King R W, Glotzer M, Kirschner M W
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mol Biol Cell. 1996 Sep;7(9):1343-57. doi: 10.1091/mbc.7.9.1343.
Mitotic cyclins are abruptly degraded at the end of mitosis by a cell-cycle-regulated ubiquitin-dependent proteolytic system. To understand how cyclin is recognized for ubiquitin conjugation, we have performed a mutagenic analysis of the destruction signal of mitotic cyclins. We demonstrate that an N-terminal cyclin B segment as short as 27 residues, containing the 9-amino-acid destruction box, is sufficient to destabilize a heterologous protein in mitotic Xenopus extracts. Each of the three highly conserved residues of the cyclin B destruction box is essential for ubiquitination and subsequent degradation. Although an intact destruction box is essential for the degradation of both A- and B-type cyclins, we find that the Xenopus cyclin A1 destruction box cannot functionally substitute for its B-type counterpart, because it does not contain the highly conserved asparagine necessary for cyclin B proteolysis. Physical analysis of ubiquitinated cyclin B intermediates demonstrates that multiple lysine residues function as ubiquitin acceptor sites, and mutagenic studies indicate that no single lysine residue is essential for cyclin B degradation. This study defines the key residues of the destruction box that target cyclin for ubiquitination and suggests there are important differences in the way in which A- and B-type cyclins are recognized by the cyclin ubiquitination machinery.
有丝分裂周期蛋白在有丝分裂末期通过细胞周期调控的泛素依赖性蛋白水解系统被突然降解。为了解周期蛋白是如何被识别用于泛素结合的，我们对有丝分裂周期蛋白的破坏信号进行了诱变分析。我们证明，一个短至27个残基的N端周期蛋白B片段，包含9个氨基酸的破坏框，足以使非洲爪蟾有丝分裂提取物中的异源蛋白不稳定。周期蛋白B破坏框的三个高度保守的残基中的每一个对于泛素化和随后的降解都是必不可少的。虽然完整的破坏框对于A类和B类周期蛋白的降解都是必不可少的，但我们发现非洲爪蟾周期蛋白A1的破坏框不能在功能上替代其B类对应物，因为它不包含周期蛋白B蛋白水解所需的高度保守的天冬酰胺。对泛素化周期蛋白B中间体的物理分析表明，多个赖氨酸残基作为泛素受体位点起作用，诱变研究表明没有单个赖氨酸残基对于周期蛋白B的降解是必不可少的。这项研究确定了破坏框中靶向周期蛋白进行泛素化的关键残基，并表明A类和B类周期蛋白被周期蛋白泛素化机制识别的方式存在重要差异。
