Akt/PKB signaling pathways, a MEK inhibitor (UO126) and PI3K inhibitors (Wortmannin, LY294002) were used to block these pathways and observe the effect of BRAFV600E on RASSF1A-induced FoxO3 transactivation. As shown in Fig. 1C, the BRAFV600E-mediated suppression of p21 and p27 induction by RASSF1A was not reversed by the MEK inhibitor U0126 or the PI3K inhibitors Wortmannin and LY294002, showing that BRAFV600E inhibits the RASSF1A-induced p21 and p27 upregu- lation independently from the ERK and PI3K-Akt/PKB signaling pathways. Recently, RASSF1A was shown to interact with MST1 via its SARAH domain and increase MST1 kinase activity [20], suggesting that RASSF1A-MST1-FoxO3 might be operational in response to oncogenic signals. To verify this hypothesis, MST1 expression was silenced using siRNA, and the RASSF1A induction of p21 and p27 was assessed. Fig. 1D shows that RASSF1A did not induce p21 and p27 expression in siMST1-treated cells, indicating that MST1 plays an important role in the p21 and p27 induction by RASSF1A. BRAFV600E represses FoxO3 transactivation by the RASSF1A-MST1 signaling pathway The transcriptional activity of FoxO3 is regulated by posttrans- lational modifications such as phosphorylation, acetylation, nitrosylation and sumoylation [33,34,35].Among these modifica- tions, the MST1-mediated serine 207 phosphorylation of FoxO3 is critical for its nuclear translocation [19,36]. To investigate whether the RASSF1A-MST1 signal increased FoxO3 transactivation, a 3XIRS reporter system was used, consisting of a 3XIRS promoter construct containing three insulin response sequences/FoxO response elements (CAAAA[C/T]AA, the 3XIRS promoter) [26,29]. Transient expression of MST1 significantly increased 3XIRS reporter activities in a dose-dependent manner (Fig. S1). Evaluation of the effect of RASSF1A on FoxO3 reporter activity showed that RASSF1A remarkably increased 3XIRS reporter activity (Fig. 2A). Moreover, this RASSF1A-induced FoxO3 reporter activation was completely abolished by MST1 silencing, indicating that RASSF1A effectively increased FoxO3 transactiva- tion through a MST1-dependent pathway (Fig. 2B). Because BRAFV600E inhibited RASSF1A-induced p21 and p27 upregulation, the effect of BRAFV600E on FoxO3 transactivation by RAASF1A was assessed. As shown in Fig. 2C, BRAFV600E abrogated the RASSF1A-induced FoxO3 transactivation. Fur- thermore, MST1-induced FoxO3 transactivation was also re- pressed by BRAFV600E, indicating that BRAFV600E inhibits the RASSF1A-MST1-FoxO3 pathway (Fig. 2D). Because the transcriptional activity of FoxO3 depends on its nuclear translocation, a FoxO3-GFP vector was constructed to confirm the data obtained with the 3XIRS reporter by immunofluorescence imaging. As shown in Fig. 2E, GFP- fluorescence was predominantly observed in the cytoplasm in control samples; introduction of MST1 caused a shift in GFP- fluorescence detection to the nucleus. When BRAFV600E was co- transfected with MST1, nuclear GFP fluorescence was barely detected. Interestingly, BRAFV600E was exclusively localized in the cytoplasm (blue), MST1 showed dominant cytoplasmic localiza- tion (red), and merged images revealed a combined color (lilac) that suggests the co-localization of BRAFV600E and MST1. To quantify FoxO3-fluorescence, the nuclear GFP-fluorescence signal intensity was analyzed using the BioApplication of CellomicsAr- rayScan, following the manufacturer’s protocol. In agreement with the immunofluorescence images, MST1 increased intensity of the nuclear GFP-fluorescence signal, and BRAFV600E inhibited the nuclear detection of GFP-fluorescence (Fig. 2F). Furthermore, we performed fractionation assay to define the subcellular localization Cross-Regulation between MST1 and BRAFV600E of FoxO3-GFP and observed that MST1 increased nuclear FoxO3-GFP and BRAFV600E could reverse this MST1-induced FoxO3 nuclear localization (Fig. S2).Taken together, these results indicate that BRAFV600E inhibits nuclear translocation of FoxO3 and suggest that this inhibition might be mediated by direct interaction between BRAFV600E and MST1. BRAFV600E represses FoxO3 transactivation via MST1 kinase-dependent pathway RAF-1 is an homologue of BRAF that interacts with MST2, suppressing MST2-mediated apoptosis [37,38], which may be important for the regulation of certain biological processes [39]. In our study, however, FoxO3 inhibition by BRAFV600E was not altered by RAF-1 silencing (Fig. S3). Furthermore, this inhibitory effect of BRAFV600E was not reversed by the inhibition of MEK or PI3-kinase (Fig. S4), confirming that BRAFV600E is able to suppress FoxO3 transactivation through a mechanism indepen- dent from MEK/ERK and PI3-kinase signaling. The inhibition of RASSF1A or MST1-induced FoxO3 transactivation and nuclear translocation by BRAFV600Eled to the hypothesis that BRAFV600E might regulate FoxO3 transactiva- tion via a MST1-dependent pathway. To verify this hypothesis, reporter experiments were carried out in siMST1-treated cells. Remarkably, following knockdown of MST1, FoxO3 could not induce full activation of the 3XIRS reporter and BRAFV600E no longer had an effect on FoxO3 induced activation of 3XIRS reporter (Fig. 3A). These observations suggest that MST1 plays a role in FoxO3 transactivation and that BRAFV600E inhibits FoxO3 transactivation by suppressing MST1 activity. The reporter assay was also used to test whether there is signal-crosstalk between MST1 and BRAFV600E on FoxO3 transactivation. As expected, BRAFV600E inhibited MST1-induced 3XIRS reporter activation in a dose-dependent manner and MST1 abolished the inhibitory effect of BRAFV600E on FoxO3 transactivation (Fig. 3B & 3C). These reporter assays indicated that increased levels of MST1 could prevent BRAFV600E-induced FoxO3 inactivation. Taken together, these data suggest that the cross-regulation between BRAFV600E and MST1 determines the degree of FoxO3 transactivation. BRAFV600E shows mutation-specific binding with the C- terminal of MST1 and inhibits MST1 kinase activity in vitro Because immunofluorescence images and reporter assays suggested that BRAFV600E and MST1 might be colocalized and have competitive effects on FoxO3 transactivation, immunopre- cipitation (IP) assays were carried out to define the direct interaction of BRAFV600E and MST1. As shown in Fig. 4A, BRAFV600E was co-precipitated with Flag-tagged MST1 in lysates from 293T cells transfected with BRAFV600E and Flag-tagged MST1 and vice versa. To verify the region of MST1 important for the interaction with BRAFV600E, IP assays using MST1 deletion constructs, such as MST1 N-terminal (residues 1–326) and C- terminal (327–487) constructs (Fig. 4B), were carried out. These IP assays consistently demonstrated that BRAFV600E binds to the C- terminal region of MST1 (Fig. 4C), which contains of regulatory binding domain with the Sav/RASSF1/Hpo (SARAH) domain of RASSF1A [40,41,42]. To verify theBRAFV600Emutation specific binding with MST1, we performed additional IP assays using BRAFWT and BRAFV600E constructs. Furthermore, as shown in Fig. 4D, BRAFV600Ewas consistently co-precipitated with Flag- tagged MST1 whereas wild-type BRAF did not. Taken together, these results show that BRAFV600E can bind to the C-terminal domain of MST1 via mutation-specific protein-protein interaction. PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16180