CUL4-DDB1-CDT2 E3 Ligase Regulates the Molecular Clock Activity by Promoting Ubiquitination-Dependent Degradation of the Mammalian CRY1.
CUL4-DDB1-CDT2 E3 Ligase Regulates the Molecular Clock Activity by Promoting Ubiquitination-Dependent Degradation of the Mammalian CRY1.
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DOI：
10.1371/journal.pone.0139725
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Yin L
中科院分区：
文献类型：
--
作者：
Tong X;Zhang D;Guha A;Arthurs B;Cazares V;Gupta N;Yin L
CUL4-DDB1 E3连接酶复合体在细胞增殖、DNA损伤修复和细胞周期进程等多种细胞过程中起着重要的调节作用。然而，这种E3连接酶复合体是否调节哺乳动物细胞中的时钟蛋白周转和分子时钟活性尚不清楚。在这里，我们证明了CUL4-DDB1-CDT2 E3连接酶泛素化CRY1，并在体外和体内促进其降解。这种E3连接酶复合体的主要成分，包括DDB1，CDT2和CDT2辅助因子增殖细胞核抗原的耗尽，导致CRY1在培养细胞或小鼠肝脏中的稳定。CUL4A-DDB1-CDT2 E3连接酶针对CRY1蛋白C-末端区域的赖氨酸585，表现为CRY1 585KA突变体对CUL4A-DDB1复合体介导的泛素化和降解的抵抗。令人惊讶的是，DDB1的耗尽和Cry1-585KA突变体的过表达都增强了BMal1启动子活性的振荡幅度，而不改变其周期长度，这表明CUL4A-DDB1-CDT2 E3针对CRY1进行降解，并降低昼夜幅度。总之，我们发现了CUL4A-DDB1-CDT2 E3连接酶的一个新的生物学作用，它通过促进CRY1泛素化依赖的降解来调节分子的昼夜行为。
The CUL4-DDB1 E3 ligase complex serves as a critical regulator in various cellular processes, including cell proliferation, DNA damage repair, and cell cycle progression. However, whether this E3 ligase complex regulates clock protein turnover and the molecular clock activity in mammalian cells is unknown. Here we show that CUL4-DDB1-CDT2 E3 ligase ubiquitinates CRY1 and promotes its degradation both in vitro and in vivo. Depletion of the major components of this E3 ligase complex, including Ddb1, Cdt2, and Cdt2-cofactor Pcna, leads to CRY1 stabilization in cultured cells or in the mouse liver. CUL4A-DDB1-CDT2 E3 ligase targets lysine 585 within the C-terminal region of CRY1 protein, shown by the CRY1 585KA mutant’s resistance to ubiquitination and degradation mediated by the CUL4A-DDB1 complex. Surprisingly, both depletion of Ddb1 and over-expression of Cry1-585KA mutant enhance the oscillatory amplitude of the Bmal1 promoter activity without altering its period length, suggesting that CUL4A-DDB1-CDT2 E3 targets CRY1 for degradation and reduces the circadian amplitude. All together, we uncovered a novel biological role for CUL4A-DDB1-CDT2 E3 ligase that regulates molecular circadian behaviors via promoting ubiquitination-dependent degradation of CRY1.