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Monitoring regulated protein-protein interactions using split TEV
.com/nature methods
Michael C Wehr1,3, Rico Laage2,3, Ulrike Bolz2, Tobias M Fischer1, Sylvia Grnewald2, Sigrid Scheek2, Alfred Bach2, Klaus-Armin Nave1 & Moritz J Rossner1,2
Signaling cascades integrate extracellular stimuli primarily through regulated protein-protein interactions (PPIs).Intracellular signal transduction strictly depends on PPIs occurring at the membrane and in the cytosol. To monitor constitutive and regulated protein interactions within living mammalian cells, we have developed a biological assay termed split TEV. We engineered inactive fragments of the NIa protease from the tobacco etch virus (TEV protease) that regain activity only when coexpressed as fusion constructs with interacting proteins. Functional reconstitution of TEV protease fragments can be monitored with proteolysis-only reporters, which can be previously silent uorescent and luminescent reporter proteins. Additionally, proteolytically cleavable inactive transcription factors can be combined with any downstream reporter gene of choice to yield transcription-coupled reporter systems. Thus, split TEV combines the advantages of split enzyme and reporter genemediated assays, and provides full exibility with regard to the nal readout. In a rst biological application, we monitored neuregulin-induced ErbB2/ErbB4 receptor tyrosine kinase heterodimerization.
Many cellular processes are controlled by specically interacting proteins forming dynamic networks regulated by external stimuli1,2. A promising approach for the development of new drugs is to interfere with pivotal steps in cellular signaling events mediated by regulated PPIs through the application of small molecules3,4. Development of intracellularly acting drugs lags behind those directed against extracellular targets. Constitutive PPIs usually rely on contacts of large hydrophobic surface regions not well suited for pharmacological intervention5. In
contrast, stimulus-regulated PPIs that depend on small post-translational modications (such as phosphorylation) represent more promising targets for drug development5. Therefore, the availability of methods for detection and quantication of transient stimulus-dependent interaction events in intact mammalian cells is essential.
Biochemical approaches are well established, but they are not optimally suited for high-throughput assays or for use with primary cells because these methods are labor- and material-
intensive6. Screening for transient interactions is limited and requires prior knowledge of the interaction kinetics, or has to be performed in a series of separate experiments. The rst method relying on PPI-dependent protein-fragment complementation was the yeast two-hybrid system7. Large-scale screens based on this technique have been published recently8 but...