Analysis of Transient Phosphorylation-Dependent Protein-Protein Interactions in Living Mammalian Cells Using Split-TEV
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 rely mainly on small posttranslational 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 potentially suited for pharmacological perturbation most likely remain to be identified. Therefore, the availability of methods to easily identify and quantify stimulus-dependent, potentially also transient, interaction events is essential.
These assays should be applicable to intact mammalian cells, optimally also to primary cultured ones.
Results: In this study, we adapted the split-TEV system to quantify phosphorylation-dependent and transient PPIs that occur in the cytosol of living mammalian cells. Split-TEV is based on a PPI-induced functional complementation of two previously inactive TEV protease fragments fused to interaction partners of choice.
Genetically encoded transcription-coupled and proteolysis-only TEV reporter systems were used to transfer the reconstituted TEV activity into an easily quantifiable readout. We measured the phosphorylation-dependent interaction between the pro-apoptotic protein Bad and the adapter proteins 14-3-3 and 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 could simultaneously determine the anti-apoptotic effect of the Bad/14-3-3 interactions in the same assay.
Finally, we measured the phosphorylation-dependent Bad/14-3-3 interactions mediated by endogenous and transient Akt-1 activity.
Conclusion: Split-TEV assays are well suited to measure phosphorylation-dependent and transient PPIs that occur specifically 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.
Author: Michael C Wehr, Lisa Reinecke, Anna Botvinnik and Moritz J Rossner
Credits/Source: BMC Biotechnology 2008, 8:55
Published on: 2008-07-13
Copyright by the authors listed above - made available via BioMedCentral (Open Access). Please
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