F-box protein complex FBXL19 regulates TGFβ1-induced E-cadherin down-regulation by mediating Rac3 ubiquitination and degradation

Abstract

BackgroundRac3 is a small GTPase multifunctional protein that regulates cell adhesion, migration, and differentiation. It has been considered as an oncogene in breast cancer; however, its role in esophageal cancer and the regulation of its stability have not been studied. F-box proteins are major subunits within the Skp1-Cullin-1-F-box (SCF) E3 ubiquitin ligases that recognize particular substrates for ubiquitination and proteasomal degradation. Recently, we have shown that SCFFBXL19 targets Rac1 and RhoA, thus regulating Rac1 and RhoA ubiquitination and degradation. Here, we demonstrate the role of FBXL19 in the regulation of Rac3 site-specific ubiquitination and stability. Expression of TGFβ1 is associated with poor prognosis of esophageal cancer. TGFβ1 reduces tumor suppressor, E-cadherin, expression in various epithelial-derived cancers. Here we investigate the role of FBXL19-mediated Rac3 degradation in TGFβ1-induced E-cadherin down-regulation in esophageal cancer cells.MethodsFBXL19-regulated endogenous and over-expressed Rac3 stability were determined by immunoblotting and co-immunoprecipitation. Esophageal cancer cells (OE19 and OE33) were used to investigate TGFβ1-induced E-cadherin down-regulation by Immunoblotting and Immunostaining.ResultsOverexpression of FBXL19 decreased endogenous and over-expressed Rac3 expression by interacting and polyubiquitinating Rac3, while down-regulation of FBXL19 suppressed Rac3 degradation. Lysine166 within Rac3 was identified as an ubiquitination acceptor site. The FBXL19 variant with truncation at the N-terminus resulted in an increase in Rac3 degradation; however, the FBXL19 variant with truncation at the C-terminus lost its ability to interact with Rac3 and ubiquitinate Rac3 protein. Further, we found that Rac3 plays a critical role in TGFβ1-induced E-cadherin down-regulation in esophageal cancer cells. Over-expression of FBXL19 attenuated TGFβ1-induced E-cadherin down-regulation and esophageal cancer cells elongation phenotype.ConclusionsCollectively these data unveil that FBXL19 functions as an antagonist of Rac3 by regulating its stability and regulates the TGFβ1-induced E-cadherin down-regulation. This study will provide a new potential therapeutic strategy to regulate TGFβ1 signaling, thus suppressing esophageal tumorigenesis.

New Insights into the Role of Podoplanin in Epithelial–Mesenchymal Transition

“…The most studied mechanism for loss of E-cadherin is transcriptional repression by the core TFs. But, as mentioned above, other mechanisms besides transcriptional repression can be involved, like temporal variations in expression of the core TFs (Tran et al, 2011), E-cadherin promoter methylation, which has been observed in a wide variety of mammary tumors (Lombaerts et al, 2006), and posttranslational regulation by endocytosis and degradation by the proteasome (Yang et al, 2006;Dong et al, 2014;Hartsock and Nelson, 2012) We propose the existence of two superimposed mechanisms (figure 7). One is the increase in cellular motility driven by podoplanin interacting with the ERM proteins-Rho GTPases axis; the second is what we call "effective cohesiveness" in the cells.…”

Regulating Rho GTPases and their regulators

“…Interestingly, RAC1 is polyubiquitylated on Lys147 by IAPs and HACE1, whereas SCF FBXL19 polyubiquitylates RAC1 and its close relative RAC3 on Lys166 (REFS 65,66), suggesting that different E3 ligases ubiquitylate RAC1 depending on the subcellular localization and/or stimulus, as for RhoA. Ubiquitylation by these three E3 ligases targets RAC1 for degradation, which affects cell morphology and migration 59,62,65 .…”

F-box protein interactions with the hallmark pathways in cancer

“…Since SCF(Skp2) destabilizes p27, another effect of over-expression of Skp2 would be to alter cell migration and increase formation of stress fibres and focal adhesions. Another negative regulator of RhoA and also its related family members Rac1 and Rac3, is SCF(Fbxl19) (204)(205)(206). Underscoring the importance of this pathway, at least two other E3 ubiquitin ligases target RhoA for ubiquitination: Smurf1 and Cullin3(BACURD) (207,208).…”