Mutations in protein-binding hot-spots on the hub protein Smad3 differentially affect its protein interactions and Smad3-regulated gene expression
| Autor: | F. Michael Hoffmann, Chateen Krueger, Sara E. Stauber, Michael A. Newton, Steven J. Darnell, Kenneth A. Satyshur, Norman R. Drinkwater, Tami L. Peterson, Michelle M. Schiro |
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| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Models
Molecular Protein Conformation Gene Expression lcsh:Medicine Smad2 Protein Plasma protein binding Kidney Biochemistry Immunoenzyme Techniques Myoblasts Mice 0302 clinical medicine Protein structure Transforming Growth Factor beta Molecular Cell Biology Gene expression Signaling in Cellular Processes Luciferases lcsh:Science Cells Cultured Oligonucleotide Array Sequence Analysis Smad4 Protein Regulation of gene expression 0303 health sciences Multidisciplinary integumentary system Reverse Transcriptase Polymerase Chain Reaction Signaling Cascades DNA-Binding Proteins 030220 oncology & carcinogenesis biological phenomena cell phenomena and immunity Signal Transduction Research Article Ubiquitin-Protein Ligases Blotting Western Nerve Tissue Proteins Biology Real-Time Polymerase Chain Reaction Molecular Genetics 03 medical and health sciences GTP-binding protein regulators GTP-Binding Proteins Genetic Mutation Proto-Oncogene Proteins Genetics Animals Humans Protein Interaction Domains and Motifs Gene Regulation RNA Messenger Smad3 Protein Protein Interactions Cell Proliferation 030304 developmental biology Reporter gene Smad Signaling Gene Expression Profiling lcsh:R Proteins Computational Biology Transforming growth factor beta Molecular biology Gene Expression Regulation Mutagenesis Mutation Trans-Activators biology.protein lcsh:Q Carrier Proteins Biomarkers |
| Zdroj: | PLoS ONE, Vol 6, Iss 9, p e25021 (2011) PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | Background Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. Methodology/Principal Findings We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Conclusions/Significance Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for specific biological responses. |
| Databáze: | OpenAIRE |
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