Figure S2. RASSF1A interacts with RhoA. A, No interaction between RASSF1A and active Ras proteins. HeLa cells were transfected with HA-tagged H-Ras-G14V, K-Ras-G14V, or N-Ras-Q61R. Immunoprecipitation and immunoblots were performed as indicated. B, Characterization of RASSF1A-binding region of RhoA. C, No RhoA inhibition activity of RASSF1A-R269F. Cells transfected with WT or mutant (R269F) RASSF1A were treated with EGF (10 ng/ml) for 6 h. D, Construction of exon 2γ-deleted RASSF1C mutant (Δ2γ). E, IP assay showing RhoA interaction with RASSF1C-Δ2γ but not with WT-RASSF1C.
ARTICLE ABSTRACT
RASSF1A is a tumor suppressor implicated in many tumorigenic processes; however, the basis for its tumor suppressor functions are not fully understood. Here we show that RASSF1A is a novel antagonist of protumorigenic RhoA activity. Direct interaction between the C-terminal amino acids (256–277) of RASSF1A and active GTP-RhoA was critical for this antagonism. In addition, interaction between the N-terminal amino acids (69-82) of RASSF1A and the ubiquitin E3 ligase Smad ubiquitination regulatory factor 1 (Smurf1) disrupted GTPase activity by facilitating Smurf1-mediated ubiquitination of GTP-RhoA. We noted that the RhoA-binding domain of RASSF1A displayed high sequence homology with Rho-binding motifs in other RhoA effectors, such as Rhotekin. As predicted on this basis, RASSF1A competed with Rhotekin to bind RhoA and to block its activation. RASSF1A mutants unable to bind RhoA or Smurf1 failed to suppress RhoA-induced tumor cell proliferation, drug resistance, epithelial–mesenchymal transition, migration, invasion, and metastasis. Clinically, expression levels of RASSF1A and RhoA were inversely correlated in many types of primary and metastatic tumors and tumor cell lines. Collectively, our findings showed how RASSF1A may suppress tumorigenesis by intrinsically inhibiting the tumor-promoting activity of RhoA, thereby illuminating the potential mechanistic consequences of RASSF1A inactivation in many cancers. Cancer Res; 76(7); 1847–59. ©2016 AACR.
