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posted on 2023-03-31, 00:04 authored by Min-Goo Lee, Seong-In Jeong, Kyung-Phil Ko, Soon-Ki Park, Byung-Kyu Ryu, Ick-Young Kim, Jeong-Kook Kim, Sung-Gil Chi Figure S4. RASSF1A blocks Rhotekin interaction with and activation of RhoA. A, Enhanced RhoA-Rhotekin interaction in RASSF1A-depeletd cells. HeLa cells transfected with siControl or siRASSF1A (20 nM) were maintained in the absence of serum for 24 h. At 12 h after serum addition, immunoprecipitation and immunoblot were performed as indicated. B, Rhotekin suppression of RASSF1A-induced RhoA ubiquitination. A549 cells were co-transfected with WT-RASSF1A and Myc-Rhotekin and immunoprecipitation assays were performed to measure ubiquitinated RhoA level. C, RASSF1A inhibition of Rhotekin's protective effect on RhoA ubiquitination. D, No effect of RASSF1A-R269F on RhoA ubiquitination. E, IP assay showing RhoA interaction with Rhotekin and S100A4. F, No effect of RASSF1A on S100A4 protein level. G, RASSF1A suppression of S100A4 activation of RhoA signaling.
Funding
Korean Health Technology R&D Project
National Research Foundation of Korea
History
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.
