Detection of Protein Kinase-Binding Partners by the Yeast Two-Hybrid Analysis

Abstract

One mechanism by which protein kinases are regulated and function is via their interaction with other cellular proteins (reviewed in ref. 1). Thus, identifying proteins that interact with kinases will reveal important mechanisms of kinase action. The two-hybrid method is a genetic assay for detecting protein-protein interactions in vivo in yeast (2). This approach has advantages over other available methods for studying protein interactions. The assay can be used to define interactions between two known proteins or to search genomic or cDNA libraries for proteins that interact with a bait protein. For the latter approach, the method not only identifies the interacting protein but also provides the gene for the protein. Or, the method can be used to identify specific domains or specific amino acids involved in protein-protein interactions. The assay is performed in vivo; thus, the interacting proteins are more likely to be in their native conformations. The method can detect transient and weak interactions, which is probably how many interactions occur in signaling complexes in vivo. This approach avoids the need to purify interacting proteins or to generate antibodies in order to identify the proteins. The assay is sensitive, efficient, and rapid. A disadvantage of the method is generation of false positives. Thus, care must be taken to eliminate false positives and to verify protein interactions that are detected in the screen. Nonetheless, the yeast two-hybrid analysis has been used successfully to identify many important protein interactions.