Pervanadate-induced nuclear factor-kappaB activation requires tyrosine phosphorylation and degradation of IkappaBalpha. Comparison with tumor necrosis factor-alpha.
ABSTRACT Tumor necrosis factor activates nuclear transcription factor kappaB (NF-kappaB) by inducing serine phosphorylation of the inhibitory subunit of NF-kappaB (IkappaBalpha), which leads to its ubiquitination and degradation. In contrast, pervanadate (PV) activates NF-kappaB and induces tyrosine phosphorylation of IkappaBalpha (Singh, S., Darney, B. G., and Aggarwal, B. B. (1996) J. Biol. Chem. 271, 31049-31054; Imbert, V., Rupec, R. A., Antonia, L., Pahl, H. L., Traenckner, E. B.-M., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auderger, P., Baeuerle, P. A., and Peyron, J.-F. (1996) Cell 86, 787-798). Whether PV also induces IkappaBalpha degradation and whether degradation is required for NF-kappaB activation are not understood. We investigated the effect of PV-induced tyrosine phosphorylation on IkappaBalpha degradation and NF-kappaB activation. PV activated NF-kappaB, as determined by DNA binding, NF-kappaB-dependent reporter gene expression, and phosphorylation and degradation of IkappaBalpha. Maximum degradation of IkappaBalpha occurred at 180 min, followed by NF-kappaB-dependent IkappaBalpha resynthesis. N-Acetylleucylleucylnorlucinal, a proteasome inhibitor, blocked both IkappaBalpha degradation and NF-kappaB activation, suggesting that the IkappaBalpha degradation is required for NF-kappaB activation. PV did not induce serine phosphorylation of IkappaBalpha but induced phosphorylation at tyrosine residue 42. Unlike tumor necrosis factor (TNF), PV did not induce ubiquitination of IkappaBalpha. Like TNF, however, PV induced phosphorylation and degradation of IkappaBalpha, and subsequent NF-kappaB activation, which could be blocked by N-tosyl-L-phenylalanine chloromethyl ketone, calpeptin, and pyrrolidine dithiocarbomate, suggesting a close link between PV-induced NF-kappaB activation and IkappaBalpha degradation. Overall, our studies demonstrate that PV activates NF-kappaB, which, unlike TNF, requires tyrosine phosphorylation of IkappaBalpha and its degradation.
- SourceAvailable from: Maria Ibarra-Sanchez[Show abstract] [Hide abstract]
ABSTRACT: NF-κB transcription factor has been associated with cancer development and chemoresistance. We studied the signaling pathway activated by doxorubicin (DOX) leading to NF-κB activation in breast cancer cells. NF-κB activity was evaluated by electrophoretic mobility shift in T47D, ZR75.30 and primary culture (MBCDF) from a ductal infiltrating carcinoma. Cell viability was measured by crystal violet. Western blotting was performed to check the expression and phosphorylation of IκBα Ser-32/36. c-Abl was inhibited with Imatinib or by overexpressing a dominant negative form of c-Abl (K290R). We found a correlation between sensitivity to DOX and amplitude of NF-κB activation. In cells least sensitive to DOX, NF-κB remained activated for longer time (T47D and MBCDF). The opposite effect was observed in cells sensitive to DOX (ZR75.30). DOX did not induce IκBα degradation or Ser-32/36 phosphorylation. Instead, there were modifications in the levels of IκBα tyrosine phosphorylation, suggesting an atypical NF-κB activation. In DOX-resistant cells, Imatinib treatment reduced IκBα tyrosine phosphorylation and NF-κB activity. The Imatinib-DOX combination significantly enhanced cell death of T47D and MBCDF breast cancer cells. Overexpression of c-Abl K290R in T47D and MBCDF cells reduced basal and DOX-induced NF-κB activation as well as IκBα tyrosine phosphorylation. In c-Abl K290R cells, DOX treatment did not mimic the combination Imatinib-DOX-induced cell death. Inhibition of c-Abl inactivated IκBα/NF-κB pathway is associated with IκBα tyrosine phosphorylation in breast cancer cells. These results also raise the potential use of a combined therapy with Imatinib and DOX for breast cancer patients.Journal of Cancer Research and Clinical Oncology 07/2013; · 2.91 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: PSMD9 is a PDZ-domain containing chaperone of proteasome assembly. Based on the ability of PDZ-like domains to recognize C-terminal residues in its interactors, we recently predicted and identified hnRNPA1 as one of the novel interacting partners of PSMD9. Contingent on the reported role of hnRNPA1 in NF-κB activation, we tested the role of human PSMD9 and hnRNPA1 in NF-κB signaling. We demonstrated in HEK293 cells that, PSMD9 influences both basal and TNF-α mediated NF-κB activation through IκBα proteasomal degradation. PSMD9 mediates the degradation through specific domain-motif interaction involving its PDZ-domain and a Short Linear Sequence Motif in C-terminus of hnRNPA1. Point mutations in the PDZ domain or deletion of C-terminal residues in hnRNPA1 disrupt interaction between the two proteins which has a direct influence on NF-κB activity. hnRNPA1 interacts with IκBα directly, whereas PSMD9 interacts only through hnRNPA1. Furthermore hnRNPA1 shows increased association with the proteasome upon TNF-α treatment which however has no such effect in the absence of PSMD9. On the other hand endogenous and trans-expressed PSMD9 are found associated with the proteasome complex. This association is unaffected by PDZ-mutations or TNF-α treatment. Collectively, these interactions between IκBα, hnRNPA1 and proteasome bound PSMD9 illustrates a potential mechanism by which ubiqutinated IκBα are recruited on proteasome for degradation. In this process, hnRNPA1 may act as a shuttle receptor and PSMD9 as a subunit acceptor. The interaction sites of PSMD9 and hnRNPA1 may emerge as a vulnerable drug target in cancer cells which require consistent NF-κB activity for survival.This article is protected by copyright. All rights reserved.FEBS Journal 04/2014; · 3.99 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: NF-κB regulates liver cell death during development, regeneration, and neoplastic transformation. For example, we showed that oncogenic Ras- or Raf-mediated transformation of rat liver epithelial cells (RLEs) led to altered NF-κB regulation through IKK complex activation, which rendered these cells more resistant to TGF-β1-induced apoptosis. Thus, based on these findings, we sought to determine whether NF-κB could also be involved in tumor growth of liver cells in vivo. Hepatocellular carcinomas (HCCs) derived from bitransgenic mice harboring TGF-α and c-myc transgenes targeted specifically to the liver were compared with HCCs from c-myc single transgenic mice. Tumors from bitransgenic mice are characterized by a higher frequency of appearance, lower apoptotic index, and a higher rate of cell proliferation. Here we show that NF-κB is activated in HCCs of double TGF-α/c-myc transgenic mice, but not of c-myc single transgenic mice, suggesting that TGF-α mediates induction of NF-κB. Activation of the IKK complex was observed in the HCCs of double TGF-α/c-myc transgenic mice, implicating this pathway in NF-κB induction. Lastly, activation of the Akt/protein kinase B (PKB), which has recently been implicated in NF-κB activation by PDGF, TNF-α, and Ras, was also observed. Importantly, human HCC cell lines similarly displayed NF-κB activation. Thus, these studies elucidate an anti-apoptotic mechanism by a TGF-α-Akt/PKB-IKK pathway, which likely contributes to survival and proliferation, thereby accelerating c-myc-induced liver neoplastic development in vivo. (HEPATOLOGY 2001;34:32-41.)Hepatology 01/2001; 34(1):32-41. · 11.19 Impact Factor