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.
O'NEILL, LUKE ANTHONY JOHN
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Citation:Mahon TM, O'Neill LA. `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? in Journal of Biological Chemistry, 270, (48), 1995, pp 28557 - 28564
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.
Publisher:The American Society for Biochemistry and Molecular Biology
Series/Report no:Journal of Biological Chemistry