Phosphorylation- and Skp1-independent in vitro ubiquitination of E2F1 by multiple ROC-cullin ligases.
Phosphorylation- and Skp1-independent in vitro ubiquitination of E2F1 by multiple ROC-cullin ligases.
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DOI：
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发表时间：
2001-02
期刊：
影响因子：
11.2
通讯作者：
Tomohiko Ohta;Yue Xiong
中科院分区：
文献类型：
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作者：
Tomohiko Ohta;Yue Xiong
泛素依赖性蛋白水解在许多细胞过程的控制中起关键作用，并且由涉及泛素活化（El）、缀合（E2）和连接（E3）活性的酶级联介导。Cullin 1/CDC 53作为E3连接酶通过与RING指蛋白ROC 1相互作用并募集磷酸化底物而发挥功能。我们在这里报告，E2 F1转录因子可以在体外和体内泛素化的多个ROC-cullin连接酶。在体外，E2 F1可以被E2/Ubc 5泛素化，但不能被E2/CDC 34泛素化，依赖于催化活性的ROC 1，并受到Rb蛋白的保护。与SKP 1-Cullin 1-F盒（SCF）复合物的底物相反，通过CUL 1-ROC 1连接酶体外泛素化E2 F1不需要E2 F1磷酸化，不受F盒蛋白SKP 2的过表达刺激，并且不受SKP 1的免疫耗竭或CUL 1中破坏SKPI结合的突变的影响。这些结果表明，一种新的，SKP 1独立的机制，针对E2 F1泛素化。
Ubiquitin-dependent proteolysis plays a critical role in the control of many cellular processes and is mediated by a cascade of enzymes involving ubiquitin activating (El), conjugating (E2), and ligating (E3) activities. Cullin 1/CDC53 functions as an E3 ligase by interacting with RING finger protein ROC1 and recruiting phosphorylated substrate. We report here that E2F1 transcription factor can be ubiquitinated in vitro and in vivo by multiple ROC-cullin ligases. In vitro, E2F1 can be ubiquitinated by E2/Ubc5 but not by E2/CDC34, is dependent on catalytically active ROC1, and is protected by the Rb protein. In contrast to substrates of the SKP1-Cullin 1-F box (SCF) complexes, in vitro ubiquitination of E2F1 by CUL1-ROC1 ligase does not require E2F1 phosphorylation, is not stimulated by overexpression of F box protein SKP2, and is not affected by immunodepletion of SKP1 or mutations in CUL1 disrupting SKPI binding. These results suggest a novel, SKP1-independent mechanism for targeting E2F1 ubiquitination.