A blocking peptide stabilizes lysophosphatidic acid receptor 1 and promotes lysophosphatidic acid-induced cellular responses.

G protein-coupled receptors regulate a variety of cellular responses and have been considered as therapeutic targets for human diseases. Lysophosphatidic acid receptor 1 (LPA1) is a receptor for bioactive lysophospholipid, LPA. LPA/LPA1-mediated signaling contributes to inflammatory and fibrotic responses in lung diseases; thus understanding regulation of LPA1 stability is important for modulating LPA/LPA1 signaling. Our previous study has shown that LPA1 is degraded in the Nedd4 like (Nedd4L) E3 ubiquitin ligase-mediated ubiquitin-proteasome system. In the current study, we attempt to identify a peptide that stabilizes LPA1 through disrupting LPA1 association with Nedd4L. LPA treatment induces both endogenous and overexpressed LPA1 degradation, which is attenuated by a proteasome inhibitor, suggesting that LPA1 is degraded in the proteasome. LPA increases phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and I-κB kinase in lung epithelial cells, and this effect is promoted by overexpression of a peptide (P1) that mimics C-terminal of LPA1. P1, not a control peptide, attenuates LPA-induced LPA1 ubiquitination and degradation, suggesting that P1 stabilizes LPA1. Further, P1 diminishes Nedd4L-mediated degradation of LPA1 and Nedd4L/LPA1 association. In addition to increasing LPA1 signaling, P1 enhances LPA-induced cell migration and gene expression of Elafin, matrix metallopeptidase 1, and serpin family B member 2 in lung epithelial cells. These data suggest that disruption of LPA1 interaction with Nedd4L by P1 increases LPA1 stability and LPA/LPA1 signaling.

A new mechanism of RhoA ubiquitination and degradation: roles of SCF(FBXL19) E3 ligase and Erk2.

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, SCF(FBXL19) 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 Lys(135). A RhoA(K135R) mutant protein was resistant to SCF(FBXL19)-mediated ubiquitination and degradation and exhibited a longer lifespan. Protein kinase Erk2-mediated phosphorylation of RhoA was both sufficient and required for SCF(FBXL19)-mediated RhoA ubiquitination and degradation. Thus, SCF(FBXL19) 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, SCF(FBXL19) 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.

Overexpression of USP14 protease reduces I-κB protein levels and increases cytokine release in lung epithelial 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.

Lipopolysaccharide-induced phosphorylation of c-Met tyrosine residue 1003 regulates c-Met intracellular trafficking and lung epithelial barrier function.

c-Met, the receptor tyrosine kinase whose natural ligand is hepatocyte growth factor, is known to have a key role in cell motility. We have previously shown that lysophosphatidic acid (LPA) induced a decrease in c-Met activation via serine phosphorylation of c-Met at cell-cell contacts. Here, we demonstrate that lipopolysaccharide (LPS) treatment of human bronchial epithelial cells induced internalization of c-Met via phosphorylation at its tyrosine residue 1003. In addition, it induced epithelial barrier dysfunction as evidenced by a decrease in transepithelial resistance (TER) in a time-dependent manner. Pretreatment with a c-Met inhibitor (PHA-665752) or inhibition of protein kinase C (PKC)-α attenuated the LPS-mediated phosphorylation of c-Met and its internalization. LPS-induced c-Met tyrosine 1003 phosphorylation, activation of PKCα, and c-Met internalization were, however, reversed by pretreatment of cells with LPA, which increased c-Met accumulation at cell-cell contacts. Inhibition of LPS-mediated c-Met tyrosine (Y1003) phosphorylation and internalization by prior treatment with PHA-665752, inhibition of PKCα, or overexpression of c-MetY1003A mutant attenuated LPS-induced reduction of TER. Furthermore, we found that c-Met accumulation at cell-cell contacts contributed to LPA-enhanced epithelial barrier integrity, since downregulation of c-Met by specific small-interfering RNA attenuated LPA-increased TER. The data reveal a novel biological function of c-Met in the regulation of lung epithelial barrier integrity.

SCF E3 ligase F-box protein complex SCF(FBXL19) regulates cell migration by mediating Rac1 ubiquitination and degradation.

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. Lysine(166) within Rac1 was identified as a polyubiquitination acceptor site. Rac1(S71A) and Rac1(K166R) 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) mutant-overexpressing 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.

F-box protein FBXL19-mediated ubiquitination and degradation of the receptor for IL-33 limits pulmonary inflammation.

The ST2L receptor for interleukin 33 (IL-33) mediates pulmonary inflammation and immune system-related disorders, such as asthma and rheumatoid arthritis. At present, very little is known about the molecular regulation of ST2L expression. Here we found that FBXL19, an 'orphan' member of the Skp1-Cullin-F-box family of E3 ubiquitin ligases, selectively bound to ST2L to mediate its polyubiquitination and elimination in the proteasome. Degradation of ST2L involved phosphorylation of ST2L at Ser442 catalyzed by the kinase GSK3ß. Overexpression of FBXL19 abrogated the proapoptotic and inflammatory effects of IL-33 and lessened the severity of lung injury in mouse models of pneumonia. Our results suggest that modulation of the IL-33-ST2L axis by ubiquitin ligases might serve as a unique strategy for lessening pulmonary inflammation.