpression inhibits WNT1, DNLRP6, and DVl, but not BIO, or b-catenin mediated b-catenin responsive reporter activation in HEK293T cells. HEK293T cells stably expressing a b-catenin responsive luciferase reporter were transfected or treated with WNT1, DNLRP6, DVL, BIO, or b-catenin along with GFP or Bili. Error bars represent STDEV. (D) Bili colocalizes with LRP6. HEK293T cells were transfected with flag-hBili and hLRP6-eGFP and imaged. (*p,0.05, **p,0.005, ***p,0.0005) doi:10.1371/journal.pone.0006129.g004 in the presence of BIO (6-bromoindirubin-39-oxime), a GSK3 inhibitor [19], or co-expressed with other pathway activators including WNT1, DNLRP6, Dishevelled (DVL) or b-catenin. Bili gain-of-function reduced reporter activation by WNT1, DNLRP6, and DVL (Fig. 4C). In contrast, hBili overexpression did not affect reporter activation by overexpression of b-catenin or BIO treatment (Fig. 4C). These results place the function of hBili between the Wnt receptor complex and the b-catenin degradation complex. Consistent with these epistasis studies, and the observed membrane associated localization of other FERM domain containing proteins, we found that dBili localizes in a concentric ring, adjacent to Arm protein at the plasma membrane in fly cells (Fig. S3E and S3E’). Based on these results we predicted that Bili might regulate signaling from the Wnt co-receptors LRP5/6 or Frizzled. In direct support of this prediction, tagged hBili colocalizes with tagged LRP6 as detected by immunocytochemistry (ICC) (Fig. 4D). Furthermore, hBili-FLAG pulls down tagged-LRP6 in co-immu- noprecipitation (co-IP) assays (Fig. 5A). The association of Bili and LRP6 was also confirmed in Drosophila S2R+ cells where immunoprecipitated tagged-DNLRP6 pulled down tagged-dBili (Fig. 5B). Collectively, these data suggest that Bili functions upstream of b-catenin stabilization and associates with the LRP6 receptor complex. Bili antagonizes the recruitment of Axin to LRP6 During Wnt signaling, LRP6 is phosphorylated in a Wnt- dependent manner by CSNK1G1 and GSK3B, which then facilitates the binding of AXIN to LRP6 [4,5]. Consistent with this model, overexpression of GSK3B consistently and robustly mimics the ability of WNT to induce the binding of AXIN to LRP6 ([20,21]. Our observations that Bili negatively regulates Wnt/b- catenin signaling upstream of the destruction complex and associates with LRP6 lead us to hypothesize that Bili may regulate the recruitment of AXIN to LRP6. To address this hypothesis we employed co-immunoprecipitation to monitor the association of AXIN and LRP6 following gain-of- function (Fig. 5C) or loss-of-function (Fig. 5D) of Bili. HEK293T cells stably expressing LRP6-EGFP were transiently transfected with AXIN-MYC. As expected LRP6 co-immunoprecipitated AXIN in the presence of overexpressed GSK3B (Fig. 5C & 5D, lane 1 compared to lane 3). This interaction of LRP6 and AXIN was attenuated approximately 2-fold in the presence of overex- pressed hBili (Fig. 5C, lane 2 compared to lane 1) and enhanced more than 2-fold when hBili levels were knocked down (Figure 5D, lane 2 compared to lane 1). Furthermore, even in the absence of GSK3B, knockdown of hBili enhanced the association of AXIN and LRP6 approximately 4-fold (Fig. 5D, lane 4 compared to lane 3). Together these data suggest that Bili negatively regulates the recruitment of AXIN to LRP6 thereby providing a mechanism for how Bili negatively regulates Wnt/b-catenin signaling. Discussion Many aspects of Wnt/b-catenin signal transduction remain poorly understood. One aspect in particular is the transduction PLoS ONE | www.plosone.org 6 July 2009 | Volume 4 | Issue 7 | e6129