title: Analysis of the STAT3 interactome using in-situ biotinylation and SILAC.
abstract: Signal transducer and activator of transcription 3 (STAT3) is activated by a variety of cytokines and growth factors. To generate a comprehensive data set of proteins interacting specifically with STAT3, we applied stable isotope labeling with amino acids in cell culture (SILAC). For high-affinity pull-down using streptavidin, we fused STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag), which did not affect STAT3 functions. By this approach, 3642 coprecipitated proteins were detected in human embryonic kidney-293 cells. Filtering using statistical and functional criteria finally extracted 136 proteins as putative interaction partners of STAT3. Both, a physical interaction network analysis and the enrichment of known and predicted interaction partners suggested that our filtering criteria successfully enriched true STAT3 interactors. Our approach identified numerous novel interactors, including ones previously predicted to associate with STAT3. By reciprocal coprecipitation, we were able to verify the physical association between STAT3 and selected interactors, including the novel interaction with TOX4, a member of the TOX high mobility group box family. Applying the same method, we next investigated the activation-dependency of the STAT3 interactome. Again, we identified both known and novel interactions. Thus, our approach allows to study protein-protein interaction effectively and comprehensively.
  
  
  CONCLUSIONS
  The location, activity, function, degradation, and synthesis of proteins are significantly regulated by interactions of proteins with other proteins, biopolymers and small molecules. Thus, the comprehensive characterization of interactions of proteins in a given proteome is the next milestone on the path to understanding the biochemistry of the cell. In order to generate a comprehensive interactome dataset of proteins specifically interacting with a selected bait protein, we fused our bait protein STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag). This bio-tag allows an affinity pull-down using streptavidin but affected neither the activation of STAT3 by tyrosine phosphorylation nor its transactivating potential. We combined SILAC for accurate relative protein quantification, subcellular fractionation to increase the coverage of interacting proteins, high-affinity pull-down and a stringent filtering method to successfully analyze the interactome of STAT3. With our approach we confirmed several already known and identified numerous novel STAT3 interactors. The approach applied provides a rapid and effective method, which is broadly applicable for studying protein-protein interactions and their dependency on post-translational modifications.
authors.affiliation: Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany; Fraunhofer Institute for Cell Therapy and Immunology, Perlickstrasse 1, 04103 Leipzig, Germany.
authors.forename: Conny
authors.initials: C
authors.lastname: Blumert
authors.forename: Stefan
authors.initials: S
authors.lastname: Kalkhof
authors.forename: Katja
authors.initials: K
authors.lastname: Brocke-Heidrich
authors.forename: Tibor
authors.initials: T
authors.lastname: Kohajda
authors.forename: Martin
authors.initials: M
authors.lastname: von Bergen
authors.forename: Friedemann
authors.initials: F
authors.lastname: Horn
mesh_terms: D019921:Biotinylation; D057809:HEK293 Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C482285:TOX4 protein, human
keywords: CDK; Epo; IL-6; In-situ biotinylation; Interactome; LC–MS; SILAC; STAT3; cyclin-dependent kinase; erythropoietin; interleukin-6; liquid chromatography–mass spectrometry; signal transducer and activator of transcription 3; stable isotope labeling with amino acids in cell culture