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.
"As with any inhibitor study, the specificity of the inhibitors is key to interpreting results. Of the inhibitors used in the present study, orthovanadate (Gordon, 1991) and peroxyvanadate (Ruff et al., 1997; Mukhopadhyay et al., 2000; Touyz et al., 2001; Shimizu et al., 2001; Mariner et al., 2001) are probably the most specific and have been used extensively for in vivo studies. Calpeptin has recently been identified as a specific PTPase inhibitor (Schoenwaelder and Burridge, 1999; Whitehouse et al., 2000) suitable for use with intact cells. "
[Show abstract][Hide abstract] ABSTRACT: Fertilization involves the activation of Src-family protein kinases which play a role at multiple stages of the egg activation process. The objective of the present study was to determine the mechanism by which one of these kinases, the Fyn kinase, is activated in response to fertilization of the zebrafish egg. Inhibitor studies demonstrated that many aspects of egg activation, including Fyn activation, require phosphotyrosyl phosphatase activity. A phosphotyrosyl phosphatase was found to be tightly associated with Fyn kinase and this interaction was mapped to the SH2 domain of Fyn. Coimmunoprecipitation studies identified rPTPalpha as a phosphatase that is complexed with Fyn in the egg, raising the possibility that rPTPalpha is part of the regulatory mechanism responsible for activating Fyn at fertilization.
[Show abstract][Hide abstract] ABSTRACT: Background: Resveratrol (a naturally occurring phytoalexin found in grapes and wine) has cardiovascular protective effects that suggest the antiatherogenic (ie, antiinflammatory) activities of the compound on endothelial cells. Objective: The antiinflammatory activity of resveratrol could be mediated by its interference with nuclear factor-� B (NF-� B)-dependent transcription. Thus, we studied the in vitro influence of physiologic concentrations of resveratrol (≤ 1 � mol/L) on the NF-� B signaling pathway after tumor necrosis factor � (TNF-� ) stimulation of endothelial cells. Design: The effects of a 30-min (acute) and an overnight incu- bation of resveratrol on the nuclear appearance of p50-NF-� B and p65-NF-� B on serine and tyrosine phosphorylation of the inhibitory subunitB � (IB� ), cytoplasmic concentrations of IB� , NF-� B phosphorylation or nitrosylation, the reduction of the mitotic inhibitor p21, and the activation of peroxisome proliferator-activated receptorwere evaluated. Results: The nuclear appearance of p50-NFB and p65-NFB acutely induced by TNF-� was not modified by resveratrol but was increased after overnight incubation with resveratrol alone or in combination with TNF-� . Acute treatment with resveratrol did not modify TNF-� -induced cytoplasmic IBserine phos- phorylation but did increase IBtyrosine phyophorylation. Resveratrol increased the tyrosine phosphorylation (but not nitro- sylation) of immunoprecipitated NF-� B, did not decrease cellu- lar p21, and did not increase peroxisome proliferator-activated receptoractivity. Conclusions: Acute resveratrol treatment does not inhibit the nuclear appearance of NF-� B in human umbilical vein endothelial cells, but overnight treatment does. The increase in tyrosine phos- phorylation of IB� , p50-NF-� B, and p65-NF-� B suggests the involvement of such alterations in the modulation of NF-� B tran- scription activity. Am J Clin Nutr 2003;77:1220-8.
[Show abstract][Hide abstract] ABSTRACT: More than 70 separate genes have now been identified, mutation of which underlie various immunodeficiency syndromes and the dissection of the function of these genes provides outstanding examples of the power of molecular medicine. In this chapter, we will focus on cytokines whose receptors comprise the common gamma chain, gc, which signal through the protein tyrosine kinase, Jak3.
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