Email updates

Keep up to date with the latest news and content from BMC Cancer and BioMed Central.

Open Access Research article

Keratin 23, a novel DPC4/Smad4 target gene which binds 14-3-3ε

Sven-T Liffers13, Abdelouahid Maghnouj2, Johanna B Munding3, René Jackstadt2, Ulrike Herbrand2, Thomas Schulenborg1, Katrin Marcus1, Susanne Klein-Scory4, Wolff Schmiegel45, Irmgard Schwarte-Waldhoff4, Helmut E Meyer1, Kai Stühler1 and Stephan A Hahn2*

Author Affiliations

1 Medizinisches Proteom-Center, Ruhr-University Bochum - Zentrum fuer Klinische Forschung, Universitaetsstr. 150, 44780 Bochum, Germany

2 Department of Internal Medicine, Molecular GI-Oncology, MGO, Ruhr-University Bochum - Zentrum fuer Klinische Forschung, Universitaetsstr. 150, 44780 Bochum, Germany

3 Department of Pathology, Kliniken Bergmannsheil, University of Bochum, Buerkle-de-la-Camp-Platz 1, 44789 Bochum, Germany

4 Department of Internal Medicine, Knappschaftskrankenhaus, IMBL, Ruhr-University Bochum, In der Schornau 23 - 25, 44892 Bochum, Germany

5 Department of Gastroenterology and Hepatology, Kliniken Bergmannsheil, University of Bochum, Buerkle-de-la-Camp-Platz 1, 44789 Bochum, Germany

For all author emails, please log on.

BMC Cancer 2011, 11:137  doi:10.1186/1471-2407-11-137

Published: 14 April 2011

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.

Conclusion

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.