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

Abstract

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.

Keywords: Cell proliferation; Phosphorylation; Protein stability; Small GTPase protein; Stress fiber; Ubiquitin-proteasome system.

Figure 1. RhoA degrades in the proteasome system in lung epithelial cells

A. MLE12 cells were transfected with V5 tagged RhoA wild type (RhoA-V5), RhoA active form (RhoAV14-V5), or RhoA inactive form (RhoAN19-V5) plasmids for 48 h. Cells were treated with cycloheximide (CHX, 20 μg/ml) for indicated times and then cell lysates were analyzed for over-expressed RhoA and β-actin by immunoblotting with V5 tag and β-actin antibodies. B. RhoAV14-V5 over-expressed MLE12 cells were treated with 20 μg/ml of CHX with or without MG-132 (20 μM) or leupeptin (100 μM) for 2 h. Cell lysates were analyzed for RhoAV14-V5 and β-actin by immunoblotting with V5 tag and β-actin antibodies. C. RhoAN19-V5 over-expressed MLE12 cells were treated with 20 μg/ml of CHX with or without MG-132 (20 μM) or leupeptin (100 μM) for 2 h. Cell lysates were analyzed for RhoAN19-V5 and β-actin by immunoblotting with V5 tag and β-actin antibodies. Shown are representative blots from three independent experiments.

Figure 2. FBXL19 regulates RhoA stability

A. MLE12 cells were transfected with three distinct FBXL19 shRNA plasmids (#1 - #3) for 72 h. RhoA, FBXL19, and β-actin expression were analyzed by immunoblotting. B. MLE12 cells were co-transfected with FBXL19-V5 plasmid and RhoA-V5 or RhoAV14-V5, or RhoAN19-V5 plasmids. Cell lysates were analyzed for RhoA-V5 or RhoAV14-V5, RhoAN19-V5, FBXL19-V5, and β-actin by immunoblotting with V5 tag and β-actin antibodies. C. RhoAN19-V5 over-expressed MLE12 cells were co-transfected with vector alone, shFBXL19, or shFBXL19 + FBXL19-V5 plasmids and then cells were treated with CHX (20 μg/ml) for 0 - 4 h. Cell lysates were analyzed for RhoAN19-V5, FBXL19-V5, FBXL19, and β-actin by immunoblotting with V5 tag, FBXL19, and β-actin antibodies. Shown are representative blots from three independent experiments.

Figure 3. FBXL19 interacts with RhoA

A. MLE12 cells were co-transfected with RhoAN19-V5 and FBXL19-HA plasmids. Cell lysates were subjected to immunoprecipitation with HA tag, followed by V5 tag immunoblotting. Input lysates were analyzed by immunoblotting with V5 tag and HA tag antibodies. Shown are representative blots from three independent experiments. B. MLE12 cell lysates were subjected to immunoprecipitation with FBXL19 antibody, followed by RhoA immunoblotting. Input lysates were analyzed by immunoblotting with RhoA and β-actin antibodies. Shown are representative blots from three independent experiments. C. MLE12 cells grown on glass-bottom dishes were transfected with FBXL19-GFP plasmid (2 μg) for 48 h followed by MG-132 (20 μM, 18 h) treatment. Localization of FBXL19-GFP (green), RhoA (red), and nuclei (blue) were examined by immunofluorescence staining. Arrows show co-localization of FBXL19 and RhoA. Shown are representative images from three independent experiments. Scale bars represent 10 μm.

Figure 4. Lysine 135 within RhoA is the ubiquitin acceptor site for FBXL19

A. MLE12 cells were co-transfected with FBXL19-V5 and RhoAN19-V5 or lysine mutant plasmids. Cell lysates were analyzed for RhoAN19-V5, mutants, FBXL19-V5, and β-actin by immunoblotting with antibodies to V5 tag and β-actin. B. MLE12 cells were transfected with RhoAN19^K135R-V5 or RhoAN19^K140R-V5 plasmid and then cells were treated with CHX for 0, 2, and 4 h. Cell lysates were analyzed for V5 tagged RhoAN19 mutants and β-actin by immunoblotting with V5 tag and β-actin antibodies. C. FBXL19-HA, RhoAN19-V5, and RhoAN19^K135R–V5 were synthesized by a TnT system, and in vitro ubiquitinations were measured by incubation with E1, E2, ubiquitin, Cullin1, Skp1, and ATP, followed by V5 tag immunoblotting. Input lysates were analyzed for RhoAN19-V5, mutant, and FBXL19-HA by immunoblotting. Shown are representative blots from three independent experiments.

Figure 5. Erk2 regulates RhoA phosphorylation

A. MLE12 cells were transfected with Erk2-V5 plasmid for 48 h. Cell lysates were subjected to immunoprecipitation with a RhoA antibody. The precipitates were probed with phospho-serine, phospho-threonine, and RhoA antibodies. Input cell lysates were subjected to V5 tag and β-actin immunoblotting. B. MLE12 cells were transfected with shCont or shErk2 plasmid for 72 h. Cell lysates were subjected to immunoprecipitation with a RhoA antibody. The precipitates were probed with phospho-serine and RhoA antibodies. Input cell lysates were subjected to Erk2 and β-actin immunoblotting. Shown are representative blots from three independent experiments.

Figure 6. Erk2 regulates RhoA stability

A. RhoAN19-V5 over-expressing MLE12 cells were pretreated with DMSO or PD98059 (10 μM, 1 h) prior to CHX treatment (20 μg/ml, 4 h) and then cell lysates were analyzed for RhoAN19-V5 and β-actin by immunoblotting with V5 tag and β-actin antibodies. B. MLE12 cells were co-transfected with RhoAN19-V5 and Erk2-V5 plasmids and then cell lysates were analyzed for RhoAN19-V5, Erk2-V5, and β-actin by immunoblotting with V5 tag and β-actin antibodies. C. MLE12 cells were transfected with Erk2-V5 plasmids (0 – 4 μg) and then cell lysates were analyzed for RhoA, Erk2-V5, and β-actin by immunoblotting with RhoA, V5 tag, and β-actin antibodies. Shown are representative blots from three independent experiments. D. MLE12 cells were transfected with FBXL19-V5 plasmid and then cells were treated with DMSO or PD98059 (5 μM, 16 h). Cell lysates were analyzed for RhoA, FBXL19-V5, and β-actin by immunoblotting with RhoA, V5 tag, and β-actin antibodies. Shown are representative blots from three independent experiments. E. FBXL19-V5 over-expressing MLE12 cells were pretreated with DMSO or PD98059 (5 μM, 16 h) prior to immunoprecipitation with ubiquitin antibody. The immunoprecipitated complex was analyzed for RhoA by immunoblotting. Input lystes were analyzed for RhoA, FBXL19-V5, and β-actin by immunoblotting with RhoA, V5 tag, and β-actin antibodies. Right panel is a light exposure image.

Figure 7. FBXL19 regulates cell proliferation

A. MLE12 cells were transfected with shCont and shFBXL19 plasmid for 72 h. Cell proliferation were measured. *p<0.01, compared to shCont. B. MLE12 cells were transfected with FBXL19-V5 plasmid and then cells were treated with 2 % of FBS for 30 min. Cell lysates were analyzed for FBXL19-V5, phospho-p27, and β-actin by immunoblotting with V5 tag, p-p27, and β-actin antibodies. C. MLE12 cells were co-transfected with RhoA-V5 and FBXL19-HA. Cell numbers were accounted for 0 – 3 days. D. MLE12 cells were transfected with shCont, shRhoA, RhoA-V5, or RhoA^K135R plasmids as indicted. Cells were treated with or without 2% of FBS for 48 h and cell proliferation were measured. Cell proliferation was normalized to the cells without FBS (veh). *p<0.01, compared to untransfected cells; **p<0.05, compared to shRhoA transfected cells.

Figure 8. FBXL19 regulates stress fiber formation

A. MLE12 cells were transfected with FBXL19-V5 plasmid and then cells were treated with LPA (5 μM) for 5 min. Cell lysates were analyzed for phospho-MLC, FBXL19-V5, and β-actin by immunoblotting with p-MLC, V5 tag, and β-actin antibodies. B. MLE12 cells were transfected with empty vector (a and b) or FBXL19-V5 plasmid (c-h) and then cells were treated with LPA (5 μM, 10 min) (b, f-h). Cells were fixed and actin filaments were stained with phalloidin (red), FBXL19-V5 was stained with a V5 tag antibody (green), and nuclei were stained with DAPI (blue). Shown are representative images from three independent experiments. Scale bar represents 2 μm.