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首页> 外文期刊>Molecular and Cellular Biology >A Bifunctional Regulatory Element in Human Somatic Wee1 Mediates Cyclin A/Cdk2 Binding and Crm1-Dependent Nuclear Export
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A Bifunctional Regulatory Element in Human Somatic Wee1 Mediates Cyclin A/Cdk2 Binding and Crm1-Dependent Nuclear Export

机译：人类体细胞Wee1中​​的双功能调节元素介导细胞周期蛋白A / Cdk2绑定和Crm1依赖核出口。

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Sophisticated models for the regulation of mitotic entry are lacking for human cells. Inactivating human cyclin A/Cdk2 complexes through diverse approaches delays mitotic entry and promotes inhibitory phosphorylation of Cdk1 on tyrosine 15, a modification performed by Wee1. We show here that cyclin A/Cdk2 complexes physically associate with Wee1 in U2OS cells. Mutation of four conserved RXL cyclin A/Cdk binding motifs (RXL1 to RXL4) in Wee1 diminished stable binding. RXL1 resides within a large regulatory region of Wee1 that is predicted to be intrinsically disordered (residues 1 to 292). Near RXL1 is T239, a site of inhibitory Cdk phosphorylation in Xenopus Wee1 proteins. We found that T239 is phosphorylated in human Wee1 and that this phosphorylation was reduced in an RXL1 mutant. RXL1 and T239 mutants each mediated greater Cdk phosphorylation and G2/M inhibition than the wild type, suggesting that cyclin A/Cdk complexes inhibit human Wee1 through these sites. The RXL1 mutant uniquely also displayed increased nuclear localization. RXL1 is embedded within sequences homologous to Crm1-dependent nuclear export signals (NESs). Coimmunoprecipitation showed that Crm1 associated with Wee1. Moreover, treatment with the Crm1 inhibitor leptomycin B or independent mutation of the potential NES (NESm) abolished Wee1 nuclear export. Export was also reduced by Cdk inhibition or cyclin A RNA interference, suggesting that cyclin A/Cdk complexes contribute to Wee1 export. Somewhat surprisingly, NESm did not display increased G2/M inhibition. Thus, nuclear export of Wee1 is not essential for mitotic entry though an important functional role remains likely. These studies identify a novel bifunctional regulatory element in Wee1 that mediates cyclin A/Cdk2 association and nuclear export.
机译：人类细胞缺乏用于控制有丝分裂进入的复杂模型。通过多种途径使人细胞周期蛋白A / Cdk2复合物失活，可延迟有丝分裂进入并促进酪氨酸15上Cdk1的抑制磷酸化作用，这是Wee1进行的修饰。我们在这里显示，cyclin A / Cdk2复合物与U2OS细胞中的Wee1物理关联。 Wee1中​​四个保守的RXL细胞周期蛋白A / Cdk结合基序（RXL1至RXL4）的突变减少了稳定的结合。 RXL1驻留在Wee1的大调控区域内，该区域被预测为内在无序的（残基1至292）。在RXL1附近的是T239，它是非洲爪蟾Wee1蛋白中抑制Cdk磷酸化的位点。我们发现，T239在人Wee1中​​被磷酸化，而该磷酸化在RXL1突变体中减少。 RXL1和T239突变体均比野生型介导更大的Cdk磷酸化和G 2 / M抑制作用，表明细胞周期蛋白A / Cdk复合物通过这些位点抑制人Wee1。 RXL1突变体还独特地显示出增加的核定位。 RXL1嵌入与Crm1依赖性核输出信号（NESs）同源的序列中。免疫共沉淀显示Crm1与Wee1相关。此外，用Crm1抑制剂瘦霉素B或潜在NES（NESm）的独立突变治疗可以消除Wee1核输出。 Cdk抑制或细胞周期蛋白A RNA干扰也减少了出口，这表明细胞周期蛋白A / Cdk复合物有助于Wee1的出口。令人惊讶的是，NESm没有显示出增加的G 2 / M抑制作用。因此，尽管有重要的功能性作用，但Wee1的核输出对于有丝分裂进入并不是必不可少的。这些研究确定了Wee1中​​介导细胞周期蛋白A / Cdk2缔合和核输出的新型双功能调节元件。

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