Cross-Regulation between MST1 and BRAFV600E Figure 2. BRAFV600E represses FoxO3 transactivation induced by RASSF1A-MST1 signal. 293T cells were transfected as indicated and luciferase-based reporter assays were conducted to observe 3XIRS reporter activity. For each sample, firefly luciferase activity was normalized to Renilla luciferase activity and expressed as the relative-fold change compared to basal luciferase activity. (a) Co-transfection with 3X-IRS Luc (100 ng/ well), FoxO3 (0.5 mg/well), and RASSF1A (0.5 mg/well) was performed as indicated, and incubation lasted for 24 h. Total lysates were immunoblotted with anti-Myc, anti-HA, or anti-Actin antibodies. (b) Co-transfection with 3XIRS Luc (100 ng/well), FoxO3 (0.5 mg/well), RASSF1A (0.5 mg/well), and SiMST1 (20 mM/well StealthTM RNA) was performed as indicated, and incubation lasted for 24 h. Total lysates were immunoblotted with anti-Myc, anti-HA, anti-MST, or anti-Actin antibodies. (c) Co-transfection with 3X-IRS Luc (100 ng/well), FoxO3 (0.5 mg/well), RASSF1A (0.5 mg/well), and BRAFV600E (0.5 mg/well) was performed as indicated, and incubation lasted for 24 hours. Total lysates were immunoblotted with anti-Myc, anti-HA, anti-BRAF, or anti-Actin antibodies. (d) Co-transfection with 3X-IRS Luc (100 ng/well), MST1(0.5 mg/well), and BRAFV600E (0.5 mg/well) was performed as indicated, and incubation lasted for 24 hours. All data are presented as mean6SD. Total lysates were immunoblotted with anti-Flag, anti-BRAF, or anti-Actin antibodies. (e) BRAFV600E inhibits MST1-induced nuclear translocation of FoxO1. 293T cells were cultured in a six-well dish until they reached 80% confluence and co-transfected with FoxO3-GFP (0.5 mg/well), Flag-MST1 (0.5 mg/well), and Myc-BRAFV600E (0.5 mg/well) as indicated. Twenty-four hours after transfection, the cells were fixed and incubated with antibodies against Myc or Flag, washed, and incubated with Cy5- conjugated anti-Rabbit for Myc-BRAFV600E and TRITC-conjugated anti-Mouse for Flag-MST1. (f) Fluorescence was observed using a Cellomics ArrayScanV HCS reader. To calculate FoxO3 distribution, at least one thousand cells in each experiment were counted and analyzed by Cellomics BioApplication. All data are presented as mean6SD: (*) P,0.01 between two groups, Abbreviations: CTL; control. doi:10.1371/journal.pone.0016180.g002 Because RASSF1A also binds to the C-terminal domain of MST1 and increases MST1 kinase activity, the interaction between BRAFV600E and MST1 might affect MST1 kinase activity by interrupting the heterotypic interaction of MST1 with RASSF1A. To verify this, the effect of BRAFV600E on the kinase activity of MST1 was examined in vitro using 293T cells, which were transfected with Flag-MST1 and BRAFV600E as indicated. The immunoprecipitates obtained with an antibody to Flag were used to assay for MST1 kinase activity using histone H2B as a substrate. As seen in Fig. 4E, the expression of BRAFV600E completely abolished MST1 kinase activity. These data suggest that BRAFV600E can negatively regulate MST1 kinase through direct interaction with its C-terminus. BRAFV600E generates an anti-apoptotic effect via repression of p21 and p27 induction FoxO3 acts as a cell-cycle regulator by inducing the transcription of genes such as p21 and p27 [43], the promoters of which contain IRS elements. The results of the assessment of the regulatory effect of BRAFV600E on the p27 reporter are shown in Fig. 5A. FoxO3 increased p27 reporter activity and MST1 potentiated this FoxO3-induced p27 activation. In addition, MST1-FoxO3 mediated p27 reporter activation was abrogated by co-transfection with BRAFV600E. To confirm that BRAFV600E can affect p27 reporter activity, the inhibitory effect of BRAFV600E on p21 and p27 induction was investigated by Western blot analysis (Fig. 5B). The results confirmed the MST1-FoxO3 PLoS ONE | www.plosone.org 5 January 2011 | Volume 6 | Issue 1 | e16180