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首页> 外文期刊>Cell cycle >Ubiquitination of cyclin-dependent kinase inhibitor, Xic1, is mediated by the Xenopus F-box protein xSkp2.
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Ubiquitination of cyclin-dependent kinase inhibitor, Xic1, is mediated by the Xenopus F-box protein xSkp2.

机译：Xenopus F-box蛋白xSkp2介导细胞周期蛋白依赖性激酶抑制剂Xic1的泛素化。

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摘要

In the frog, Xenopus laevis, the Cip/Kip-type cyclin-dependent kinase (CDK) inhibitor, Xic1, inhibits DNA replication in interphase egg extracts through the binding of CDK2-cyclins and Proliferating Cell Nuclear Antigen (PCNA). During DNA polymerase switching in the replicating Xenopus egg extract, Xic1 is targeted for ubiquitination and degradation when localized to chromatin through its binding to PCNA. To date, the machinery responsible for Xic1 ubiquitination is unknown and although it is predicted that the E3 called SCF may mediate Xic1 ubiquitination, characterization of the SCF in Xenopus is lacking. In this study, we describe the identification and characterization of Xenopus Skp2 (xSkp2) and the role of xSkp2 in the ubiquitination of Xic1. Our results indicate that the expression of xSkp2 appears to be developmentally regulated with low protein levels found in the egg and increased levels found in the developing embryo. We also demonstrate that when ectopically expressed, a xSkp2 F-box deletion mutant inhibits the initiation of DNA replication suggesting a role for the SCF in the onset of S phase in Xenopus egg extracts. We further show that xSkp2 binds to C-terminal residues of Xic1 and when coexpressed with Skp1, promotes the proteolysis of Xic1 in the egg extract. Moreover, the xSkp2 F-box deletion mutant inhibits the DNA-dependent ubiquitination and proteolysis of Xic1 when added to the interphase egg extract. Importantly, our studies demonstrate that SCF(xSkp2) supports the ubiquitination of Xic1 in a reconstituted in vitro ubiquitination assay and that this Xic1 ubiquitination does not require either CDK2-cyclins or Cks1. These studies provide the first characterization of the SCF in Xenopus and its role in the ubiquitination of CDK inhibitor, Xic1, during DNA replication initiation.
机译：在青蛙中，非洲爪蟾（Xenopus laevis）是Cip / Kip型细胞周期蛋白依赖性激酶（CDK）抑制剂Xic1，通过CDK2细胞周期蛋白和增殖细胞核抗原（PCNA）的结合来抑制相间卵提取物中的DNA复制。在复制非洲爪蟾卵提取物中的DNA聚合酶转换过程中，当Xic1通过与PCNA结合而定位于染色质时，其目标是泛素化和降解。迄今为止，尚不清楚负责Xic1泛素化的机制，尽管据预测，称为SCF的E3可能介导Xic1泛素化，但缺乏非洲爪蟾中SCF的表征。在这项研究中，我们描述了非洲爪蟾Skp2（xSkp2）的鉴定和特征以及xSkp2在Xic1泛素化中的作用。我们的结果表明，xSkp2的表达似乎受到鸡蛋中低蛋白水平和发育中胚胎中水平升高的发育调控。我们还证明，当异位表达时，xSkp2 F-box缺失突变体抑制DNA复制的启动，这提示非洲爪蟾在爪蟾卵提取物中S期的发作中起作用。我们进一步表明xSkp2结合Xic1的C末端残基，当与Skp1共表达时，促进卵提取物中Xic1的蛋白水解。此外，xSkp2 F-box缺失突变体添加到相间卵提取物中时，会抑制Xic1的DNA依赖性泛素化和蛋白水解。重要的是，我们的研究表明SCF（xSkp2）在重构的体外泛素化测定中支持Xic1的泛素化，并且该Xic1泛素化不需要CDK2-cyclins或Cks1。这些研究提供了非洲爪蟾中SCF的第一个特征及其在DNA复制起始过程中CDK抑制剂Xic1泛素化中的作用。

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