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-κB 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 IκB-α degradation, and in unstimulated cells, it modified NF-κB prior to chemical dissociation with sodium deoxycholate. Because herbimycin A is thiol-reactive, we suspected that the target was the p50 subunit of NF-κB, 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-κB 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-κB-regulated gene) in EL4.NOB-1 cells. These data therefore suggest that herbimycin A inhibits NF-κB by modifying the p50 subunit on cysteine 62 in the NF-κB complex, which blocks DNA binding and NF-κB-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-κB-dependent gene transcription.