P27Kip1 ubiquitination and degradation is regulated by the SCFSkp2 complex through phosphorylated Thr187 in p27
P27Kip1 ubiquitination and degradation is regulated by the SCFSkp2 complex through phosphorylated Thr187 in p27
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DOI：
10.1016/s0960-9822(99)80290-5
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发表时间：
1999-06-17
期刊：
影响因子：
9.2
通讯作者：
Zhang, H
中科院分区：
文献类型：
--
作者：
Tsvetkov, LM;Yeh, KH;Zhang, H
许多致瘤过程通过影响细胞周期蛋白依赖性激酶抑制剂p27（Kip 1）的水平来影响细胞周期进程[1，2]。p27的磷酸化和泛素化依赖性蛋白水解与细胞周期中G1-S转换的控制有关[3-6]。为了确定控制p27稳定性的因素，我们建立了一种无细胞提取物测定法，该测定法重现了p27的降解。降解也依赖于SCFSkp 2，这是一种涉及靶向磷酸化蛋白以进行泛素化的蛋白质复合物[7-10]。从提取物中免疫耗竭复合物Cul-1，Skp 1或Skp 2-的组分废除了p27降解，而将纯化的SCF Skp 2添加到Skp 2耗竭的提取物中恢复了降解p27的能力。在Skp 2和含有磷酸化Thr 187的p27羧基末端肽之间观察到特异性结合，但在Skp 2和非磷酸化肽之间没有观察到特异性结合。还检测到Skp 1或Cul-1与p27磷酸肽之间的Skp 2依赖性关联。分离的SCFSkp 2含有对p27的E3泛素连接酶活性，因此我们的数据表明，SCFSkp 2特异性靶向p27在细胞周期进程中降解。(C)爱思唯尔科学有限公司ISSN 0960-9822。
Many tumorigenic processes affect cell-cycle progression by their effects on the levels of the cyclin-dependent kinase inhibitor p27(Kip1) [1,2]. The phosphorylation- and ubiquitination-dependent proteolysis of p27 is implicated in control of the G1-S transition in the cell cycle [3-6]. To determine the factors that control p27 stability, we established a cell-free extract assay that recapitulates the degradation of p27, Phosphorylation of p27 at Thr187 was essential for its degradation. Degradation was also dependent on SCFSkp2, a protein complex implicated in targeting phosphorylated proteins for ubiquitination [7-10]. Immunodepletion of components of the complex Cul-1, Skp1, or Skp2 - from the extract abolished p27 degradation, while addition of purified SCFSkp2 to Skp2-depleted extract restored the capacity to degrade p27, A specific association was observed between Skp2 and a p27 carboxy-terminal peptide containing phosphorylated Thr187, but not between Skp2 and the nonphosphorylated peptide. Skp2-dependent associations between Skp1 or Cul-1 and the p27 phosphopeptide were also detected. Isolated SCFSkp2 contained an E3 ubiquitin ligase activity towards p27, Our data thus suggest that SCFSkp2 specifically targets p27 for degradation during cell-cycle progression. (C) Elsevier Science Ltd ISSN 0960-9822.