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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 S1. RASSF1A stimulates GTP-RhoA degradation. A, RASSF1A inhibition of RhoA-induced membrane ruffling and protrusion. GFP-expressing cells were transfected with RASSF1A and/or RhoA-G14V. B, RASSF1A suppression of RhoA-mediated FAK, MLC, and LIMK1/2 phosphorylation. C, No effect of RASSF1A on Ras activation of Raf-Mek-Erk signaling. A549 cells were co-transfected with RASSF1A-V5 and either HA-tagged H-Ras-G14V, K-Ras-G14V, or N-Ras-Q61R. Immunoblots were performed at 48 h after transfection. D. Effect of RASSF1A depletion on EGF (10 ng/ml) induction of GTP-RhoA. E. RASSF1A suppression of serum- or LPA-mediated GTP-RhoA induction. Cells transfected with either RASSF1A or RASSF1C were maintained in the absence of serum for 24 h and then exposed to serum or LPA. GTP-RhoA level was determined by GTPase pull-down assay. F, No effect of RASSF1A on Rho-GAP and Rho-GEF levels. G, A CHX assay showing reduction of the RhoA protein half-life by RASSF1A. RhoA intensities were measured by densitometric scanning of the immunoblot gels (Fig. 1H) and adjusted for Tubulin.
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.
