Schematic of an integrated, multi-dimensional approach to identify signaling pathway components. Genes harboring cell signaling-associated domains were randomly selected and then used to generate libraries of corresponding Flag-tagged cDNAs and siRNAs. These were then used in three high-throughput screens to map protein–protein interactions by LUMIER and to examine the effect of altering protein expression levels on a transcriptional reporter. The data from the three screens were integrated to derive a combined pathway score (CPS), which predicts the likelihood that a protein is a component of the signaling pathway being studied.

High-throughput screening of protein–protein interactions of the Wnt pathway. (A) The LUMIER assay involves coexpression of a Flag-tagged prey and Firefly luciferase (FF-Luc)-tagged bait in mammalian cells. An interaction is detected by α-Flag immunoprecipitation followed by measurement of luciferase activity. (B) Results of the LUMIER screen of the Wnt pathway. Luciferase-tagged baits (left) were screened against 640 Flag-tagged cDNAs (numbered across top) in the absence (−) or presence (+) of Wnt3A-conditioned media. The mLIR score for each interaction is represented by colors according to the indicated scale. (C) The Wnt interactome as defined by LUMIER. Interactions with an mLIR >2 in at least 2 of 3 runs or in the single Wnt3A run, and with a total average mLIR of >2 are visualized with Cytoscape (Shannon et al, 2003). The complete network is shown as a circular representation with baits represented by large yellow nodes, preys as small nodes and interactions by edges.

RNAi and overexpression screens for Wnt pathway modulators. Modulation of TOPflash activity by overexpression or RNA-mediated abrogation of expression of specific genes was used to identify regulators of the Wnt pathway. Data are shown as scatterplot comparisons of fold over median values from two runs with the indicated concentrations of cDNAs (A) or replicate siRNA screens (B) conducted in the absence (left panels) or presence of Wnt3A (right panels).

Integration of data from multiple screens. (A) A proportional Venn diagram (prepared with www.cs.kent.ac.uk/people/staff/pjr/EulerVennCircles/EulerVennApplet.html), indicating the number of hits in the LUMIER (as defined in Figure 2), RNAi and cDNA overexpression (where overexpression or knockdown gave an average fold over median >1.5 or <0.6 in the presence or absence of Wnt3A) and the degree of overlap. (B) Distribution of combined pathway scores (CPSs) of integrated screen data. The percentage of core (Group 1) and known (Group 2) components of the Wnt pathway and all tested genes (All) corresponding to the indicated CPS thresholds are shown. (C) Receiver operating characteristic (ROC) curves assessing screen and CPS performance. The sensitivity (true positives) versus 1-specificity (false positives) is plotted for the integrated (CPS) and individual screens (cDNA, LUMIER and RNAi).

Top hits as defined by CPS. Scores for individual screens and the CPS are shown. Shading indicates known Group 1 (green), Group 2 (yellow) and putative novel (blue) pathway components. Full length and a splice variant of Dvl1 are shown.

Ube2m is a Wnt pathway component. (A) Endogenous β-catenin interacts with 3XFlag-Ube2m. HEK293T cells were transfected with Flag-tagged Ube2m or empty vector. Interactions were detected by anti-Flag immunoprecipitation followed by anti-β-catenin immunoblotting. (B) Ube2m overexpression increases TOPflash activity. HEK293T cells were transfected with TOPflash and the indicated amounts of Ube2m. Promoter activity was measured by luciferase assay and data are shown as the mean of triplicate samples ±standard deviation. (C) Ube2m overexpression increases β-catenin levels. HEK293T cells were transfected with increasing amounts of Flag-Ube2m cDNA and β-catenin levels were determined by anti-β-catenin, anti-phospho-β-catenin or anti-active-β-catenin immunoblotting. Actin levels were detected by immunoblotting as a loading control. (D, E) HEK293T cells were transfected with TOPflash and the indicated amounts of myc-tagged Nedd8, Smad1 or cofilin in the absence (D) or presence (E) of Ube2m. Cells were treated with or without Wnt3A overnight. Promoter activity was measured by luciferase assay and the data are shown as the mean of duplicate (D) or triplicate (E) samples±standard deviation. (F) Model of the role of Ube2m in the degradation of β-catenin by the β-TrCP/SCF complex.

Nkd1 cooperates with Axin1 to inhibit the Wnt pathway. (A) Nkd1 interacts with Axin. HEK293T cells were transfected with luciferase-tagged Nkd1 (Nkd1-FF-Luc) and 3XFlag-tagged Axin1 or Axin2. Interactions were detected by luciferase assay following anti-flag immunoprecipitation. Data are shown as the mean of two samples ±standard deviation. (B) Endogenous Axin1 interacts with 3XFlag-Nkd1. HEK293T cells were transfected with Flag-tagged Nkd1 or empty vector. Interactions were detected by anti-Flag immunoprecipitation followed by immunoblotting. The migration of endogenous Axin was confirmed in total cell lysates by comparison with transiently transfected Axin1 (left two lanes). ^* Indicates a nonspecific band. (C) Schematic of Nkd1 deletion mutants and the amino acid sequence of the His-rich carboxy-terminus is shown. (D) Axin1 interacts with the C-terminal His-rich tail of Nkd1. HEK293T cells were transfected with luciferase-tagged Axin1 (Axin1-FF-Luc) and the indicated Flag-Nkd1 constructs. Cell lysates were subject to anti-Flag immunoprecipitation and the presence of Axin1 was assessed by luciferase assay. Data are expressed as the mean of two samples±range. (E) Multiple regions of Nkd1 are required for efficient inhibition of TOPflash activity. HEK293T cells were transfected with TOPflash and the indicated Flag-Nkd1 constructs. Cells were treated overnight with or without Wnt3A and TOPflash activity was measured by luciferase assay. Data are shown as the mean of two samples ±standard deviation.

Nkd1 cooperates with Axin1 to inhibit the Wnt pathway. (A) Nkd1 and Axin1 cooperate to inhibit TOPflash. HEK293T cells were transfected with TOPflash, the indicated amount of 3XFlag-Nkd1 and 20 nM control or Axin1-targeting siRNA. Cells were treated overnight with or without Wnt3A and TOPflash activity was measured by luciferase assay. Data are shown as the mean of three samples (left panel) or percentage of control (right panel) ±standard deviation. (B) Model of inhibition of Wnt3A signaling by Nkd1 and Axin1 cooperation.