Effective Mammalian Two Hybrid Screening Approach

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DESCRIPTION (provided by applicant): This application, Effective Mammalian Two Hybrid Screening Approach, is in response to RFA-CA-07-001. Identification of novel protein-protein interactions is a fundamental step to understanding protein function and signaling networks allowing efficient implementation of targeted cancer therapy. The majority of protein-protein interactions are currently identified using yeast two hybrid (Y2H), co-immunoprecipitation and mass spectrometry or protein libraries. Each of these approaches has its own set of major limitations, failing to mimic native physiological conditions (Y2H and protein libraries) or to efficiently identify protein interactions on the cytoskeleton or membrane, either due to the location of the interaction (Y2H) or due to difficulties in co-immunoprecipitation of cytoskeletal or membrane proteins. Furthermore, conventional Y2H approaches yield false positive signals with transcription factors precluding screening. Therefore, a novel screening method that efficiently identifies biologically-relevant protein interactions, bypassing the limitations of present screening methods, would have wide applicability. We propose to develop and validate a readily applicable, context-dependent, subcellular localization-, cDNA library- and cell type-independent retrovirus-based mammalian two hybrid (ReMTH) screen method for identification of novel protein-protein interactions, including cytoskeletal and membrane proteins, in allowing native protein folding and post-translational modifications in mammalian cells. The resultant cells will be reagents for the study of the localization and function of the novel protein-protein interaction complex as well as resources for high content drug or siRNA screening. The fully developed technology will identify functional protein-protein interactions more efficiently than present methods and identify interactions not discoverable by present methods, particularly in context-dependent mammalian screens. Furthermore, the proposed ReMTH screen has the unique potential to stabilize or trap transient/weak interactions such as enzyme/substrate interactions, allowing identification of components of signaling pathways and networks in cancers previously undetectable. We have completed an initial proof-of-concept screen in HeLa cells for identification of interaction partners of the oncogene AKT1. We identified a series of previously known AKT1 interaction partners and substrates, as well as novel interaction partners, including cytoskeleton and membrane proteins. We have confirmed that one novel interaction partner, ACTN4, interacts physically and functionally with AKT1. Thus the technology will uncover functional protein-protein interactions not detectable by other approaches and advance our understanding of protein functions and signaling networks in cancer.