title: Keratin 23, a novel DPC4/Smad4 target gene which binds 14-3-3ε.
abstract: BACKGROUND
  Inactivating mutations of SMAD4 are frequent in metastatic colorectal carcinomas. In previous analyses, we were able to show that restoration of Smad4 expression in Smad4-deficient SW480 human colon carcinoma cells was adequate to suppress tumorigenicity and invasive potential, whereas in vitro cell growth was not affected. Using this cellular model system, we searched for new Smad4 targets comparing nuclear subproteomes derived from Smad4 re-expressing and Smad4 negative SW480 cells.
  
  
  METHODS
  High resolution two-dimensional (2D) gel electrophoresis was applied to identify novel Smad4 targets in the nuclear subproteome of Smad4 re-expressing SW480 cells. The identified candidate protein Keratin 23 was further characterized by tandem affinity purification. Immunoprecipitation, subfractionation and immunolocalization studies in combination with RNAi were used to validate the Keratin 23-14-3-3ε interaction.
  
  
  RESULTS
  We identified keratins 8 and 18, heat shock proteins 60 and 70, plectin 1, as well as 14-3-3ε and γ as novel proteins present in the KRT23-interacting complex. Co-immunoprecipitation and subfractionation analyses as well as immunolocalization studies in our Smad4-SW480 model cells provided further evidence that KRT23 associates with 14-3-3ε and that Smad4 dependent KRT23 up-regulation induces a shift of the 14-3-3ε protein from a nuclear to a cytoplasmic localization.
  
  
  CONCLUSIONS
  Based on our findings we propose a new regulatory circuitry involving Smad4 dependent up-regulation of KRT23 (directly or indirectly) which in turn modulates the interaction between KRT23 and 14-3-3ε leading to a cytoplasmic sequestration of 14-3-3ε. This cytoplasmic KRT23-14-3-3 interaction may alter the functional status of the well described 14-3-3 scaffold protein, known to regulate key cellular processes, such as signal transduction, cell cycle control, and apoptosis and may thus be a previously unappreciated facet of the Smad4 tumor suppressive circuitry.
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum-Zentrum fuer Klinische Forschung, Universitaetsstr. 150, 44780 Bochum, Germany.
authors.forename: Sven-T
authors.initials: ST
authors.lastname: Liffers
authors.forename: Abdelouahid
authors.initials: A
authors.lastname: Maghnouj
authors.forename: Johanna B
authors.initials: JB
authors.lastname: Munding
authors.forename: René
authors.initials: R
authors.lastname: Jackstadt
authors.forename: Ulrike
authors.initials: U
authors.lastname: Herbrand
authors.forename: Thomas
authors.initials: T
authors.lastname: Schulenborg
authors.forename: Katrin
authors.initials: K
authors.lastname: Marcus
authors.forename: Susanne
authors.initials: S
authors.lastname: Klein-Scory
authors.forename: Wolff
authors.initials: W
authors.lastname: Schmiegel
authors.forename: Irmgard
authors.initials: I
authors.lastname: Schwarte-Waldhoff
authors.forename: Helmut E
authors.initials: HE
authors.lastname: Meyer
authors.forename: Kai
authors.initials: K
authors.lastname: Stühler
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Hahn
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D057809:HEK293 Cells; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D053538:Keratin-18 / Q000378:metabolism; D053533:Keratin-8 / Q000378:metabolism; D053530:Keratins, Type I / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D051190:Plectin / Q000378:metabolism; D011485:Protein Binding; D034622:RNA Interference; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D048948:14-3-3 Proteins; D018834:Chaperonin 60; D018840:HSP70 Heat-Shock Proteins; C420247:KRT23 protein, human; D053538:Keratin-18; D053533:Keratin-8; D053530:Keratins, Type I; C495332:PLEC protein, human; D051190:Plectin; C497796:SMAD4 protein, human; D051901:Smad4 Protein