Wei, S

Wei, S. in the ubiquitin-proteasome system. FBXL19 can also be acetylated, and enhancing acetylation of FBXL19 by a deacetylase inhibitor reduces FBXL19 ubiquitination levels. Acetylation-mimic FBXL19 mutant exhibits a longer half-life than wild type. An acetyltransferase CBP catalyzes acetylation of FBXL19. Inhibition or down-regulation of CBP reduces FBXL19 stability, whereas it is Astragaloside II increased in CBP-overexpressing cells. Taken together, the data indicate that CBP-mediated acetylation reduces ubiquitination and stabilizes FBXL19. Further, we demonstrate that FBXL19 Astragaloside II targets small GTPase Cdc42 for its ubiquitination and degradation, whereas this effect is reversed by inhibition of CBP, suggesting that CBP increases the effect of SCF FBXL19 E3 ligase through acetylation and stabilization of FBXL19. Our study reveals a new molecular model for regulation of SCF E3 ligase function by acetylation and stabilization of its subunit F-box protein.Wei, J., Dong, S., Yao, K., Martinez, M. F. Y. M., Fleisher, P. R., Zhao, Y., Ma, H., Zhao, J. Histone acetyltransferase CBP promotes function of SCF FBXL19 ubiquitin E3 ligase Astragaloside II by acetylation and stabilization of its F-box protein subunit. its F-box domain and substrate binding motif, respectively (5C7). Although the biological roles of F-box proteins in many cellular responses have been demonstrated, the regulation of F-box protein stability remains unclear. Increasing evidence suggests that F-box proteins are autoubiquinated (8C10) or ubiquitinated by other ubiquitin E3 ligases (11), thus leading to their degradation in the proteasome system. Galan and Peter (8) revealed that 3 F-box proteins (Grr1p, Cdc4p, and Met30p) were degraded in the proteasome by an autocatalytic mechanism. Chen (11) and Liu (20) demonstrated that acetylation reduces p53 ubiquitination through competing for the same lysine residues at the C terminus of p53. Protein acetylation is catalyzed by acetyltransferases, such as P300, CBP, and PCAF. P300 stabilizes Smad7 (21) and Skp2 (22) by inducing their ubiquitination. CBP/P300-mediated acetylation stabilizes p53 (20). We have shown that Astragaloside II CBP is a substrate of SCF FBXL19 E3 ligase (14). Overexpression of FBXL19 reduces CBP stability and mitigates CBP-mediated histone acetylation (14). In this study, Astragaloside II we demonstrate that FBXL19 is unstable and is degraded in the ubiquitin-proteasome system. FBXL19 stability is increased by CBP-mediated acetylation. Further, we show that FBXL19 targets Cdc42 for its degradation. This study reveals a new molecular mechanism by which a substrate stabilizes the F-box protein post-translational modification. MATERIALS AND METHODS Cells and reagents Murine lung epithelial (MLE)12 cells [American Type Culture Collection (ATCC), Manassas, MA, USA] were cultured with medium supplemented with hydrocortisone, insulin, transferrin, estrogen, selenium; 10% fetal bovine serum; and antibiotics at 37C in a 5% CO2 incubator. A549 cells (ATCC) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum and antibiotics. V5 antibody, mammalian expressional plasmid pcDNA3.1/His-V5-topo, and Top10 competent cells were from Thermo Fisher Scientific, Waltham, MA, USA. Antibodies against acetylated lysine and ubiquitin were from Cell Signaling Technology (Danvers, MA, USA). FBXL19 antibody was purchased from Abgent (San Diego, CA, USA). Cycloheximide, leupeptin, wild type was inserted into pCDNA3.1/V5-His-Topo vector as described elsewhere (13). Site-directed mutagenesis was performed to generate FBXL19 mutants according to the manufacturers instructions (Agilent Technologies, Santa Clara, CA, USA). Overexpression of plasmids or cellular transfection of shRNA in MLE12 cells was performed using the Lonza Nucleofector System according to the manufacturers protocol. Immunoblotting and immunoprecipitation Cells grown on 6-well plates or 100-mm dishes PRKM8IPL were washed with cold PBS, collected in the cell lysis buffer, and subjected to sonication. An equal amount of cell lysates (20 g) was subjected to 10% SDS-PAGE, electrotransferred to membranes, and immunoblotted as standard protocol. For immunoprecipitation, equal amounts of cell lysates (1 mg) were incubated with an antibody overnight at 4C followed by the addition of 40 l of protein A/G agarose and incubation for 2 h at 4C. The immunoprecipitated complex was washed 3 times with 1% Triton X-100 in ice-cold PBS and analyzed by immunoblotting. ubiquitin assay A modified immunoprecipitation (IP) protocol under denaturing conditions was performed. After cells were treated under the indicated conditions, cells were washed and collected with cold PBS. After centrifuging at 1000 rpm for 5 min, the supernatant was removed, and 50C80 l of 2% SDS lysis buffer containing 1 l of ubiquitin aldehyde and 1 l of test. A value of 0.05 was considered statistically significant. RESULTS FBXL19 degradation is mediated in the ubiquitin-proteasome system F-box is a component of SCF E3 ligase, which plays a critical role in the recognition of specific substrates (6). Our previous.