From the Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan and CREST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan.
From the Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan and CREST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan.
J Biol Chem. 2013 Nov 8;288(45):32766-32776. doi: 10.1074/jbc.M113.511303. Epub 2013 Sep 30.
The Skp1-Cul1-F-box protein (SCF) complex is one of the most well characterized types of ubiquitin ligase (E3), with the E3 activity of the complex being regulated in part at the level of complex formation. Fbxl3 is an F-box protein that is responsible for the ubiquitylation and consequent degradation of cryptochromes (Crys) and thus regulates oscillation of the circadian clock. Here we show that formation of the SCF(Fbxl3) complex is regulated by substrate binding in vivo. Fbxl3 did not associate with Skp1 and Cul1 to a substantial extent in transfected mammalian cells. Unexpectedly, however, formation of the SCF(Fbxl3) complex was markedly promoted by forced expression of its substrate Cry1 in these cells. A mutant form of Fbxl3 that does not bind to Cry1 was unable to form an SCF complex, suggesting that interaction of Cry1 with Fbxl3 is essential for formation of SCF(Fbxl3). In contrast, recombinant Fbxl3 associated with recombinant Skp1 and Cul1 in vitro even in the absence of recombinant Cry1. Domain-swap analysis revealed that the COOH-terminal leucine-rich repeat domain of Fbxl3 attenuates the interaction of Skp1, suggesting that a yet unknown protein associated with the COOH-terminal domain of Fbxl3 and inhibited SCF complex formation. Our results thus provide important insight into the regulation of both SCF ubiquitin ligase activity and circadian rhythmicity.
Skp1-Cul1-F-box 蛋白（SCF）复合物是最具特征的泛素连接酶（E3）之一，复合物的 E3 活性部分受复合物形成水平的调节。Fbxl3 是一种 F-box 蛋白，负责 cryptochromes（Cry）的泛素化和随后的降解，从而调节生物钟的振荡。在这里，我们表明 SCF（Fbxl3）复合物的形成受体内底物结合的调节。在转染的哺乳动物细胞中，Fbxl3 没有与 Skp1 和 Cul1 大量结合。然而，出乎意料的是，其底物 Cry1 的强制表达显著促进了这些细胞中 SCF（Fbxl3）复合物的形成。不能与 Cry1 结合的 Fbxl3 突变体形式无法形成 SCF 复合物，这表明 Cry1 与 Fbxl3 的相互作用对于形成 SCF（Fbxl3）复合物至关重要。相比之下，重组 Fbxl3 即使在没有重组 Cry1 的情况下，也能与重组 Skp1 和 Cul1 在体外结合。结构域交换分析表明，Fbxl3 的 COOH 端富含亮氨酸重复结构域减弱了 Skp1 的相互作用，这表明与 Fbxl3 的 COOH 端结构域相关的未知蛋白与抑制 SCF 复合物形成有关。我们的研究结果为 SCF 泛素连接酶活性和昼夜节律性的调节提供了重要的见解。
