Ras signaling pathway for analysis of protein-protein interactions

Abstract

This chapter discusses the Ras signalling pathway for analysis of protein-protein interactions. When protein-protein interaction occurs between the bait and the prey proteins it is expected to result in homolog for Cdc25 (hSos) translocation and thereby Ras activation. This system, designated the Sos recruitment system (SRS), is efficient in detection of protein-protein interaction among known proteins as well as for screening cDNA expression libraries fused to a membrane localization signal. The Ras proteins are found to efficiently complement the Cdc25-2 mutations by virtue of bypassing the requirement for a functional exchange factor. This inherent problem of the system could be partially overcome by expression of the mammalian GTPase-activating protein simultaneously. An improved protein recruitment system has been developed that overcomes several problems of the SRS. This system, designated the Ras recruitment system (RRS), is based on a similar approach; however, it has an absolute requirement that the Ras protein be localized to the inner leaflet of the plasma membrane for its function. The RRS exhibits several advantages over the SRS, including effector size, lower rate of self-activation, and reduced number of predicted “false positives,” and it provides all the advantages of the SRS over the two-hybrid approach. The RRS, similar to the SRS, can be used to test known and novel protein-protein interactions. The chapter describes in detail the technical aspects of the study of protein-protein interactions, using the SRS and RRS.