Analysis of transient phosphorylation-dependent protein-protein interactions in living mammalian cells using split-TEV

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Abstract

Background: Regulated protein-protein interactions (PPIs) are pivotal molecular switches that are important for the regulation of signaling processes within eukaryotic cells. Cellular signaling is altered in various disease conditions and offers interesting options for pharmacological interventions. Constitutive PPIs are usually mediated by large interaction domains. In contrast, stimulus-regulated PPIs often depend on small post-translational modifications and are thus better suited targets for drug development. However, the detection of modification-dependent PPIs with biochemical methods still remains a labour- and material-intensive task, and many pivotal PPIs that are potentially suited for pharmacological intervention most likely remain to be identified. The availability of methods to easily identify and quantify stimulus-dependent, potentially also transient interaction events, is therefore essential. The assays should be applicable to intact mammalian cells, optimally also to primary cells in culture. Results: In this study, we adapted the split-TEV system to quantify phosphorylation-dependent and transient PPIs that occur at the membrane and in the cytosol of living mammalian cells. Split-TEV is based on a PPI-induced functional complementation of two inactive TEV protease fragments fused to interaction partners of choice. Genetically encoded transcription-coupled and proteolysis-only TEV reporter systems were used to convert the TEV activity into an easily quantifiable readout. We measured the phosphorylation-dependent interaction between the pro-apoptotic protein Bad and the adapter proteins 14-3-3ε and ζ in NIH-3T3 fibroblasts and in primary cultured neurons. Using split-TEV assays, we show that Bad specifically interacts with 14-3-3 isoforms when phosphorylated by protein kinase Akt-1/PKB at Ser136. We also measured the phosphorylation-dependent Bad/14-3-3 interactions mediated by endogenous and transient Akt-1 activity. We furthermore applied split-TEV assays to measure the phosphorylation-dependent interactions of Neuregulin-1-stimulated ErbB4 receptors with several adapter proteins. Conclusion: Split-TEV assays are well suited to measure phosphorylation-dependent and transient PPIs that occur specifically at the membrane and in the cytosol of heterologous and primary cultured mammalian cells. Given the high sensitivity of the split-TEV system, all assays were performed in multi-plate formats and could be adapted for higher throughput to screen for pharmacologically active substances. © 2008 Wehr et al; licensee BioMed Central Ltd.

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Wehr, M. C., Reinecke, L., Botvinnik, A., & Rossner, M. J. (2008). Analysis of transient phosphorylation-dependent protein-protein interactions in living mammalian cells using split-TEV. BMC Biotechnology, 8. https://doi.org/10.1186/1472-6750-8-55

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