Rac1, a member of the Rho family of GTPases, regulates diverse cellular functions, including cytoskeleton reorganization and cell migration. F‐box proteins are major subunits within the Skp1‐Cul1‐F‐box (SCF) E3 ubiquitin ligases that recognize specific substrates for ubiquitination. The role of F‐box proteins in regulating Rac1 stability has not been studied. Mouse lung epithelial (MLE12) cells were used to investigate Rac1 stability and cell migration. Screening of an F‐box protein library and in vitro ubiquitination assays identified FBXL19, a relatively new member of the F‐box protein family that targets Rac1 for its polyubiquitination and proteasomal degradation. Overexpression of FBXL19 decreased both Rac1 active and inactive forms and significantly reduced cellular migration. Protein kinase AKT‐mediated phosphorylation of Rac1 at serine ⁷¹ was essential for FBXL19‐mediated Rac1 ubiquitination and depletion. Lysin ¹⁶⁶ within Rac1 was identified as a polyubiquitination acceptor site. Rac1 ^S71A and Rac1K166R mutant proteins were resistant to FBXL19‐mediated ubiquitination and degradation. Further, ectopically expressed FBXL19 reduced cell migration in Rac1‐overexpressing cells ( P <0.01, Rac1 cells vs. FBXL19+Rac1 cells), but not in Rac1 lysine ¹⁶⁶ mutantoverexpressing cells. FBXL19 diminished formation of the migratory leading edge. Thus, SCF ^FBXL19 targets Rac1 for its disposal, a process regulated by AKT. These findings provide the first evidence of an F‐box protein targeting a small G protein for ubiquitination and degradation to modulate cell migration.—Zhao, J., Mialki, R. K., Wei, J., Coon, T. A., Zou, C., Chen, B. B., Mallampalli, R. K., Zhao, Y. SCF E3 ligase F‐box protein complex SCF regulates cell migration by mediating Rac1 ubiquitination and degradation. FASEB J. 27, 2611‐2619 (2013). www.fasebj.org

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.

The ubiquitin-proteasome system is the major pathway of non-lysosomal intracellular protein degradation, playing an important role in a variety of cellular responses including cell division, proliferation, and apoptosis. Ubiquitin-specific protease 14 (USP14) is a component of proteasome regulatory subunit 19 S that regulates deubiquitinated proteins entering inside the proteasome core 20 S. The role of USP14 in protein degradation is still controversial. Several studies suggest that USP14 plays an inhibitory role in protein degradation. Here, in contrast, overexpression of USP14 induced I-κB degradation, which increased cytokine release in lung epithelial cells. Overexpression of HA-tagged USP14 (HA-USP14) reduced I-κB protein levels by increasing the I-κB degradation rate in mouse lung epithelial cells (MLE12). I-κB polyubiquitination was reduced in HA-USP14-overexpressed MLE12 cells, suggesting that USP14 regulates I-κB degradation by removing its ubiquitin chain, thus promoting the deubiquitinated I-κB degradation within the proteasome. Interestingly, we found that USP14 was associated with RelA, a binding partner of I-κB, suggesting that RelA is the linker between USP14 and I-κB. Lipopolysaccharide (LPS) treatment induced serine phosphorylation of USP14 as well as further reducing I-κB levels in HA-USP14-overexpressed MLE12 cells as compared with empty vector transfected cells. Further, overexpression of HA-USP14 increased the LPS-, TNFα-, or Escherichia coli-induced IL-8 release in human lung epithelial cells. This study suggests that USP14 removes the ubiquitin chain of I-κB, therefore inducing I-κB degradation and increasing cytokine release in lung epithelial cells.

The transforming growth factorβ-1 (TGFβ-1) signaling pathway plays a central role in the pathogenesis of pulmonary fibrosis. Two TGFβ-1 receptors, TβRI and TβRII, mediate this pathway. TβRI protein stability, as mediated by the ubiquitin/de-ubiquitination system, has been well studied; however, the molecular regulation of TβRII still remains unclear. Here we reveal that a de-ubiquitinating enzyme, USP11, promotes TGFβ-1 signaling through de-ubiquitination and stabilization of TβRII. We elucidate the role that mitoxantrone (MTX), an USP11 inhibitor, has in the attenuation of TGFβ-1 signaling. Inhibition or downregulation of USP11 results in increases in TβRII ubiquitination and reduction of TβRII stability. Subsequently, TGFβ-1 signaling is greatly attenuated, as shown by the decreases in phosphorylation of SMAD2/3 levels as well as that of fibronectin (FN) and smooth muscle actin (SMA). Overexpression of USP11 reduces TβRII ubiquitination and increases TβRII stabilization, thereby elevating phosphorylation of SMAD2/3 and the ultimate expression of FN and SMA. Further, elevated expression of USP11 and TβRII were detected in lung tissues from bleomycin-challenged mice and IPF patients. Therefore, USP11 may contribute to the pathogenesis of pulmonary fibrosis by stabilization of TβRII and promotion of TGFβ-1 signaling. This study provides mechanistic evidence for development of USP11 inhibitors as potential antifibrotic drugs for pulmonary fibrosis.

Lysophosphatidic acid (LPA), a bioactive phospholipid, plays an important role in lung inflammation by inducing the release of chemokines and lipid mediators. Our previous studies have shown that LPA induces the secretion of interleukin-8 and prostaglandin E₂ in lung epithelial cells. Here, we demonstrate that LPA receptors contribute to lipopolysaccharide (LPS)-induced inflammation. Pretreatment with LPA receptor antagonist Ki16425 or downregulation of LPA receptor 1 (LPA₁) by small-interfering RNA (siRNA) attenuated LPS-induced phosphorylation of p38 MAPK, I-κB kinase, and I-κB in MLE12 epithelial cells. In addition, the blocking of LPA₁ also suppressed LPS-induced IL-6 production. Furthermore, LPS treatment promoted interaction between LPA₁ and CD14, a LPS coreceptor, in a time- and dose-dependent manner. Disruption of lipid rafts attenuated the interaction between LPA₁ and CD14. Mice challenged with LPS increased plasma LPA levels and enhanced expression of LPA receptors in lung tissues. To further investigate the role of LPA receptors in LPS-induced inflammation, wild-type, or LPA₁-deficient mice, or wild-type mice pretreated with Ki16425 were intratracheally challenged with LPS for 24 h. Knock down or inhibition of LPA₁ decreased LPS-induced IL-6 release in bronchoalveolar lavage (BAL) fluids and infiltration of cells into alveolar space compared with wild-type mice. However, no significant differences in total protein concentration in BAL fluids were observed. These results showed that knock down or inhibition of LPA₁ offered significant protection against LPS-induced lung inflammation but not against pulmonary leak as observed in the murine model for lung injury.