title: Mutations in protein-binding hot-spots on the hub protein Smad3 differentially affect its protein interactions and Smad3-regulated gene expression.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin Carbone Cancer Center, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, United States of America.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Schiro
authors.forename: Sara E
authors.initials: SE
authors.lastname: Stauber
authors.forename: Tami L
authors.initials: TL
authors.lastname: Peterson
authors.forename: Chateen
authors.initials: C
authors.lastname: Krueger
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Darnell
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Satyshur
authors.forename: Norman R
authors.initials: NR
authors.lastname: Drinkwater
authors.forename: Michael A
authors.initials: MA
authors.lastname: Newton
authors.forename: F Michael
authors.initials: FM
authors.lastname: Hoffmann
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007124:Immunoenzyme Techniques; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism; D051900:Smad3 Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
chemical_list: D015415:Biomarkers; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C491124:SARA protein, mouse; C491561:Sip1 protein, mouse; C512653:Ski protein, mouse; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D008156:Luciferases; C533325:Smurf2 protein, mouse; D044767:Ubiquitin-Protein Ligases; D019204:GTP-Binding Proteins