High-throughput approaches to dissecting MAPK signaling pathways.
With the development of genome-wide RNAi libraries, it is now possible to screen for novel components of mitogen-activated protein kinase (MAPK) pathways in cell culture. Although genetic dissection in model organisms and biochemical approaches in mammalian cells have been successful in identifying the core signaling cassettes of these pathways, high-throughput assays can yield unbiased, functional genomic insight into pathway regulation. We describe general high-throughput approaches to assaying MAPK signaling and the receptor tyrosine kinase (RTK)/extracellular signal-regulated kinase (ERK) pathway in particular using a phospho-specific antibody-based readout of pathway activity. We also provide examples of secondary validation screens and methods for managing large datasets for future in vivo functional characterization.
Friedman
Adam
A
Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
Perrimon
Norbert
N
D000818:Animals; D000918:Antibody Specificity; D002478:Cells, Cultured; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D004330:Drosophila / Q000166:cytology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results
D012330:RNA, Double-Stranded; D020794:Receptor Protein-Tyrosine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases