Abstract
The 14-3-3 proteins form a highly conserved family of dimeric proteins that interact with various signal transduction proteins and regulate cell cycle, apoptosis, stress response, and malignant transformation. We previously demonstrated that the beta isoform of 14-3-3 proteins promotes tumorigenicity and angiogenesis of rat hepatoma K2 cells. In this study, to analyze the mechanism of 14-3-3beta-induced malignant transformation, yeast two-hybrid screening was performed, and a novel 14-3-3beta-binding factor, FBI1 (fourteen-three-three beta interactant 1), was identified. In vitro binding and co-immunoprecipitation analyses verified specific interaction of 14-3-3beta with FBI1. The strong expression of FBI1 was observed in several tumor cell lines but not in non-tumor cell lines. Forced expression of antisense FBI1 in K2 cells inhibited anchorage-independent growth but had no significant effect on cell proliferation in monolayer culture. Down-regulation of FBI1 also inhibited tumorigenicity and metastasis accompanying a decrease in MMP-9 (matrix metalloproteinase-9) expression. In addition, the duration of ERK1/2 activation was curtailed in antisense FBI1-expressing K2 cells. A luciferase reporter assay revealed that the FBI1-14-3-3beta complex could act as a transcriptional silencer, and MKP-1 (MAPK phosphatase-1) was one of the target genes of the FBI1-14-3-3beta complex. Moreover, chromatin immunoprecipitation analysis demonstrated that FBI1 and 14-3-3beta were presented on the MKP-1 promoter. These results indicate that FBI1 promotes sustained ERK1/2 activation through repression of MKP-1 transcription, resulting in promotion of tumorigenicity and metastasis.
Highlights
In contrast to 14-3-3, which is known as a tumor suppressor gene [5,6,7], we previously reported that 14-3-3 is implicated in the positive regulation of cell cycle progression and tumorigenesis [8]. 14-3-3 was overexpressed in various cancer cell lines, including aflatoxin B1-induced rat hepatocellular carcinoma K1 and K2 cells [9, 10]
Interaction of FBI1 with 14-3-3 in Vivo—In order to identify novel 14-3-3-interacting proteins, K2 cell cDNA and rat brain cDNA libraries were subjected to yeast two-hybrid screening using 14-3-3 as a bait, and three novel cDNA clones (1-1, 2-3, and 2-6) were obtained
Overexpression of FBI1 mRNA in Various Tumor Cell Lines— To examine the tissue specificity of FBI1 mRNA expression, total RNAs were extracted from various adult rat tissues and analyzed by Northern blotting with the 1.3-kb fragment of pACT2/FBI1 as a probe
Summary
In antisense FBI1 K2 transfectants, the expression level of MMP-9 (matrix metalloproteinase-9) mRNA was reduced, whereas the expression of MKP-1 (MAPK phosphatase) was substantially up-regulated by serum stimulation. Antisense FBI1 cDNA expression vector was introduced into K2 cells, and two stable clones, K2A1 and K2A2, were selected by Northern blotting (Fig. 3C). To confirm expression level of FBI1 protein in transfectants, cell lysates were analyzed by Western blotting with anti-FBI1 antibody.
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