Application of an integrated physical and functional screening approach to identify inhibitors of the Wnt pathway.
Large-scale proteomic approaches have been used to study signaling pathways. However, identification of biologically relevant hits from a single screen remains challenging due to limitations inherent in each individual approach. To overcome these limitations, we implemented an integrated, multi-dimensional approach and used it to identify Wnt pathway modulators. The LUMIER protein-protein interaction mapping method was used in conjunction with two functional screens that examined the effect of overexpression and siRNA-mediated gene knockdown on Wnt signaling. Meta-analysis of the three data sets yielded a combined pathway score (CPS) for each tested component, a value reflecting the likelihood that an individual protein is a Wnt pathway regulator. We characterized the role of two proteins with high CPSs, Ube2m and Nkd1. We show that Ube2m interacts with and modulates beta-catenin stability, and that the antagonistic effect of Nkd1 on Wnt signaling requires interaction with Axin, itself a negative pathway regulator. Thus, integrated physical and functional mapping in mammalian cells can identify signaling components with high confidence and provides unanticipated insights into pathway regulators.
Miller
Bryan W
BW
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
Lau
Garnet
G
Grouios
Chris
C
Mollica
Emanuela
E
Barrios-Rodiles
Miriam
M
Liu
Yongmei
Y
Datti
Alessandro
A
Morris
Quaid
Q
Wrana
Jeffrey L
JL
Attisano
Liliana
L
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D060466:Axin Protein; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D051379:Mice; D008954:Models, Biological; D011485:Protein Binding; D025941:Protein Interaction Mapping; D034622:RNA Interference; D012097:Repressor Proteins / Q000378:metabolism; D015398:Signal Transduction; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D051153:Wnt Proteins / Q000037:antagonists & inhibitors*
D048868:Adaptor Proteins, Signal Transducing; D060466:Axin Protein; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; C449091:NKD1 protein, human; D012097:Repressor Proteins; D051153:Wnt Proteins; D044763:Ubiquitin-Conjugating Enzymes; C000596156:UBE2M protein, human