The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase
The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase
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DOI：
10.1002/j.1460-2075.1996.tb00913.x
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发表时间：
1996-10-01
期刊：
影响因子：
11.4
通讯作者：
Hunt, T
中科院分区：
文献类型：
--
作者：
Brandeis, M;Hunt, T
我们研究了小鼠成纤维细胞中B型细胞周期蛋白的周期特异性振荡是如何产生的。一种由B型细胞周期蛋白的N-末端与细菌氯霉素乙酰转移酶（CAT）融合的报告酶在有丝分裂结束时像内源性细胞周期蛋白一样被降解。该结构的破坏盒中的点突变完全消除了其有丝分裂不稳定性。当可破坏的报告基因由细胞周期蛋白B2启动子驱动时，CAT活性模仿内源性细胞周期蛋白B2水平的振荡。当报道基因从SV 40启动子组成型转录时，这些振荡在很大程度上是保守的。脉冲追踪实验或加入蛋白酶体抑制剂lactacystin和ALLN表明，细胞周期蛋白的合成在有丝分裂结束后继续进行。细胞周期蛋白的破坏盒特异性降解通常在S期开始时停止，并在G（0）和分化的C2成肌细胞中活跃。我们在体外同步化细胞提取物中重现了这种蛋白水解，G（1）细胞提取物降解cyclin B1，而p27（Kip 1）是稳定的，相反，cyclin B1保持稳定，而p27（Kip 1）在S期细胞提取物中被降解。
We have studied how the cell cycle-specific oscillations of mitotic B-type cyclins are generated in mouse fibroblasts, A reporter enzyme comprising the N-terminus of a B-type cyclin fused to bacterial chloramphenicol acetyl transferase (CAT) was degraded at the end of mitosis like endogenous cyclins. Point mutations in the destruction box of this construct completely abolished its mitotic instability. When the destructible reporter was driven by the cyclin B2 promoter, CAT activity mimicked the oscillations in the level of the endogenous cyclin B2. These oscillations were largely conserved when the reporter was transcribed constitutively from the SV40 promoter. Pulse-chase experiments or addition of the proteasome inhibitors lactacystin and ALLN showed that cyclin synthesis continued after the end of mitosis, The destruction box-specific degradation of cyclins normally ceases at the onset of S phase, and is active in fibroblasts arrested in G(0) and in differentiated C2 myoblasts. We were able to reproduce this proteolysis in vitro in extracts of synchronized cells, Extracts of G(1) cells degraded cyclin B1 whereas p27(Kip1) was stable, in contrast, cyclin B1 remained stable and p27(Kip1) was degraded in extracts of S phase cells.