Mori S, Tanaka K, Kanaki H, Nakao M, Anan T, Yokote K, Tamura K, Saito Y
Second Department of Internal Medicine, Chiba University School of Medicine, Japan.
Eur J Biochem. 1997 Aug 1;247(3):1190-6. doi: 10.1111/j.1432-1033.1997.01190.x.
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.
一些受体酪氨酸激酶，如表皮生长因子（EGF）受体和血小板衍生生长因子受体，在配体结合后会发生多聚泛素化。用赫曲霉素A处理表皮生长因子受体也会使其泛素化，赫曲霉素A是一种广泛用作酪氨酸激酶抑制剂的安莎霉素类抗生素。为了研究受体泛素化的机制，我们建立了一个体外分析赫曲霉素A诱导的泛素化过程的检测系统。我们现在表明，用赫曲霉素A处理纯化的表皮生长因子受体可在兔网织红细胞裂解物中诱导该受体的多聚泛素化。网织红细胞裂解物中的DEAE未吸附物质（组分I）和高盐洗脱物（组分II）协同参与泛素化过程，其中泛素结合酶UBC4可在功能上替代组分I。通过DEAE阴离子交换色谱从组分II中部分纯化得到的一种类泛素-蛋白连接酶活性也与UBC4协同发挥作用。然而，赫曲霉素A诱导表皮生长因子受体泛素化的确切机制尚未完全明了，不过我们目前的研究结果表明，与赫曲霉素A的直接相互作用会导致受体发生某种修饰，这种修饰可被兔网织红细胞裂解物中的泛素结合系统识别。
