LABELING OF V-SRC AND BCR-ABL TYROSINE KINASES WITH [C-14] HERBIMYCIN-A AND ITS USE IN THE ELUCIDATION OF THE KINASE INACTIVATION MECHANISM

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 [C-14]herbimycin A. p60(v-src) and p210(BCR-ABL) in immune complexes were labeled with [C-14]herbimycin A, demonstrating that the antibiotic binds directly to tyrosine kinases. Digestion of [C-14]herbimycin A-labeled p60(v-src) with Staphylococcus aureus V8 protease revealed that the herbimycin A binding site is within the C-terminal 26-kDa fragment of p60(v-src), which contains the tyrosine kinase domain. Herbimycin A treatment inhibited labeling of p60(v-src) by [C-14]fluorosulfonylbenzoyl adenosine, an affinity labeling reagent of nucleotide binding sites, indicating that herbimycin A-modified p60(v-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.