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

Abstract

BackgroundInactivating 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.MethodsHigh 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.ResultsWe 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.ConclusionBased 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.

Identification of Putative Immunologic Targets for Colon Cancer Prevention Based on Conserved Gene Upregulation from Preinvasive to Malignant Lesions

“…Upregulation of CDH3 in cancer is associated with increased proliferation (45). KRT23 is responsible for the structural integrity of epithelial cells, and important in modulating and controlling cellular signaling processes and apoptosis (46). KRT23 expression differentiates between microsatellite-stable (MSS) and microsatellite-instable (MSI) colon cancers (47), with 88% of MSI tumors negative for KRT23 and 70% of MSS tumors with KRT23 over-expression.…”

Keratin function and regulation in tissue homeostasis and pathogenesis

“…In a cell culture model, increase in the tumor suppressor SMAD4, acting downstream of TGF-β , coincides with elevated K23 expression. The concomitant identifi cation of 14-3-3 β as a K23-binding protein and its cytoplasmic re-localization upon K23 expression suggests a mechanism for K23 as tumor suppressor (13) .…”

“…Experimentally confi rmed sites in K8, K18 are marked with 1 and 2 in brackets, respectively. Site(s) in keratins 7,9,13,14, and 17 were not identifi ed so far. In addition, amino acid sequence comparisons of types I and II keratins for which O-glycosylation sites are predicted or confi rmed are provided.…”

The Study on Biological Function of Keratin 26, a Novel Member of Liaoning Cashmere Goat Keratin Gene Family

“…In addition, some keratins were also related to the function of epithelial cells, for example, keratin 8 and keratin 18 could guarantee rigidity and structure integrity of extracellular matrix of epithelial cells, affect intracellular transportation mechanism and signaling pathway, and might inhibit the apoptosis of epithelial cells [6–8]; keratin 8 could play a role in infection activity of transferable cells of epithelium [9]. In addition, keratins also had other important function on cells, for example, keratin 18 played a significant role in estrogen receptor α signaling pathway regulation [10]; keratin 23 could regulate functional status of 14-3-3ε scaffolding protein in cytoplasm, and then regulate signal transduction, cell cycle, cell apoptosis and other processes [11]. In the early time, bioinformatics analysis and gene localization analysis of keratin 26 in Liaoning cashmere goat were conducted by Jin Mei, Xing Mengxin and other people, and they found that the relationship between keratin 26 expression and skin [12].…”