The ras recruitment system, a novel approach to the study of protein-protein interactions

Abstract

The yeast two-hybrid system represents one of the most efficient approaches currently available for identifying and characterizing protein-protein interactions [1-4]. Although very powerful, this procedure exhibits several problems and inherent limitations [5]. A new system, the Sos recruitment system (SRS), was developed recently [6] based on a different readout from that of the two-hybrid system [6-8]. SRS overcomes several of the limitations of the two-hybrid system and thus serves as an attractive alternative for studying protein-protein interactions between known and novel proteins. Nevertheless, we encountered a number of problems using SRS and so have developed an improved protein recruitment system, designated the Ras recruitment system (RRS), based on the absolute requirement that Ras be localized to the plasma membrane for its function [9-10]. Ras membrane localization and activation can be achieved through interaction between two hybrid proteins. We have demonstrated the effectiveness of the novel RRS system using five different known protein-protein interactions and have identified two previously unknown protein-protein interactions through a library screening protocol. The first interaction (detailed here) is between JDP2, a member of the basic leucine zipper (bZIP) family, and C/EBPgamma, a member of the CCAAT/enhancer-binding protein (C/EBP) family. The second interaction is between the p21-activated protein kinase Pak65 and a small G protein (described in the accompanying paper by Aronheim et al. [11]). The RRS system significantly extends the usefulness of the previously described SRS system and overcomes several of its limitations.