Cooperative signals governing ARF-Mdm2 interaction and nucleolar localization of the complex
Cooperative signals governing ARF-Mdm2 interaction and nucleolar localization of the complex
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DOI：
10.1128/mcb.20.7.2517-2528.2000
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发表时间：
2000-04-01
影响因子：
5.3
通讯作者：
Sherr, CJ
中科院分区：
文献类型：
--
作者：
Weber, JD;Kuo, ML;Sherr, CJ
ARF肿瘤抑制蛋白通过拮抗p53的负调节因子Mdm 2（人类中为Hdm 2）来稳定p53。小鼠p19（ARF）和人p14（ARF）都与Mdm 2的中心区域（残基210至304）结合，该区域不与其N-末端p53结合结构域、核输入或输出信号或Mdm 2 E3泛素连接酶活性所需的C-末端RING结构域重叠。小鼠p19（ARF）的N-末端37个氨基酸是必需的，足以结合Mdm 2，Mdm 2的定位到核仁，和p53依赖的细胞周期阻滞。尽管核仁定位信号（NrLS）映射在人p14（ARF）蛋白的不同片段（残基82至101）内，但与Mdm 2的结合和ARF-Mdm 2复合物的核仁输入都是由小鼠或人ARF蛋白诱导的细胞周期停滞所需的。由于小鼠ARF mRNA的许多密码子不被最丰富的细菌tRNA识别，我们合成了含有优选细菌密码子的ARF小基因。使用细菌产生的ARF多肽和化学合成的肽缀合到琼脂糖凝胶，小鼠p19（ARF）的残基1至14和26至37被发现与Mdm 2的相互作用独立和合作，而残基15至25被标记为结合。特别地，小鼠p19（ARF）的残基26至37在Mdm 2不存在的情况下对于ARF核仁定位也是必需的。然而，p19（ARF）-Mdm 2复合物进入核仁的动员也需要Mdm 2 C-末端RING结构域内的隐蔽NrLS。Mdm 2 NrLS在ARF结合时被揭开，并且其缺失防止ARF-Mdm 2复合物输入到核仁中。总的来说，结果表明，ARF结合Mdm 2诱导的构象变化，有利于核仁进口的ARF Mdm 2复合物和p53依赖性细胞周期阻滞。因此，ARF-Mdm 2相互作用可以被视为双向的，每种蛋白质都能够调节另一种蛋白质的亚核定位。
The ARF tumor suppressor protein stabilizes p53 by antagonizing its negative regulator, Mdm2 (Hdm2 in humans). Both mouse p19(ARF) and human p14(ARF) bind to the central region of Mdm2 (residues 210 to 304), a segment that does not overlap with its N-terminal p53-binding domain, nuclear import or export signals, or C-terminal RING domain required for Mdm2 E3 ubiquitin ligase activity. The N-terminal 37 amino acids of mouse p19(ARF) are necessary and sufficient for binding to Mdm2, localization of Mdm2 to nucleoli, and p53-dependent cell cycle arrest. Although a nucleolar localization signal (NrLS) maps within a different segment (residues 82 to 101) of the human p14(ARF) protein, binding to Mdm2 and nucleolar import of ARF-Mdm2 complexes are both required for cell cycle arrest induced by either the mouse or human ARF proteins. Because many codons of mouse ARF mRNA are not recognized by the most abundant bacterial tRNAs, we synthesized,ARF minigenes containing preferred bacterial codons. Using bacterially produced ARF polypeptides and chemically synthesized peptides conjugated to Sepharose, residues 1 to 14 and 26 to 37 of mouse p19(ARF) were found to interact independently and cooperatively with Mdm2, while residues 15 to 25 were dispensable for binding. Paradoxically, residues 26 to 37 of mouse p19(ARF) are also essential for ARF nucleolar localization in the absence of Mdm2. However, the mobilization of the p19(ARF)-Mdm2 complex into nucleoli also requires a cryptic NrLS within the Mdm2 C-terminal RING domain. The Mdm2 NrLS is unmasked upon ARF binding, and its deletion prevents import of the ARF-Mdm2 complex into nucleoli. Collectively, the results suggest that ARF binding to Mdm2 induces a conformational change that facilitates nucleolar import of the ARF-Mdm2 complex and p53-dependent cell cycle arrest. Hence, the ARF-Mdm2 interaction can be viewed as bidirectional, with each protein being capable of regulating the subnuclear localization of the other.