THE RAS RECRUITMENT SYSTEM, A NOVEL-APPROACH TO THE STUDY OF PROTEIN-PROTEIN INTERACTIONS

The yeast two-hybrid system represents one of the most efficient appro aches currently available for identifying and characterizing protein-p rotein interactions [1-4]. Although very powerful, this procedure exhi bits several problems and inherent limitations [5]. A new system, the Sos recruitment system (SRS), was developed recently [6] based on a di fferent readout from that of the two-hybrid system [6-8]. SRS overcome s 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 numb er of problems using SRS and so have developed an improved protein rec ruitment 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 diff erent known protein-protein interactions and have identified two previ ously unknown protein-protein interactions through a library screening protocol. The first interaction (detailed here) is between JDP2, a me mber of the basic leucine zipper (bZIP) family, and C/EBP gamma, a mem ber of the CCAAT/enhancer-binding protein (C/EBP) family. The second i nteraction is between the p21-activated protein kinase Pak65 and a sma ll G protein (described in the accompanying paper by Aronheim et al. [ 11]). The RRS system significantly extends the usefulness of the previ ously described SRS system and overcomes several of its limitations. ( C) Current Biology Ltd ISSN 0960-9822.