Regulation of IκB Kinase Complex by Phosphorylation of γ-Binding Domain of IκB Kinase β by Polo-like Kinase 1
Regulation of IκB Kinase Complex by Phosphorylation of γ-Binding Domain of IκB Kinase β by Polo-like Kinase 1
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DOI：
10.1074/jbc.m806258200
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发表时间：
2008-12-19
影响因子：
4.8
通讯作者：
Zandi, Ebrahim
中科院分区：
文献类型：
--
作者：
Higashimoto, Tomoyasu;Chan, Nymph;Zandi, Ebrahim
I κ B激酶（IKK）复合物是NF-κ B通路的关键调节因子。IKK γ（NEMO）亚基与IKK β的C-末端IKK γ/NEMO结合结构域（γ BD）的信号诱导相互作用是IKK调节的重要相互作用。这种相互作用的潜在调控机制尚不清楚。已表明γ BD的磷酸化对IKK激活起调节作用。然而，尚未鉴定磷酸化γ BD的激酶。在这项研究中，我们使用IKK β的C-末端片段作为底物，并通过标准色谱法从HeLa细胞提取物中纯化Polo样激酶1（Plk 1）作为γ BD激酶。Plk 1在体外磷酸化IKK β γ BD中的丝氨酸733、740和750。在pulldown试验中，用Plk 1磷酸化γ BD降低了其对IKK γ的亲和力。我们产生的磷酸化抗体对丝氨酸740，并表明，γ BD在体内磷酸化。在哺乳动物细胞中表达组成型活性Plk 1减少了肿瘤坏死因子（TNF）诱导的IKK活化，导致内源性I κ B α磷酸化减少和NF-κ B活化减少。为了激活内源性Plk 1，用诺考达唑处理细胞，其减少TNF诱导的IKK激活，并增加γ BD的磷酸化。在哺乳动物细胞中敲低Plk 1恢复了诺考达唑处理细胞中TNF诱导的IKK激活。Plk 1的激活抑制了TNF诱导的cyclin D1的表达。在Plk 1被敲低的细胞中，TNF α增加了细胞周期蛋白D1的表达和细胞周期S期细胞的比例。总之，这项研究表明，磷酸化调节IKK β的γ BD与IKK γ的相互作用，因此对IKK激活起着关键作用。此外，我们确定Plk 1作为γ BD激酶，负调节TNF诱导的IKK激活和细胞周期蛋白D1的表达，从而影响细胞周期调控。TNF α过早激活细胞周期蛋白D1可以为TNF α参与炎症诱导的癌症提供潜在的机制。
I kappa B kinase (IKK) complex is a key regulator of NF-kappa B pathways. Signal-induced interaction of the IKK gamma ( NEMO) subunit with the C-terminal IKK gamma/NEMO-binding domain (gamma BD) of IKK beta is an essential interaction for IKK regulation. Underlying regulatory mechanism(s) of this interaction are not known. Phosphorylation of gamma BD has been suggested to play a regulatory role for IKK activation. However, a kinase that phosphorylates gamma BD has not been identified. In this study, we used a C-terminal fragment of IKK beta as substrate and purified Polo-like kinase 1 (Plk1) from HeLa cell extracts by standard chromatography as a gamma BD kinase. Plk1 phosphorylates serines 733, 740, and 750 in the gamma BD of IKK beta in vitro. Phosphorylating gamma BD with Plk1 decreased its affinity for IKK gamma in pulldown assay. We generated phosphoantibodies against serine 740 and showed that gamma BD is phosphorylated in vivo. Expressing a constitutively active Plk1 in mammalian cells reduced tumor necrosis factor (TNF)-induced IKK activation, resulting in decreased phosphorylation of endogenous I kappa B alpha and reduced NF-kappa B activation. To activate endogenous Plk1, cells were treated with nocodazole, which reduced TNF-induced IKK activation, and increased the phosphorylation of gamma BD. Knocking down Plk1 in mammalian cells restored TNF-induced IKK activation in nocodazole-treated cells. Activation of Plk1 inhibited TNF-induced expression of cyclin D1. In cells in which Plk1 was knocked down, TNF alpha increased expression of cyclin D1 and the proportion of cells in the S phase of the cell cycle. Taken together, this study shows that phosphorylation regulates the interaction of gamma BD of IKK beta with IKK gamma and therefore plays a critical role for IKK activation. Moreover, we identify Plk1 as a gamma BD kinase, which negatively regulates TNF-induced IKK activation and cyclin D1 expression, thereby affecting cell cycle regulation. Untimely activation of cyclin D1 by TNF alpha can provide a potential mechanism for an involvement of TNF alpha in inflammation-induced cancer.