title: Labeling of v-Src and BCR-ABL tyrosine kinases with [14C]herbimycin A and its use in the elucidation of the kinase inactivation mechanism.
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.
authors.affiliation: Department of Bioactive Molecules, National Institute of Health, Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Fukazawa
authors.forename: Y
authors.initials: Y
authors.lastname: Uehara
authors.forename: Y
authors.initials: Y
authors.lastname: Murakami
authors.forename: S
authors.initials: S
authors.lastname: Mizuno
authors.forename: M
authors.initials: M
authors.lastname: Hamada
authors.forename: T
authors.initials: T
authors.lastname: Takeuchi
mesh_terms: D016475:3T3 Cells; D000345:Affinity Labels; D000818:Animals; D016227:Benzoquinones; D002250:Carbon Radioisotopes; D004789:Enzyme Activation; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism*; D047029:Lactams, Macrocyclic; D051379:Mice; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011809:Quinones; D017828:Rifabutin / Q000031:analogs & derivatives
chemical_list: D000345:Affinity Labels; D016227:Benzoquinones; D002250:Carbon Radioisotopes; D047029:Lactams, Macrocyclic; D011809:Quinones; D017828:Rifabutin; C020754:herbimycin; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; D015688:Oncogene Protein pp60(v-src)