Identification of an ubiquitin-ligation system for the epidermal-growth-factor receptor--herbimycin A induces in vitro ubiquitination in rabbit-reticulocyte lysate.
Some receptor tyrosine kinases such as the receptors for epidermal-growth factor (EGF) and platelet-derived growth factor undergo polyubiquitination as a consequence of ligand binding. The EGF receptor is also ubiquitinated by treatment with herbimycin A, an ansamycin antibiotic widely used as a tyrosine kinase inhibitor. To investigate the mechanism of the receptor ubiquitination, we have established an assay system in which herbimycin-A-induced ubiquitination processes can be analyzed in vitro. We now show that herbimycin A treatment of the purified EGF receptor induces polyubiquitination of the receptor in rabbit-reticulocyte lysate. Both DEAE unadsorbed material (fraction I) and high salt eluate (fraction II) of the reticulocyte lysate are involved cooperatively in the ubiquitination process, where the ubiquitin-conjugating enzyme UBC4 can functionally substitute for fraction I. A ubiquitin-protein ligase-like activity, partially purified from fraction II by DEAE anion-exchange chromatography, also functions in concert with UBC4. The precise mechanism of herbimycin A-induced ubiquitination of the EGF receptor is not fully understood, however, our present findings suggest that direct interaction with herbimycin A results in some modification of the receptor which is recognized by the ubiquitin-conjugating system in rabbit-reticulocyte lysate.
Mori
S
S
Second Department of Internal Medicine, Chiba University School of Medicine, Japan. seimori@intmed02.m.chiba-u.ac.jp
Tanaka
K
K
Kanaki
H
H
Nakao
M
M
Anan
T
T
Yokote
K
K
Tamura
K
K
Saito
Y
Y
D000818:Animals; D016227:Benzoquinones; D002848:Chromatography, DEAE-Cellulose; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D047029:Lactams, Macrocyclic; D011809:Quinones / Q000494:pharmacology*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D017828:Rifabutin / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000378:metabolism*
D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D011809:Quinones; D014452:Ubiquitins; D017828:Rifabutin; C020754:herbimycin; D066246:ErbB Receptors