Cross-Regulation between MST1 and BRAFV600E Figure 4. BRAFV600E directly binds to the C-terminal domain of MST1 and represses MST1 kinase activity in vitro. (a–d) Co-transfection with BRAFV600E (1 mg/well), BRAFWT (1 mg/well), Flag-MST1 (1 mg/well), Flag-MST1-N (1 mg/well), and Flag-MST1-C (1 mg/well) was performed as indicated. Twenty-four hours after transfection, cells were lysed and the lysates were used for immunoprecipitation analyses with the indicated primary antibodies. The immunoprecipitates were separated by SDS-PAGE and subjected to immunoblotting with anti-Flag or anti-BRAF antibodies. (e) Co-transfection with BRAFV600E (1 mg/well) and Flag-MST1 (indicated amount/well) was performed. Twenty-four hours after transfection, immunoprecipitation of MST1 from cell lysates was performed and the washed precipitates were incubated for 30 minutes at 30uC with 1 mg histone H2B in 25 mL kinase assay buffer. The reaction was terminated by the addition of Laemmli sample buffer and phosphorylated proteins were detected by SDS-PAGE followed by autoradiography. doi:10.1371/journal.pone.0016180.g004 RASSF1A primarily interacts with small GTPases such as Ras to facilitate cell cycle arrest and apoptosis. Transcriptional silencing of RASSF1A by promoter methylation has been reported in many human cancers including thyroid cancer [46,47]. However, epigenetic alteration has been regarded as a slow and time- consuming process for gene silencing, implying that tumor suppressors such as RASSF1A are inhibited to allow the initiation and maintenance of oncogenic processes necessary for the establishment of clinically significant thyroid cancers [48]. The results of the present study suggest that BRAFV600E tumors might PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e16180