The SARAH Domain of RASSF1A and Its Tumor Suppressor Function
Figure 1
Binding studies of RASSF1, MST1, and MST2. (a) Characteristic domains of RASSF1 isoforms and SARAH deletion (ΔS) mutants are the protein kinase C conserved region (C1; blue), the ATM-kinase phosphorylation site (black), Ras-association (RalGDS/AF-6) domain (RA; yellow), and the Sav-RASSF-Hpo interaction site (SARAH; red). (b) Interaction analysis using the yeast two hybrid system. The indicated constructs were cotransformed into yeast strain PJ 69-4A. Interaction was evaluated on SD plates without alanine and histidine (interaction plate). Transformation was controlled on SD plates with alanine and histidine (control plate). (c) Quantitative interaction analysis using the ONPG assay. In three independent colonies, the activation of the β-galactosidase reporter gene was quantified with ONPG as substrate. The standard deviation is indicated. (d) Binding studies in coprecipitation. Constructs that express GST (a), GST-Flag-RASSF1A (b), GST-Flag-RASSF1AΔS (c), Flag-MST1 (d), or Flag-MST2 (e) were transfected into HEK293 cells. After two days, total protein was extracted and GST-tagged proteins were precipitated with glutathione sepharose. Samples were separated on a 10% PAGE gel and blotted. The precipitated and coprecipitated proteins were determined with anti-Flag-antibodies and anti-GST antibodies.
