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 GSK3β. 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.

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.

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.

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Background: Cortactin is essential for barrier integrity. Results: ERK inhibition attenuates cortactin phosphorylation, ubiquitination, and degradation via the ␤-Trcp-mediated ubiquitin-proteasome system. Conclusion: Cortactin stability is coordinately regulated by stress kinases and the ubiquitin-proteasomal network. Significance: Understanding cortactin stability provides new targets to regulate epithelial barrier integrity.