Analysis of phosphorylation-dependent protein-protein interactions using a bacterial two-hybrid system.
Phosphorylation-dependent protein-protein interactions provide the foundation for a multitude of intracellular signal transduction pathways. One of the goals of signal transduction research is to more precisely understand the nature of these phosphorylation-dependent interactions. Here, we describe a bacterial two-hybrid assay that allows for the rapid, efficient analysis of phosphorylation-dependent protein-protein interactions. In this system, the interacting protein domains are provided as fusion proteins in Escherichia coli. cells that contain a eukaryotic kinase. Specific phosphorylation of one of the fused protein domains results in a protein-protein interaction that can be detected as a change in the expression of a reporter gene. We also describe how this system can be modified to permit the use of cDNA libraries to identify either novel binding partners for a phosphorylated substrate or novel kinases that can induce a specific protein-protein interaction.
Shaywitz
Adam J
AJ
Program in Biological and Biomedical Sciences, Division of Medical Sciences, Harvard Medical School, and Division of Neuroscience, Children's Hospital, Boston, MA 02115, USA.
Dove
Simon L
SL
Greenberg
Michael E
ME
Hochschild
Ann
A
D010582:Bacteriophage lambda / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D017930:Genes, Reporter / Q000235:genetics; D010766:Phosphorylation; D010957:Plasmids / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D014158:Transcription, Genetic / Q000235:genetics / Q000502:physiology; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D054334:Viral Regulatory and Accessory Proteins; D001616:beta-Galactosidase / Q000096:biosynthesis; D001618:beta-Lactamases / Q000096:biosynthesis
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit; D001616:beta-Galactosidase; D001618:beta-Lactamases