Labeling of v-Src and BCR-ABL tyrosine kinases with [14C]herbimycin A and its use in the elucidation of the kinase inactivation mechanism

FEBS Lett. 1994 Mar 7;340(3):155-8. doi: 10.1016/0014-5793(94)80127-4.

Abstract

The ansamycin antibiotic, herbimycin A, selectively inactivates cytoplasmic tyrosine kinases, most likely by binding irreversibly to the reactive SH group(s) of kinases. To further investigate the mechanism of herbimycin A action, we attempted to label tyrosine kinases with [14C]herbimycin A. p60v-src and p210BCR-ABL in immune complexes were labeled with [14C]herbimycin A, demonstrating that the antibiotic binds directly to tyrosine kinases. Digestion of [14C]herbimycin A-labeled p60v-src with Staphylococcus aureus V8 protease revealed that the herbimycin A binding site is within the C-terminal 26-kDa fragment of p60v-src, which contains the tyrosine kinase domain. Herbimycin A treatment inhibited labeling of p60v-src by [14C]fluorosulfonylbenzoyl adenosine, an affinity labeling reagent of nucleotide binding sites, indicating that herbimycin A-modified p60v-src cannot interact with ATP. The results suggest that herbimycin A inactivates tyrosine kinases by binding directly to the kinase domain, thereby inhibiting access to ATP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Affinity Labels
  • Animals
  • Benzoquinones
  • Carbon Radioisotopes
  • Enzyme Activation
  • Fusion Proteins, bcr-abl / metabolism*
  • Lactams, Macrocyclic
  • Mice
  • Oncogene Protein pp60(v-src) / metabolism*
  • Protein-Tyrosine Kinases / antagonists & inhibitors*
  • Protein-Tyrosine Kinases / metabolism
  • Quinones*
  • Rifabutin / analogs & derivatives

Substances

  • Affinity Labels
  • Benzoquinones
  • Carbon Radioisotopes
  • Lactams, Macrocyclic
  • Quinones
  • Rifabutin
  • herbimycin
  • Protein-Tyrosine Kinases
  • Fusion Proteins, bcr-abl
  • Oncogene Protein pp60(v-src)