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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.
J Biol Chem. 1995 Dec 01; 270(48):28557-64.JB

Abstract

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.

Authors+Show Affiliations

Department of Biochemistry, Trinity College, Dublin, Ireland.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

7499370

Citation

Mahon, T M., and L A. O'Neill. "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 Journal of Biological Chemistry, vol. 270, no. 48, 1995, pp. 28557-64.
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. J Biol Chem. 1995;270(48):28557-64.
Mahon, T. M., & O'Neill, L. A. (1995). 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 Journal of Biological Chemistry, 270(48), 28557-64.
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. J Biol Chem. 1995 Dec 1;270(48):28557-64. PubMed PMID: 7499370.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Mahon,T M, AU - O'Neill,L A, PY - 1995/12/1/pubmed PY - 1995/12/1/medline PY - 1995/12/1/entrez SP - 28557 EP - 64 JF - The Journal of biological chemistry JO - J Biol Chem VL - 270 IS - 48 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/7499370/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_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(18)87805-X DB - PRIME DP - Unbound Medicine ER -