RhoA is a small GTPase multifunctional protein that regulates cell proliferation and cytoskeletal reorganization. Regulation of its protein stability plays an important role in its biological functions. We have shown that a Skp1-Cul1-F-box (SCF) FBXL19 E3 ubiquitin ligase targets Rac1, a related member of the Rho family for ubiquitination and degradation. Here, SCFFBXL19 mediates RhoA ubiquitination and proteasomal degradation in lung epithelial cells. Ectopically expressed FBXL19 decreased RhoA wild type, active, and inactive forms. Cellular depletion of FBXL19 increased RhoA protein levels and extended its half-life. FBXL19 bound the small GTPase in the cytoplasm leading to RhoA ubiquitination at Lys135. A RhoAK135R mutant protein was resistant to SCFFBXL19-mediated ubiquitination and degradation and exhibited a longer lifespan. Protein kinase Erk2-mediated phosphorylation of RhoA was both sufficient and required for SCFFBXL19-mediated RhoA ubiquitination and degradation. Thus, SCFFBXL19 targets RhoA for its disposal, a process regulated by Erk2. Ectopically expressed FBXL19 reduced phosphorylation of p27 and cell proliferation, a process mediated by RhoA. Further, FBXL19 cellular expression diminished lysophosphatidic acid (LPA)-induced phosphorylation of myosin light chain (MLC) and stress fiber formation. Hence, SCFFBXL19 functions as a RhoA antagonist during cell proliferation and cytoskeleton rearrangement. These results provide the first evidence of an F-box protein targeting RhoA thereby modulating its cellular lifespan that impacts cell proliferation and cytoskeleton rearrangement.

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.

Cyclic adenosine monophosphate (cAMP) response element–binding protein (CREB)–binding protein (CBP) is a histone acetyltransferase that plays a pivotal role in the control of histone modification and the expression of cytokine-encoding genes in inflammatory diseases, including sepsis and lung injury. We found that the E3 ubiquitin ligase subunit FBXL19 targeted CBP for site-specific ubiquitylation and proteasomal degradation. The ubiquitylation-dependent degradation of CBP reduced the extent of lipopolysaccharide (LPS)–dependent histone acetylation and cytokine release in mouse lung epithelial cells and in a mouse model of sepsis. Furthermore, we demonstrated that the deubiquitylating enzyme USP14 (ubiquitin-specific peptidase 14) stabilized CBP by reducing its ubiquitylation. LPS increased the stability of CBP by reducing the association between CBP and FBXL19 and by activating USP14. Inhibition of USP14 reduced CBP protein abundance and attenuated LPS-stimulated histone acetylation and cytokine release. Together, our findings delineate the molecular mechanisms through which CBP stability is regulated by FBXL19 and USP14, which results in the modulation of chromatin remodeling and the expression of cytokine-encoding genes.

. Serum starvation regulates E-cadherin upregulation via activation of c-Src in non-small-cell lung cancer A549 cells. Am J Physiol Cell Physiol 307: C893-C899, 2014. First published August 27, 2014 doi:10.1152/ajpcell.00132.2014.-E-cadherin is essential for the integrity of adherens junctions between lung epithelial cells, and the loss of E-cadherin allows cell motility and is thought to promote lung cancer metastasis. While the downregulation of E-cadherin expression has been well characterized and is seen with transforming growth factor-␤1 (TGF-␤1) exposure, few studies have focused on E-cadherin upregulation. Here, we show that serum starvation causes increased E-cadherin expression via the activation of c-Src kinase in non-small-cell lung cancer A549 cells. Serum starvation increased E-cadherin protein levels in a time-and dose-dependent manner. E-cadherin mRNA transcripts were unchanged with starvation, while protein translation inhibition with cycloheximide attenuated E-cadherin protein induction by starvation, suggesting that E-cadherin is regulated at the translational level by serum starvation. c-Src is a nonreceptor tyrosine kinase known to regulate protein translation machinery; serum starvation caused early and sustained activation of c-Src in A549 cells followed by Ecadherin upregulation. Furthermore, overexpression of a dominant negative c-Src attenuated the induction of E-cadherin by serum deprivation. Finally, we observed that TGF-␤1 treatment attenuated the serum activation of c-Src as well as E-cadherin expression when cells were deprived of serum. In conclusion, our data demonstrate that the c-Src kinase is activated by serum starvation to increase Ecadherin expression in A549 cells, and these phenomena are antagonized by TGF-␤1. These novel observations implicate the c-Src kinase as an upstream inducer of E-cadherin protein translation with serum starvation and TGF-␤1 diametrically regulating c-Src kinase activity and thus E-cadherin abundance in A549 cells.

Rationale: CREB‐binding protein (CBP), a versatile acetyltransferase, plays a critical role in the pathology of Acute Lung Injury (ALI) via regulation of proinflammatory gene expression. However, molecular regulation of CBP at the level of protein stability has not been well studied. Three enzyme complexes (E1, E2, and E3) are involved in linking ubiquitin chains onto target proteins. F‐box protein is a substrate‐recognition component within the SCF E3 ligase complex. Here, we investigate the role of F‐box protein, FBXL19, in the regulation of CBP ubiquitination and degradation. Methods and Results: We found that FBXL19‐V5 over‐expression reduced both endogenous and over‐expressed CBP in mouse lung epithelial cells (MLE12). Further, over‐expression of FBXL19 induced CBP ubiquitination, which was reduced in FBXL19 knock‐down cells. Co‐immunoprecipitation and co‐immunostaining studies showed that FBXL19 is associated with CBP. To investigate the role of FBXL19 in CBP‐mediated histone acetylation and proinflammatory gene expression, we transfected human lung epithelial cells with FBXL19‐V5 plasmid prior to bacterial endotoxin treatment. FBXL19‐V5 attenuated LPS‐induced histone H4 acetylation at lysine residue 8 (H4K8) and IL‐8 release. Further, we found that over‐expression of FBXL19 in mouse lungs by intratracheal lentiviral vector gene delivery system attenuated intratracheal LPS‐induced acetylation of H4K8 in mouse lung tissues and IL‐6 in bronchoalveolar lavage fluids. Conclusions: SCFFBXL19 targets CBP for its ubiquitination and degradation. Over‐expression of FBXL19 reduces CBP‐mediated histone acetylation and cytokine release. FBXL19 protects against lung inflammation in bacterial endotoxin‐induced murine model of acute lung injury. These data may provide a potential therapeutic strategy to target FBXL19 / CBP pathway to lessen the severe of lung inflammatory diseases. Grant Funding Source: The study is supported by NIH RO1 HL091916 and HL112791 (to YZ) and American Heart Association Scien