ABSTRACT
Mitogen-activated protein kinase (MAPK) pathways form the backbone of signal transduction in the mammalian cell. Here we applied a systematic experimental and computational approach to map 2,269 interactions between human MAPK-related proteins and other cellular machinery and to assemble these data into functional modules. Multiple lines of evidence including conservation with yeast supported a core network of 641 interactions. Using small interfering RNA knockdowns, we observed that approximately one-third of MAPK-interacting proteins modulated MAPK-mediated signaling. We uncovered the Na-H exchanger NHE1 as a potential MAPK scaffold, found links between HSP90 chaperones and MAPK pathways and identified MUC12 as the human analog to the yeast signaling mucin Msb2. This study makes available a large resource of MAPK interactions and clone libraries, and it illustrates a methodology for probing signaling networks based on functional refinement of experimentally derived protein-interaction maps.
Subject(s)
MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinases/metabolism , Proteomics/methods , Base Sequence , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cell Line , Cell Membrane/metabolism , Cloning, Molecular , DNA, Complementary/genetics , HSP90 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Luciferases/genetics , MAP Kinase Signaling System/genetics , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/physiology , Molecular Sequence Data , Mucins/genetics , Mucins/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Small Interfering/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sodium-Hydrogen Exchanger 1 , Sodium-Hydrogen Exchangers/genetics , Sodium-Hydrogen Exchangers/metabolism , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Two-Hybrid System TechniquesABSTRACT
Large-scale functional genomics approaches are fundamental to the characterization of mammalian transcriptomes annotated by genome sequencing projects. Although current high-throughput strategies systematically survey either transcriptional or biochemical networks, analogous genome-scale investigations that analyze gene function in mammalian cells have yet to be fully realized. Through transient overexpression analysis, we describe the parallel interrogation of approximately 20,000 sequence annotated genes in cancer-related signaling pathways. For experimental validation of these genome data, we apply an integrative strategy to characterize previously unreported effectors of activator protein-1 (AP-1) mediated growth and mitogenic response pathways. These studies identify the ADP-ribosylation factor GTPase-activating protein Centaurin alpha1 and a Tudor domain-containing hypothetical protein as putative AP-1 regulatory oncogenes. These results provide insight into the composition of the AP-1 signaling machinery and validate this approach as a tractable platform for genome-wide functional analysis.
