Medizinisches Proteom-Center, Ruhr-University Bochum-Zentrum fuer Klinische Forschung, Universitaetsstr. 150, 44780 Bochum, Germany.
BMC Cancer. 2011 Apr 14;11:137. doi: 10.1186/1471-2407-11-137.
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.
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.
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.
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.
SMAD4 的失活突变在转移性结直肠癌中很常见。在之前的分析中，我们能够表明，在 SMAD4 缺陷的 SW480 人结肠癌细胞中恢复 Smad4 表达足以抑制肿瘤发生和侵袭潜能，而体外细胞生长不受影响。使用这个细胞模型系统，我们比较了 Smad4 再表达和 Smad4 阴性 SW480 细胞的核亚细胞蛋白组，寻找新的 Smad4 靶标。
高分辨率二维（2D）凝胶电泳用于鉴定 Smad4 再表达 SW480 细胞核亚细胞蛋白组中的新 Smad4 靶标。进一步通过串联亲和纯化鉴定鉴定候选蛋白角蛋白 23。免疫沉淀、亚细胞分级和免疫定位研究与 RNAi 结合用于验证角蛋白 23-14-3-3ε 相互作用。
我们鉴定了角蛋白 8 和 18、热休克蛋白 60 和 70、plectin 1 以及 14-3-3ε 和 γ 作为新型蛋白质，存在于 KRT23 相互作用复合物中。共免疫沉淀和亚细胞分级分析以及我们的 Smad4-SW480 模型细胞中的免疫定位研究提供了进一步的证据，表明 KRT23 与 14-3-3ε 结合，并且 Smad4 依赖性 KRT23 上调诱导 14-3-3ε 蛋白从核定位到细胞质定位的转移。
基于我们的发现，我们提出了一个新的调节回路，涉及 Smad4 依赖性 KRT23（直接或间接）上调，从而调节 KRT23 与 14-3-3ε 之间的相互作用，导致 14-3-3ε 的细胞质隔离。这种细胞质 KRT23-14-3-3 相互作用可能改变了众所周知的 14-3-3 支架蛋白的功能状态，该蛋白已知可调节关键的细胞过程，如信号转导、细胞周期控制和细胞凋亡，因此可能是 Smad4 肿瘤抑制回路的一个以前未被重视的方面。
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