F-box protein complex FBXL19 regulates TGFβ1-induced E-cadherin down-regulation by mediating Rac3 ubiquitination and degradation
- Su Dong1, 2, 3,
- Jing Zhao3,
- Jianxin Wei3,
- Rachel K Bowser3,
- Andrew Khoo3,
- Zhonghui Liu†1,
- James D Luketich4,
- Arjun Pennathur4,
- Haichun Ma†2 and
- Yutong Zhao†3Email author
© Dong et al.; licensee BioMed Central Ltd. 2014
Received: 6 March 2014
Accepted: 25 March 2014
Published: 1 April 2014
Rac3 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.
FBXL19-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.
Overexpression 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.
Collectively 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.
KeywordsSmall GTPase protein Protein stability E3 ligase Ubiquitin-proteasome system TGFβ1 E-cadherin
Rac3 (ras-related C3 botulinum toxin substrate 3) is a member of the RhoGTPase family. In its active form, it is bound to GTP, whereas it is inactive in its GDP-bound form. It has been well studied that activation of RhoGTPases is controlled by guanidine activating proteins (GEFs) that exchange bound GDP to GTP and by GTPase activating proteins (GAPs) that promote GTP hydrolysis. Rac3 is enriched in the brains but it also expressed in a wide range of tissues . Aberrant activation of Rac3 has been recognized to be important in tumor proliferation in both breast cancer and prostate cancer [2, 3]. Rac3 regulates a variety of cellular functions including adhesion, cell migration, and differentiation. The controversial effects of Rac3 on cell migration have been reported. Rac3 negatively regulates diapedesis of prostate cancer cell PC3, since knockdown of Rac3 using Rac3 specific siRNA increased migration of PC3 cells through a bone marrow endothelial cell layer . In contrast, overexpression of Rac3 promotes estrogen-induced breast cancer cell migration . Rac3 was found to be involved in breast cancer cell aggressiveness through the activation of NF-κB and Erk2. Inhibition of Rac3 caused an increase in TNFα-induced apoptosis . However, the role of Rac3 in the pathogeneses of esophageal cancer has not been studied.
Many posttranslational modifications, including ubiquitination, expand the size of the proteome exponentially and are pivotal in the regulation of protein stability [6–9]. The ubiquitin proteasome system (UPS) regulates protein ubiquitination, and therefore degradation and turnover, of many proteins vital of cellular regulation and function . The UPS depends upon the action of three enzyme complexes. The E1 enzyme functions as an activator by creating a thioester bond between a cysteine of the E1 enzyme and the ubiquitin molecule via ATP hydrolysis. E2, known as the conjugating enzyme, then accepts the ubiquitin protein onto an active site cysteine. Finally, the E3 ligating enzyme complex is responsible for attachment of this ubiquitin protein to a lysine of the target protein . Among the families of E3 ubiquitin ligases, the Skp1-Cullin-1-F-box protein (SCF) ligases complex is one of the largest. In this complex, the F-box protein is the substrate-recognition component [12, 13]. F-box proteins have two main domains: an F-box motif and a substrate binding motif. F-box proteins use their F-box motif to bind to Skp1 and assemble the SCF ligase complex, whereas the substrate-binding motif is used for recognition and interaction with phosphorylated substrates . Through an in silico search, the ‘orphan’ F-box protein FBXL19 has been identified and verified as an SCF E3 subunit . Recently, we demonstrated that FBXL19 regulates interleukin (IL)-33 signaling by targeting its cognate receptor ST2L, for ubiquitination, which, in turn, triggers its proteasomal degradation to alter the innate immune response . In addition to ST2L, we also found that Rac1 and RhoA are targets for FBXL19 and revealed new functions of FBXL19 in regulating cell migration, proliferation and cytoskeleton rearrangement [17, 18].
E-cadherin, a type I classical cadherin, is a key component in the formation of cell-cell adherens-type junctions in epithelial tissues [19–21]. A variety of studies in cancers, including hepatocellular carcinoma, squamous cell carcinomas of the skin, head and neck, and pancreatic cancer, have demonstrated that E-cadherin plays a critical role as a tumor suppressor [22–25]. E-cadherin is often down-regulated during carcinoma progression and metastatic spread of tumors [26, 27]. Loss of E-cadherin changes cancer cell phenotype and facilitates the initial invasive behaviors of epithelial-derived cancer . Transforming growth factor β (TGFβ), a pleiotropic cytokine comprised of three isoforms in mammalian cells, function as a tumor promoting mediator in the later stages of cancers [29, 30]. TGFβ1 signaling has been shown to play an important role in down-regulation of E-cadherin. It appears, that many epithelial tumors escape growth inhibition by TGFβ1, and TGFβ1 secretion by cancer may contribute to late tumor progression [31, 32]. It has been shown that TGFβ1 expression is higher in esophageal cancer tissues, compared to normal squamous epithelium and non-malignant Barrett’s mucosa . Over-expression of TGFβ1 in esophageal cancer is associated with advanced stage of disease and poor prognosis .
In this study, we demonstrate that Rac3 is a target protein of SCFFBXL19 E3 ligase. FBXL19 regulates Rac3 stability by ubiquitinating Rac3 on lysine 166 residue. This is the first report to reveal that Rac3 is implicated in TGFβ1-induced E-cadherin down-regulation in esophageal cancer cells. Further, we found that over-expression of FBXL19 attenuates the effect of TGFβ1 on E-cadherin down-regulation. This study will provide a molecular basis for SCF E3 ligase in the regulation of esophageal tumorigenesis.
FBXL19 reduces Rac3 protein expression
FBXL19 induces Rac3 degradation in the proteasome system
FBXL19 targets Rac3 lysine166 for ubiquitination
Role of N-terminus and C-terminus of FBXL19 in Rac3 degradation
Rac3 regulates TGFβ1-induced E-cadherin down-regulation
FBXL19 regulates TGFβ1-induced E-cadherin down-regulation
Rho family GTPases are important intracellular signaling proteins that control diverse cellular functions, including actin cytoskeletal organization, migration and invasion, transcriptional regulation, cell cycle progression, apoptosis, vesicle trafficking, and cell-to-cell and cell-to-extracellular matrix adhesions [36, 37]. Of the Rho family GTPases, Rac3 is implicated in regulating cell adhesion, growth, differentiation, and autophagy . To date, little is known regarding the molecular regulation of Rac3 stability. Here, we show that Rac3 lifespan is regulated by the SCFFBXL19 E3 ligase and the proteasome system. FBXL19 targets Rac3 for ubiquitination in a specific lysine site, thus resulting in its degradation. Rac3, as an oncogene protein, plays a pivotal role in tumorgenesis of a variety type of cancers, including breast cancer and prostate cancer. This study is the first to report that Rac3 regulates TGFβ1-mediated E-cadherin down-regulation in esophageal cancer cells, indicating a critical role of Rac3 in the progress of esophageal cancer. Further, we demonstrate that FBXL19 negatively regulates Rac3-mediated TGFβ1 signaling in esophageal cancer. Here, we provide new evidence that ubiquitin E3 ligase contributes to the tumorgenesis of esophageal cancer. Targeting the ubiquitin E3 ligase will build a basis to develop a new potential therapeutic strategy to inhibit tumor growth and invasion. Despite the high homology in amino-acid sequence between Rac1 and Rac3, Rac3 differs from Rac1 in the COOH terminal region, which is involved in Rac localization and regulatory protein binding . However, most of literature addressing the role of Rac in cancer progression concern Rac1, with little mention of Rac3. The elucidation of mechanisms for control of Rac3 protein stability therefore might have important implications for metastasis.
Post-translational modifications, including ubiquitination, regulate the function of key signaling proteins by modulating their activity, localization, and protein stability. Ubiquitination of small GTPases controls their behavior in cells, including migratory ability and cell cycle progression. We established recently that FBXL19 targets RhoA and Rac1 for ubiquitination and degradation, thus regulating cell growth, stress fiber formation, and cell migration [17, 18]. F-box proteins have been shown to target multiple-substrates. For example, FBXW7 ubiquitinates multiple proteins involved in different signal pathways, such as Notch, cyclin, c-Myc and c-Jun, for ubiquination and degradation [39–41]. Here we uncover that Rac3 is a new substrate for FBXL19 and it interacts with C-terminus of FBXL19. This is the first study to investigate Rac3 ubiquitination and degradation. Over-expression of FBXL19 reduced Rac3 for disposal. In addition, we identified that FBXL19 induced Rac3 ubiquitination at lysine166. This acceptor site is similar to Rac1 ubiquitin acceptor site from FBXL19, which is distant from GTP binding site and resides within a C-terminal α-helix distinct from the polybasic tail . It has been shown that other ubiquitin E3 ligases such as IAP and HACE1 also target Rac1 for ubiquitination and degradation. Since Rac3 shares a high homology with Rac1, the future study will focus on the role of IAP or HACE1 in regulation of Rac3 stability.
In summary, the current study unveils Rac3 as a new molecular target of FBXL19. FBXL19 targets Rac3 for ubiquitination in lysine 166 and induces its proteasomal degradation. Inhibition of Rac3 by FBXL19 modulates E-cadherin expression in esophageal cancer cells. This is the first evidence to support that Rac3 plays a critical role in the tumorgenesis of esophageal cancer and that FBXL19 exhibits an anti-tumor property by down-regulation of small GTPase.
Material and methods
Cells and reagents
Esophageal adenocarcinoma (OE19 and OE33) cancer cells [Sigma-Aldrich (St. Louis, MO, USA)] were cultured with RPMI 1640 medium containing 2 mM glutamine and 10% FBS. HEK293 cells [Invitrogen (Carlsbad, CA, USA)] were cultured with DMEM medium containing 10% FBS. Murine lung epithelia (MLE12) cells [American Type Culture Collection (ATCC), Manassas, VA, USA] were cultured with HITES medium containing 10% FBS. All the cells were cultured at 37°C in 5% CO2. V5 antibody, E-cadherine antibody, mammalian expressional plasmid pcDNA3.1D/His V5 TOPO, Escherichia coli Top 10 competent cells, and recombinant TGFβ1 were from Invitrogen (Carlsbad, CA, USA). HA tag (29 F4), Flag tag (9A3), and ubiquitin (P4D1) antibodies were from Cell Signaling Technology (Danvers, MA, USA). Rac3 antibody was from Proteintech Group (Chicago, IL, USA). FBXL19 antibody was from Abgent (San Diego, CA, USA). Leupeptin, ammonium chloride (NH4Cl), β-actin antibody, individual FBXL19 shRNAs, and scrambled shRNA were from Sigma Aldrich (St. Louis, MO, USA). MG132 and lactacystin from Calbiochem (La Jolla, CA, USA). Immunobilized protein A/G beads were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Superfect transfection reagent was from QIAGEN (Valencia, CA, USA). All materials in highest grades uses in the experiments are commercially available.
Construction of FBXL19 and Rac3 plasmids
The FBXL19 cDNA was inserted into a pcDNA3.1D/V5-His vector (Invitrogen, CA, USA). Site directed mutagenesis was performed to generate Rac3 lysine or serine mutant according to the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA, USA).
Immunoblotting and immunoprecipitation
Cells were washed with cold PBS and collected in cell lysis buffer containing 20 mM Tris HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 5 mM β glycerophosphate, 1 mM MgCl2, 1% Trison X100, 1 mM sodium orthovanadate, 10 μg/ml protease inhibitors, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. An equal amount of cell lysates (20 μg) was subjected to SDS-PAGE, electrotransferred to membranes and immunoblotted as described previously [17, 18]. For immunoprecipitation, equal amounts of cell lysates (1 mg) were incubated with specific primary antibodies overnight at 4°C, followed by the addition of 40 μl of protein A/G agarose and incubation for additional 2 h at 4°C. The immunoprecipitated complex was washed 3 times with 1% Triton X100 in ice cold phosphate-buffered saline and analyzed by immunoblotting with indicated antibodies.
OE19 cells were plated on 35 mm glass-bottom culture dishes. After treatment, cells were fixed in 3.7% formaldehyde for 20 min, followed by permeabilization with 0.1% Triton X100 for 2 min. Cells were incubated with 1:200 dilution of E-cadherin, followed by a 1:200 dilution of fluorescence-conjugated secondary antibody sequentially for immunostaining. Immunofluorescent cell imaging was performed on a Nikon confocal microscope.
HEK293 cells and OE19 cells were subcultured on 6-well plates or 35 mm plates for 24 h. Superfect transfection reagent was used for HEK293 cells and lipofectamine transfection reagent was used for OE19 cells. The transfection reagent was added to the mixture of 3 μg of plasmid and 200 μl of reduced serum medium, mixed, and incubated for 10 minutes to allow transfection reagent/DNA complexes to form. And then the mixture was added directly to the cells. The transfected cells were cultured for 48 h. shRNA plasmids were also delivered into cells with the same protocol and the cells were cultured for 72 h.
Rac3 activity assay
FBXL19-V5 transfected HEK293 and OE19 cells were cultured in 100-mm dishes and Rac3 activation was evaluated using a PBD-GTPγ-Rac3 pull down assay. Briefly, cell lysates (0.5-1 mg/ml) were loaded with 10 μg of PAK-1 p21-binding domain fusion-protein conjugated to agarose for 1 h to bind Rac3-GTP, centrifuged, and washed three times with lysis buffer. The proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with a Rac3 antibody. Total cell lysates were also probed separately with anti-Rac3 and anti-β-actin antibodies to confirm equal loading.
Esophageal cancer cells were cultured in 6-well plates or 35 mm dishes in 10% FBS medium. After 48 h of transfection, the medium was replaced with 1 ml of serum-free (blank) medium. TGFβ1 was added into the medium with the concentration range of 0, 1.25, 2.5, 5.0 10 ng/ml. After 4 days of incubation at 37°C, the cells were collected and analyzed by immunoblotting with E-cadherin antibodies.
All results were subjected to statistical analysis using two-way analysis of variance, and, wherever appropriate, analyzed by Student-Newman-Keuls test. Data are expressed as mean ± SD of triplicate samples from at least three independent experiments and values that were p < 0.01 were considered statistically significant.
YZ, HM, and ZL are the corresponding authors of this work.
Ras-related C3 botulinum toxin substrate 3
Ubiquitin proteasome system
Guanidine activating proteins
GTPase activating proteins
Transforming growth factor β
This study was supported by the US National Institutes of Health (R01 HL091916 and R01HL112791 to YZ), American Heart Association awards 12SDG9050005 (JZ).
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