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 Letters

Hamada, Masa¹  Murakami, Yuko²  Uehara, Yoshimasa²  Fukazawa, Hidesuke²  Takeuchi, Tomio¹  Mizuno, Satoshi² 

[1] Institute of Microbial Chemistry 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141, Japan;Department of Bioactive Molecules, National Institute of Health 1-23-1 Toyama, Shinjuku-ku, Tokyo 162, Japan

关键词: Tyrosine kinase;    Herbimycin A;    FSBA;    fluorosulfonylbenzoyl adenosine;    HEPES;    N-(hydroxyethyl)piperazine-N'-2-ethanesulfonic acid;    EDTA;    ethylene glycol-bis(β-aminoethyl ether)N;    N;    N';    N'-tetraacetic acid;    DMSO;    dimethyl sulfoxide;    SDS-PAGE;    sodium dodecyi sulfate-polyacrylamide gel electrophoresis;   

DOI  :  10.1016/0014-5793(94)80127-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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. p60 ^v-src and p2 10 ^BCR-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 p60 ^v-src with Staphylococcus taureus 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 [¹⁴]C]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.

【 授权许可】

Unknown