M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFβ-TrCP
M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFβ-TrCP
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DOI：
10.1073/pnas.0307700101
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发表时间：
2004-03-30
影响因子：
11.1
通讯作者：
Osada, H
中科院分区：
文献类型：
--
作者：
Watanabe, N;Arai, H;Osada, H
Wee 1是Cdc 2抑制性激酶，需要在有丝分裂开始时下调，以确保Cdc 2的快速激活。以前，我们已经表明，人体细胞Wee 1（Wee 1A）是下调蛋白磷酸化和降解，但其潜在的机制尚未阐明。在本研究中，我们已经确定了β-transducin重复包含蛋白1/2（β-TrCP 1/2）F-box蛋白包含SKP 1/Cul 1/F-box蛋白（SCF）复合物（SCF β-TrCP 1/2）作为E3泛素连接酶Wee 1A泛素化。虽然Wee 1A缺乏一个DS（p）GXXS（p）磷酸依赖性结合基序，但Wee 1A被β-TrCP识别依赖于磷酸化，并且发现Wee 1A中的两个丝氨酸残基S53和S123是β-TrCP识别的最重要的磷酸化位点。我们还发现，主要的M期激酶polo样激酶1（Plk 1）和Cdc 2分别负责S53和S123的磷酸化，并且在每种情况下，磷酸化都会产生一种非常规的磷酸降解决定子（降解信号），可以被β-TrCP识别。这些激酶对Wee 1A的磷酸化在体外协同刺激SCF β-TrCP 1/2对Wee 1A的识别和泛素化。这些残基的突变或小干扰RNA处理的β-TrCP的耗尽增加了Wee 1A在HeLa细胞中的稳定性。此外，我们的分析表明，Wee 1A的β-TrCP依赖性降解对于体内M期的正常开始是重要的。这些结果还建立了Cdc 2和Wee 1A之间的反馈回路在体细胞中的存在，这取决于泛素化和蛋白质降解，并确保Cdc 2的快速激活时，细胞准备分裂。
Wee1, the Cdc2 inhibitory kinase, needs to be down-regulated at the onset of mitosis to ensure rapid activation of Cdc2. Previously, we have shown that human somatic Wee1 (Wee1A) is downregulated both by protein phosphorylation and degradation, but the underlying mechanisms had not been elucidated. In the present study, we have identified the beta-transducin repeat-containing protein 1/2 (beta-TrCP1/2) F-box protein-containing SKP1/Cul1/F-box protein (SCF) complex (SCFbeta-TrCP1/2) as an E3 ubiquitin ligase for Wee1A ubiquitination. Although Wee1A lacks a consensus DS(p)GXXS(p) phospho-dependent binding motif for beta-TrCP, recognition of Wee1A by beta-TrCP depended on phosphorylation, and two serine residues in Wee1A, S53 and S123, were found to be the most important phosphorylation sites for beta-TrCP recognition. We have found also that the major M-phase kinases polo-like kinase 1 (Plk1) and Cdc2 are responsible for the phosphorylation of S53 and S123, respectively, and that in each case phosphorylation generates an unconventional phospho-degron (signal for degradation) that can be recognized by beta-TrCP. Phosphorylation of Wee1A by these kinases cooperatively stimulated the recognition and ubiquitination of Wee1A by SCFbeta-TrCP1/2 in vitro. Mutation of these residues or depletion of beta-TrCP by small-interfering RNA treatment increased the stability of Wee1A in HeLa cells. Moreover, our analysis indicates that beta-TrCP-dependent degradation of Wee1A is important for the normal onset of M-phase in vivo. These results also establish the existence of a feedback loop between Cdc2 and Wee1A in somatic cells that depends on ubiquitination and protein degradation and ensures the rapid activation of Cdc2 when cells are ready to divide.