Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways
Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways
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DOI：
10.1073/pnas.0500410102
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发表时间：
2005-08-16
影响因子：
11.1
通讯作者：
Osada, H
中科院分区：
文献类型：
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作者：
Watanabe, N;Arai, H;Osada, H
在M期开始时，细胞周期蛋白依赖性激酶（CDK）的抑制性激酶体细胞Wee 1（Wee 1A）的活性主要通过E3泛素连接酶SCPbeta-TrcP泛素化后的蛋白酶体依赖性降解而下调。F-box蛋白β-TrCP（含β-转导蛋白重复序列的蛋白）是泛素连接酶的底物识别组分，通过含有两个磷酸丝氨酸DpSGXXpS的保守结合基序（磷酸降解决定子）与其底物结合。虽然Wee 1A缺乏该基序，但Wee 1A的丝氨酸53和123（S53和S123）分别被polo样激酶1（Plk 1）和CDK磷酸化是结合β-TrCP所必需的。磷酸化S53（DpSAGE）周围的序列与保守的β-TrCP结合基序相似;然而，S123磷酸化（EEGFGSSpSPVK）在β-TrCP结合中的作用尚未阐明。在本研究中，我们发现CDK对S123（pS123）的磷酸化通过三种独立的机制促进Wee 1A与β-TrCP的结合。pS123不仅直接与β-TrCP的WD 40重复结构域中的碱性残基相互作用，而且还通过两种独立的蛋白激酶Plk 1和CK 2（以前的酪蛋白激酶2）引发磷酸化，从而在Wee 1A上产生两个磷酸降解决定子。在Plk 1的情况下，S123磷酸化在Wee 1A上产生了一个波罗box结构域结合基序（SpSP），以加速Plk 1对S53的磷酸化。CK 2可以磷酸化S121，但只有当S123首先被磷酸化，从而产生第二个β-TrCP结合位点（EEGFGpS 121）。使用CK 2的特异性抑制剂，我们发现Wee 1A的磷酸化依赖性降解对于有丝分裂的适当开始是重要的。
At the onset of M phase, the activity of somatic Wee1 (Wee1 A), the inhibitory kinase for cyclin-dependent kinase (CDK), is down-regulated primarily through proteasome-dependent degradation after ubiquitination by the E3 ubiquitin ligase SCPbeta-TrcP. The F-box protein beta-TrCP (beta-transducin repeat-containing protein), the substrate recognition component of the ubiquitin ligase, binds to its substrates through a conserved binding motif (phosphodegron) containing two phosphoserines, DpSGXXpS. Although Wee1A lacks this motif, phosphorylation of serines 53 and 123 (S53 and S123) of Wee1A by polo-like kinase 1 (Plk1) and CDK, respectively, are required for binding to beta-TrCP. The sequence surrounding phosphorylated S53 (DpSAFQE) is similar to the conserved beta-TrCP-binding motif; however, the role of S123 phosphorylation (EEGFGSSpSPVK) in beta-TrCP binding was not elucidated. in the present study, we show that phosphorylation of S123 (pS123) by CDK promoted the binding of Wee1A to beta-TrCP through three independent mechanisms. The pS123 not only directly interacted with basic residues in the WD40 repeat domain of beta-TrCP but also primed phosphorylation by two independent protein kinases, Plk1 and CK2 (formerly casein kinase 2), to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation created a polo box domain-binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 could phosphorylate S121, but only if S123 was phosphorylated first, thereby generating the second)beta-TrCP-binding site (EEGFGpS121). Using a specific inhibitor of CK2, we showed that the phosphorylation-dependent degradation of Wee1A is important for the proper onset of mitosis.