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.

JDP2 suppresses adipocyte differentiation by regulating histone acetylation

“…We and others reported that JDP2 is an AP-1 transcriptional repressor 15,[17][18][19][20] with histone-chaperone activity that inhibits histone acetylation by recruiting HDAC3. 24 JDP2 also inhibited the RA-dependent differentiation of embryonic carcinoma F9 cells.…”

“…12,13 Jun dimerization protein 2 (JDP2) is a DNA-binding protein that forms homodimers or heterodimers with c-Jun, ATF2 and C/EBPg. [14][15][16] JDP2 can function not only as a transcriptional repressor but also as a coactivator in various types of cell, 15,[17][18][19][20] and it is involved in a variety of biological phenomena such as proliferation and differentiation of cells and apoptosis. 14,15,17,18,[20][21][22] For example, forced expression of JDP2 represses the retinoic acid-mediated (RA-mediated) transcription of the c-jun gene and the differentiation of F9 cells in response to RA.…”

A novel in vivo assay for the analysis of protein-protein interaction

“…Recently, two novel protein-protein interaction assays were developed which complement and overcome some of the problems and limitations of the two hybrid system (11,12). These assays are based on a completely different readout to monitor a successful protein-protein interaction in yeast.…”

“…These assays are based on a completely different readout to monitor a successful protein-protein interaction in yeast. It is based on translocation of the active molecules, hSos (11) or Ras (12) to their site of action at the inner leaflet of the plasma membrane. Translocation is achieved through protein-protein interaction.…”

“…Unlike the two hybrid system, this assay is monitored by testing cell viability of an otherwise temperature-sensitive CDC25-2 yeast strain (13) at the restrictive temperature. The protein recruitment systems were proven to be useful for the identification and isolation of known and novel protein interactions (11,12,14,15). Since the detection of cell growth is mainly qualitative and cannot be quantitated easily, this assay is limited in its ability to compare interactions between different protein pairs.…”