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.