To confirm the results obtained in the synthetic TOP luciferase reporter assays, we further examined the effects of C9orf140 overexpression on the transcript levels of well-defined Wnt target genes, including [11], [12], and [13]

To confirm the results obtained in the synthetic TOP luciferase reporter assays, we further examined the effects of C9orf140 overexpression on the transcript levels of well-defined Wnt target genes, including [11], [12], and [13]. that C9orf140 mediates a negative feedback loop of Wnt/-catenin signaling by interacting with Axin1. Our results advance the current understanding of the exquisite control of Wnt/-catenin signaling cascade, and provide evidence of the new role of C9orf140. Introduction The canonical Wnt signaling pathway coordinates cell-fate specification and has a key role in normal metazoan development and survival [1]. Dysregulated Wnt signaling results in developmental deficits and contributes to multiple human malignancies, including cancer [2]. A key feature of the canonical OAC1 Wnt signaling is the regulation of -catenin stabilization by a OAC1 cytoplasmic destruction complex, composed of Axin1, adenomatous polyposis coli (APC), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3) [3]. Without Wnt signals, a low level of cytoplasmic -catenin was kept through captured by Axin1 in the destruction complex, phosphorylated by CK1 and GSK3, and then ubiquitinated by E3 ubiquitin ligase SCF-TrCP [2]. In the process, protein phosphatases (PP) such as PP2A may act as antagonists of CK1 and GSK3, dephosphorylating -catenin [4, 5]. With Wnt ligand stimulation, the SCF-TrCP -mediated ubiquitination of -catenin was suppressed, which leads to the stabilization of -catenin and the transcriptional activation of Wnt target genes [2]. Among all other core components of the destruction complex, Axin1 is the central scaffold and the rate-limiting factor, because the other components regulate Wnt/-catenin signaling by directly interacting with Axin1 [6]. Given the important role of Wnt/-catenin signaling in maintenance of cell and tissue homeostasis, novel regulators that interact with Axin1 and the regulatory mechanisms involved in their fine-tuning should be determined [6]. Here, we identified a novel Axin1-interacting protein named C9orf140, also known as and suppressor APC domain containing 2 (SAPCD2). Functional analyses in cultured cells and zebrafish demonstrate that C9orf140 negatively regulates Wnt/-catenin signaling at the destruction complex level. Moreover, the expression of C9orf140 is induced by Wnt through -catenin. Our results suggest that C9orf140 induced by Wnt functions in OAC1 a negative feedback loop by outcompeting PP2A for binding to Axin1, thus leading to a significant decrease in -catenin protein levels and the transcript levels of Wnt target genes. Results C9orf140 is a novel Axin1-interacting factor To understand how the Wnt/-catenin signaling pathway is regulated, we set out to define its molecular makeup using proteomic approaches. By coupling tandem-affinity purification with mass spectrometry analysis, we have successfully identified C9orf140, an uncharacterized protein, as a novel factor that resides in the Axin1 protein complex (Supplementary Table OAC1 1). We further carried out a reciprocal co-immunoprecipitation experiment (Fig. ?(Fig.1a)1a) and a GST pulldown experiment (Fig. ?(Fig.1b),1b), respectively, which confirmed the interaction between ectopically expressed C9orf140 and Axin1. Furthermore, we found that C9orf140 also interacted with endogenous Axin1 (Fig. ?(Fig.1c).1c). Together, these data suggested that C9orf140 is a novel Axin1-interacting protein. Open in a separate window Fig. 1 C9orf140 is a novel Axin1-interacting protein. a Confirmation of the C9orf140CAxin1 interaction by using co-immunoprecipitation. The 293T cells were co-transfected with Flag-C9orf140/Flag-PSMD4 and HA-Axin1 for 24?h. Cell lysates were subjected to immunoprecipitation with an anti-Flag antibody and an anti-HA antibody. Co-precipitating proteins were visualized by western blotting. The unrelated protein PSMD4 was used as a negative control. b The purified GST or GST-C9orf140-fusion protein bound to agarose beads was added to the lysate of 293T cells ectopically expressing Myc-Axin1. After GST affinity purification, protein complexes OAC1 were washed and detected by western blot analysis with anti-Myc or anti-GST as indicated. GST protein was used as a negative control. c HeLa cell lysates were immunoprecipitated with rabbit anti-Axin1 antibody, rabbit anti-C9orf140 antibody and control rabbit IgG, then immunoblotted with antibodies as indicated. d C9orf140 co-localizes with RUNX2 Axin1. HeLa cells were transfected with Flag-C9orf140 and Myc-Axin1. The cells were then fixed and stained with anti-Flag antibody (green), anti-Myc antibody (red), and DAPI (blue). No pre-extract: cells were first fixed and then permeabilized as described in the Materials and methods; Pre-extract: cells were first permeabilized and then fixed as described in the Materials and Methods. Bar, 20?m To support this hypothesis, we performed indirect immunofluorescence studies to examine the sub-cellular localization of C9orf140 and Axin1 through overexpressing Flag-C9orf140 and Myc-Axin1 in the cells. Without pre-extraction.