A Novel in Vitro Assay for Deubiquitination of IκBα

Strayhorn, W. David; Wadzinski, Brian E.

doi:10.1006/abbi.2002.2760

Cited by 24 publications

(21 citation statements)

References 47 publications

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“…The in vitro deubiquitination assay was performed according to published methods (29,30). Flag-tagged USP47CD or USP47CDm (5 g) was transfected into HEK293T cells in 10-cm plates for 48 h. Cell lysates were immunoprecipitated with M2 (anti-Flag antibody [Ab]-conjugated) beads and eluted with 100 l glycine buffer, pH 3.5, and then neutralized with Tris-HCl buffer, pH 8.0.…”

Section: Methodsmentioning

confidence: 99%

Deubiquitinase USP47/UBP64E Regulates β-Catenin Ubiquitination and Degradation and Plays a Positive Role in Wnt Signaling

Shi

Liu

et al. 2015

Molecular and Cellular Biology

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Wnt signaling plays important roles in development and tumorigenesis. A central question about the Wnt pathway is the regulation of ␤-catenin. Phosphorylation of ␤-catenin by CK1␣ and GSK3 promotes ␤-catenin binding to ␤-TrCP, leading to ␤-catenin degradation through the proteasome. The phosphorylation and ubiquitination of ␤-catenin have been well characterized; however, it is unknown whether and how a deubiquitinase is involved. In this study, by screening RNA interference (RNAi) libraries, we identified USP47 as a deubiquitinase that prevents ␤-catenin ubiquitination. Inactivation of USP47 by RNAi increased ␤-catenin ubiquitination, attenuated Wnt signaling, and repressed cancer cell growth. Furthermore, USP47 deubiquitinates itself, whereas ␤-TrCP promotes USP47 ubiquitination through interaction with an atypical motif in USP47. Finally, in vivo studies in the Drosophila wing suggest that UBP64E, the USP47 counterpart in Drosophila, is required for Armadillo stabilization and plays a positive role in regulating Wnt target gene expression. Wnt/␤-catenin signaling plays an essential role in animal development and tumorigenesis (1, 2). In normal cells, ␤-catenin is sequentially phosphorylated by CKI␣ and GSK-3 in a protein complex containing the tumor suppressor proteins axin and APC (3). Phosphorylated ␤-catenin is recognized by the ubiquitin (Ub) ligase ␤-TrCP, an F-box and WD40 repeat protein (4). The WD40 repeat domain of ␤-TrCP binds ␤-catenin. ␤-TrCP also binds, via its F-box, to components of the ubiquitination machinery, including Skp1, cullin 1, ring box protein 1 (Rbx1), and ubiquitin-conjugating enzyme (E2). Ubiquitinated ␤-catenin is degraded via the 26S proteasome (4-7). Upon Wnt stimulation, the Wnt protein binds its receptor, Frizzled, and coreceptor, LRP5/6; disrupts the axin complex; blocks the phosphorylation required for ␤-TrCP-mediated ubiquitination; and thus stabilizes ␤-catenin (8-10). Wnt further promotes the accumulated ␤-catenin entry into the nucleus; binding of TCF/LEF; and recruitment of transcriptional coactivators, such as Bcl9/legless, Pygopus, and CBP/p300, to activate downstream target genes (2, 11).In human cancers, particularly in colorectal cancer, ␤-catenin is stabilized by mutations in APC or ␤-catenin (12, 13). ␤-Catenin mutations prevent CKI␣ or GSK-3 phosphorylation and ␤-TrCP recognition of ␤-catenin, resulting in abnormal ␤-catenin accumulation that ultimately leads to cancer (3, 4). APC truncations inhibit both phosphorylation and ubiquitination of ␤-catenin, also resulting in ␤-catenin accumulation (14-16). Understanding the molecular mechanisms of ␤-catenin ubiquitination/degradation is essential for developing therapeutic agents targeting this important pathway for cancer prevention and therapeutics (17).The kinases and ubiquitin protein ligase for ␤-catenin phosphorylation and ubiquitination have been well studied. Phosphatase 2A (PP2A) and its regulatory subunit, PR55␣, directly interact with and inhibit ␤-catenin phosphorylation (18). Whether ␤-catenin ubiquiti...

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Section: Methodsmentioning

confidence: 99%

Deubiquitinase USP47/UBP64E Regulates β-Catenin Ubiquitination and Degradation and Plays a Positive Role in Wnt Signaling

Shi

Liu

et al. 2015

Molecular and Cellular Biology

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“…The assay for deubiquitination was carried out as described previously (26). To prepare polyubiquitinated HIF-1a, MCF-7 cells were transfected with HA-ubiquitin using LipofectAMINE 2000 (Invitrogen) and left to recover overnight.…”

Section: Western Blottingmentioning

confidence: 99%

Molecular mechanisms for the activity of PX-478, an antitumor inhibitor of the hypoxia-inducible factor-1α

Koh

Spivak-Kroizman

Venturini

et al. 2008

Molecular Cancer Therapeutics

267

188

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We have reported previously that PX-478 (S-2-amino-3-[4 ¶-N,N,-bis(chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride) has potent antitumor activity against a variety of human tumor xenografts associated with the levels of the hypoxia-inducible factor-1A (HIF-1A) within the tumor. We now report that PX-478 inhibits HIF-1A protein levels and transactivation in a variety of cancer cell lines. Hypoxia-induced vascular endothelial growth factor formation was inhibited by PX-478, whereas baseline levels of vascular endothelial growth factor in normoxia were unaffected. Studies of the mechanism of PX-478 action showed that HIF-1A inhibition occurs in both normoxia and hypoxia and does not require pVHL or p53. In addition, PX-478 decreases levels of HIF-1A mRNA and inhibits translation as determined by 35 S labeling experiments and reporter assays using the 5 ¶ untranslated region of HIF-1A. Moreover, to a lesser extent, PX-478 also inhibits HIF-1A deubiquitination resulting in increased levels of polyubiquitinated HIF-1A. The inhibitory effect of PX-478 on HIF-1A levels is primarily due to its inhibition of translation because HIF-1A translation continues in hypoxia when translation of most proteins is decreased. We conclude that PX-478 inhibits HIF-1A at multiple levels that together or individually may contribute to its antitumor activity against HIF-1A-expressing tumors. [Mol Cancer Ther 2008;7(1):90 -100]

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“…Plasmids FlagFwd1, flag-IKK1, HA-Ub, and T7-IB␣ were gifts from Dr. B.E. Wadzinski (Vanderbilt University Medical Center, Nashville, TN 15 ). All transfections were performed in 60-mm dishes with 8-g total DNA that included 1 g of HA-ubiquitin, 1 g of pcDNA3 Flag-FWD1, 1 g of flag-IKK1, and 5 g of pCMV4 T7-IB␣.…”

Section: Transfections and Immunoprecipitationsmentioning

confidence: 99%

Salmonella Effector AvrA Regulation of Colonic Epithelial Cell Inflammation by Deubiquitination

Petrof

Boone

et al. 2007

The American Journal of Pathology

191

231

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AvrA is a newly described bacterial effector existing in Salmonella. Here, we test the hypothesis that AvrA is a deubiquitinase that removes ubiquitin from two inhibitors of the nuclear factor-B (NF-B) pathway, IB␣ and ␤-catenin, thereby inhibiting the inflammatory responses of the host. The role of AvrA was assessed in intestinal epithelial cell models and in mouse models infected with AvrA-deficient and -sufficient Salmonella strains. We also purified AvrA and AvrA mutant proteins and characterized their deubiquitinase activity in a cellfree system. We investigated target gene and inflammatory cytokine expression, as well as effects on epithelial cell proliferation and apoptosis induced by AvrA-deficient and -sufficient bacterial strains in vivo. Our results show that AvrA blocks degradation of IB␣ and ␤-catenin in epithelial cells. AvrA deubiquitinates IB␣, which blocks its degradation and leads to the inhibition of NF-B activation. Target genes of the NF-B pathway, such as interleukin-6, were correspondingly down-regulated during bacterial infection with Salmonella expressing AvrA. AvrA also deubiquitinates and thus blocks degradation of ␤-catenin. Target genes of the ␤-catenin pathway, such as c-myc and cyclinD1, were correspondingly up-regulated with AvrA expression. Increased ␤-catenin further negatively regulates the NF-B pathway. Our findings suggest an important role for AvrA in regulating host inflammatory responses through NF-B and ␤-catenin pathways. (Am J Pathol

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A Novel in Vitro Assay for Deubiquitination of IκBα

Cited by 24 publications

References 47 publications

Deubiquitinase USP47/UBP64E Regulates β-Catenin Ubiquitination and Degradation and Plays a Positive Role in Wnt Signaling

Deubiquitinase USP47/UBP64E Regulates β-Catenin Ubiquitination and Degradation and Plays a Positive Role in Wnt Signaling

Molecular mechanisms for the activity of PX-478, an antitumor inhibitor of the hypoxia-inducible factor-1α

Salmonella Effector AvrA Regulation of Colonic Epithelial Cell Inflammation by Deubiquitination

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