Studies into the effect of the tyrosine kinase inhibitor herbimycin A on NF-kappa B activation in T lymphocytes. Evidence for covalent modification of the p50 subunit.
The tyrosine kinase inhibitor herbimycin A was found to block NF-kappa 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 I kappa B-alpha degradation, and in unstimulated cells, it modified NF-kappa 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-kappa 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-kappa 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-kappa B-regulated gene) in EL4.NOB-1 cells. These data therefore suggest that herbimycin A inhibits NF-kappa B by modifying the p50 subunit on cysteine 62 in the NF-kappa B complex, which blocks DNA binding and NF-kappa 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-kappa B-dependent gene transcription.
D000818:Animals; D001483:Base Sequence; D016227:Benzoquinones; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007375:Interleukin-1 / Q000494:pharmacology; D007376:Interleukin-2 / Q000096:biosynthesis; D047029:Lactams, Macrocyclic; D016399:Lymphoma, T-Cell / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology*; D017828:Rifabutin / Q000031:analogs & derivatives; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013945:Thymoma / Q000473:pathology; D013953:Thymus Neoplasms / Q000473:pathology; D051841:Transcription Factor RelB; D014157:Transcription Factors; D014407:Tumor Cells, Cultured
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D007376:Interleukin-2; D047029:Lactams, Macrocyclic; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D011809:Quinones; C497594:Relb protein, mouse; D014157:Transcription Factors; D051841:Transcription Factor RelB; D017828:Rifabutin; C020754:herbimycin; D011505:Protein-Tyrosine Kinases; D013755:Tetradecanoylphorbol Acetate