title: Visualizing Smad1/4 signaling response to bone morphogenetic protein-4 activation by FRET biosensors.
abstract: Smad proteins are the major signal transducers for the Transforming Growth Factor superfamily of cytokines and their serine/threonine kinase receptors. Smads mediate the signal from the membrane into the nucleus. Bone Morphogenetic Protein-4 stimulates phosphorylation of Smad1, which interacts with Smad4. This complex translocates into the nucleus and regulates transcription of target genes. Here, we report our development of cellular fluorescence biosensors for direct visualization of Smad signaling in live mammalian cells. Fluorescence resonance energy transfer between cyan and yellow fluorescent proteins fused to the Smad1 and Smad4 proteins was used to unravel the temporal aspects of BMP/Smad signaling. A rate-limiting delay of 2-5 min occurred between BMP activation and Smad1 activity. A similar delay was observed in the Smad1/Smad4 complexation. Further experimentation indicated that the delay is dependent on the MH1 domain and linker of Smad1. These results give new insights into the dynamics of the BMP receptor -Smad1/4 signaling process and provide a new tool for studying Smads.
authors.affiliation: Molecular Microscopy Group, Rudolf-Virchow-Center, University of Würzburg, Versbacher Str. 9, D-97078 Würzburg, Germany.
authors.forename: Kira V
authors.initials: KV
authors.lastname: Gromova
authors.forename: Mike
authors.initials: M
authors.lastname: Friedrich
authors.forename: Andrey
authors.initials: A
authors.lastname: Noskov
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Harms
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D019556:COS Cells; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D051898:Smad1 Protein / Q000737:chemistry / Q000378:metabolism*; D051901:Smad4 Protein / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: C525809:BMP4 protein, human; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D051898:Smad1 Protein; D051901:Smad4 Protein