A refined two-hybrid system reveals that SCFCdc4-dependent degradation of Swi5 contributes to the regulatory mechanism of S-phase entry
A refined two-hybrid system reveals that SCFCdc4-dependent degradation of Swi5 contributes to the regulatory mechanism of S-phase entry
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DOI：
10.1073/pnas.0806253105
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发表时间：
2008-09-23
影响因子：
11.1
通讯作者：
Nagao, Rina
中科院分区：
文献类型：
--
作者：
Kishi, Tsutomu;Ikeda, Akemi;Nagao, Rina
泛素依赖性降解涉及各种细胞调节机制。SCFCdc 4（Skp 1，Cullin/Cdc 53和F-box蛋白Cdc 4）复合物是一种泛素连接酶复合物，作为细胞周期，信号转导和转录的调节剂。这些调控机制还没有很好地定义，因为在确定泛素连接酶和它们的底物之间的相互作用的困难。为了鉴定酵母SCFCdc 4泛素连接酶复合物的底物，我们改进了酵母双杂交系统，以允许在底物稳定的条件下筛选Cdc 4-底物相互作用，并鉴定Swi 5为SCFCdc 4复合物的底物。Swi 5是Sic 1的转录激活因子，Sic 1是S期细胞周期蛋白依赖性激酶（CDKs）的抑制剂。我们发现Swi 5确实通过SCFCdc 4复合物被泛素化和降解。此外，Swi 5的SCFCdc 4依赖性降解需要在早期G（1）期终止SIC 1转录，这确保有效进入S期：在表达稳定化Swi 5的细胞中注意到SIC 1的过度积累，并且稳定化Swi 5的表达延迟S期进入，这主要被SIC 1缺失抑制。这些发现表明，SCFCdc 4复合物调节S期进入不仅通过降解Sic 1，但也通过降解Swi 5。
Ubiquitin-dependent degradation is implicated in various cellular regulatory mechanisms. The SCFCdc4 (Skp1, Cullin/Cdc53, and the F-box protein Cdc4) complex is an ubiquitin ligase complex that acts as a regulator of cell cycle, signal transduction, and transcription. These regulatory mechanisms are not well defined because of the difficulty in identifying the interaction between ubiquitin ligases and their substrates. To identify substrates of the yeast SCFCdc4 ubiquitin ligase complex, we refined the yeast two-hybrid system to allow screening Cdc4-substrate interactions under conditions of substrate stabilization, and identified Swi5 as a substrate of the SCFCdc4 complex. Swi5 is the transcriptional activator of Sic1, the inhibitor of S phase cyclin-dependent kinases (CDKs). We showed that Swi5 is indeed ubiquitinated and degraded through the SCFCdc4 complex. Furthermore, the SCFCdc4-dependent degradation of Swi5 was required to terminate SIC1 transcription at early G(1) phase, which ensured efficient entry into S phase: Hyperaccumulation of Sic1 was noted in cells expressing stabilized Swi5, and expression of stabilized Swi5 delayed S phase entry, which was dominantly suppressed by SIC1 deletion. These findings indicate that the SCFCdc4 complex regulates S phase entry not only through degradation of Sic1, but also through degradation of Swi5.