Year-end Sale % OFF 
Abstract
Dimerization in signal transduction is a dynamically regulated process and a key regulatory mechanism. Signal transducer and activator of transcription 3 (STAT3) dimerizes after tyrosine phosphorylation upon cytokine stimulation. Because only the STAT3 dimer possesses the trans-activation activity, dimerization is an indispensable process for cytokine signaling. Here we report the detection of dynamic STAT3 dimerization in living cells using the homoFluoppi system. This method allowed us to validate the presence of an intact Src homology 2 domain and STAT3 Tyr705 phosphorylation, which facilitate puncta formation and homodimerization. Puncta formation was reversible, as determined by a decreased punctate signal after washout of oncostatin M. We analyzed STAT3 mutants, which have been reported in patients with hyper IgE syndrome and inflammatory hepatocellular adenoma (IHCA). Analysis of the IHCA mutants using homoFluoppi revealed constitutive activity independent of cytokine stimulation and novel insight into kinetics of dimer dissociation process. Next, we used homoFluoppi to screen for inhibitors of STAT3 dimerization, and identified 3,4-methylenedioxy-β-nitrostyrene as a novel inhibitor. The results of this study show that homoFluoppi is a useful research tool for the analysis of proteins like STAT3 that dynamically dimerize, and is applicable for the screening of dimerization modulators.
Highlights
The dimerization of many proteins such as transcription factors or G protein-coupled receptors is an important regulatory mechanism governing cell function
In the homoFluoppi system, when proteins fused to PB1/mAG1 tags interact and form homodimers, the homodimers are observed as fluorescent puncta[23]
We were able to differentiate between endogenous Signal transducer and activator of transcription 3 (STAT3) (~88 kDa) and exogenous Fluorescent protein-protein interaction visualization (Fluoppi) (PB1 and mAG1) tagged STAT3 (~130 kDa) by western blotting because of their different molecular weights
Summary
The dimerization of many proteins such as transcription factors or G protein-coupled receptors is an important regulatory mechanism governing cell function. Despite the importance of modulating dimer formation as a potential therapeutic strategy, research methods for analyzing the dynamic dimerization process in normal and disease states, and screening methods for identifying dimerization modulators, especially in living cells, are limited. FRET and BRET assays are difficult and time-consuming for optimizing constructs such as the length of linker sequences Another method that could be used to detect STAT3 dimerization is the split-green fluorescent protein-based biomolecular fluorescence complementation (BiFC) assay, which is based on formation of a fluorescent complex when two proteins fused to non-fluorescent fragments of a fluorescent protein interact with each other. The complex that forms is irreversible, so once the dimer forms it is difficult to dissociate[21,22] To overcome these shortcomings, a novel method that enables the detection of homodimerization in living cells quantitatively, and reversibly is strongly desired
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
