Studies into the Effect of the Tyrosine Kinase Inhibitor Herbimycin A on NF-κB Activation in T Lymphocytes EVIDENCE FOR COVALENT MODIFICATION OF THE p50 SUBUNIT

Abstract

The tyrosine kinase inhibitor herbimycin A was found to block NF-B stimulation in response to interleukin-1 and phorbol 12-myristate 13-acetate in EL4.NOB-1 thymoma cells and phorbol 12-myristate 13-acetate in Jurkat T lymphoma cells. The effect appeared not to involve inhibition of tyrosine kinase activation as neither interleukin-1 nor phorbol 12-myristate 13-acetate induced major changes in tyrosine phosphorylation in EL4.NOB-1 or Jurkat cells, respectively. Herbimycin A did not interfere with IB-␣ degradation, and in unstimulated cells, it modified NF-B prior to chemical dissociation with sodium deoxycholate. Because herbimycin A is thiol-reactive, we suspected that the target was the p50 subunit of NF-B, which has a key thiol at cysteine 62. Herbimycin A inhibited DNA binding when added to nuclear extracts prepared from stimulated cells, which were shown to contain high levels of p50. Incubation of herbimycin A with 2-mercaptoethanol attenuated the effect. Herbimycin A was also shown to react directly with p50, blocking its ability to bind to the NF-B consensus sequence. However, a mutant form of p50 in which cysteine 62 was mutated to serine was insensitive to herbimycin A. Finally, we demonstrated that the compound inhibited the expression of interleukin-2 (an NF-B-regulated gene) in EL4.NOB-1 cells. These data therefore suggest that herbimycin A inhibits NF-B by modifying the p50 subunit on cysteine 62 in the NF-B complex, which blocks DNA binding and NF-B-driven gene expression. The results urge caution in the use of herbimycin A as a specific tyrosine kinase inhibitor and suggest that the development of agents that selectively modify p50 may have potential as a means of inhibiting NF-B-dependent gene transcription.

Vitamin C Inhibits NF-κB Activation by TNF Via the Activation of p38 Mitogen-Activated Protein Kinase

“…To explore this possibility for vitamin C, we incubated nuclear extracts from TNF-stimulated cells with vitamin C for 2 h at 37°C. The profile of NF-B detected in crude cytosolic extracts, and in the presence of detergent differed somewhat from that detected in nuclear extracts, in that an extra lower band seemed to be revealed This demonstrated that vitamin C was not altering the NF-B/I-B complex in a manner that would render it insensitive to stimulantinduced dissociation, as has been shown for some NF-B inhibitors (33).…”

“…2A shows that membrane integrity was not affected because there was no increase in LDH release in response to up to 100 mM vitamin C in ECV304 cells over the 2-h time course of the experiments shown in Fig. 1, A Some inhibitors of NF-B have been shown to act by directly modifying NF-B and so interfere with its binding to DNA (33,34). To explore this possibility for vitamin C, we incubated nuclear extracts from TNF-stimulated cells with vitamin C for 2 h at 37°C.…”

Simvastatin Potentiates TNF-α-Induced Apoptosis through the Down-Regulation of NF-κB-Dependent Antiapoptotic Gene Products: Role of IκBα Kinase and TGF-β-Activated Kinase-1

“…Several agents have been described that suppress NF-B binding to the DNA, e.g., caffeic acid phenethyl ester (37), herbimycin (38), plumbagin (39), ethyl caffeate (40), and tosyl-L-phenylalanine chloromethyl ketone (41). Therefore, we investigated whether simvastatin also mediates its effects through a similar mechanism.…”

Celastrol, a novel triterpene, potentiates TNF-induced apoptosis and suppresses invasion of tumor cells by inhibiting NF-κB–regulated gene products and TAK1-mediated NF-κB activation

“…[42][43][44][45] We examined whether celastrol mediates its effect through a similar mechanism. EMSA showed that celastrol did not modify the DNA-binding ability of NF-B proteins prepared from TNF-treated cells ( Figure 4E).…”