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.

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.

Figure 1

Differential proteome analysis of Smad4 re-expressing and negative SW480 cells. A) A representative example of the 2D-DIGE analyses is shown. Differentially expressed proteins are labeled by an arrow. The protein spot signals derived from the nuclear lysates of Smad4 negative cells are shown in green and those from Smad4 re-expressing cells in red. The protein names corresponding to each numbered spot are given in table 1. Spot numbers 10 and 11 mark the KRT23 spots. B) and C) Expression levels of the two KRT23 spots relative to the protein standard according to the dye swop experiment (neg., Smad4 negative SW480 cells; pos., Smad4 re-expressing SW480 cells) D) Expression of KRT23 analyzed by Northern blotting.

Figure 2

Tandem affinity purification of Keratin 23. Confocal immunofluorescence images of SW480 cells overexpressing KRT23. A) Smad4 re-expressing SW480 cells B) Smad4 negative SW480 cells. KRT23 was visualized by α-Flag M2 antibody. C) FLAG eluates from the tandem affinity purification experiments with TAP-KRT23 were separated on a standard 1D-PAGE and proteins visualized by silver staining. The protein names corresponding to each numbered band given in table 2.

Figure 3

Interaction of Keratin 23 with 14-3-3ε. A) Endogenous 14-3-3ε and 14-3-3γ expression levels of Smad4 re-expressing and Smad4 negative SW480 cells. 20 μg whole cell lysates derived from Smad4 re-expressing and Smad4 negative cells were subjected to SDS-PAGE. Flag-tagged KRT23 and VSV-G-tagged 14-3-3ε were transfected into HEK 293T cells as indicated. B) Confirmation of KRT23-14-3-3ε interaction: cell lysates were immunoprecipitated with anti-Flag antibody and blotted as indicated. C) KRT23 expression leads to cytoplasmic sequestration of 14-3-3ε: following fractionation into cytoplasmic and nuclear fractions, proteins were subjected to Western blot analysis with the indicated antibodies. Anti-lamin B and anti-β-tubulin were used as marker proteins for the purity of the fractions. C, cytoplasmic fraction, N, nuclear fraction. A representative blot from three independent experiments is shown.

Figure 4

Cellular localization of 14-3-3ε according to the Smad4 expression and KRT23 knock-down status of SW480 cells. A) Confocal immunofluorescence images of SW480 cells. The cellular localization of 14-3-3ε was analyzed in Smad4 negative and Smad4 re-expressing SW480 cells. KRT23 knock-down was achieved by stable overexpression of shKRT1010 in Smad4 re-expressing cells. The cells were double stained with antibodies to 14-3-3ε (red) and the cytosolic marker tubulin (green). B) Validation of Keratin 23 knock-down efficiency. The Keratin 23 expression was monitored by quantitative RT-PCR in stably sh-KRT23 overexpressing SW480 cells as indicated. Each condition was measured in triplicates and normalized to B2M.