Cysteine-179 of IκB kinase β plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines
Cysteine-179 of IκB kinase β plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines
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DOI：
10.1038/emm.2006.64
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发表时间：
2006-10-31
影响因子：
12.8
通讯作者：
Jue, Dae-Myung
中科院分区：
文献类型：
--
作者：
Byun, Mi-Sun;Choi, Jin;Jue, Dae-Myung
IKK β复合物的I κ B激酶β（IKKO）亚基对于响应各种促炎信号的NF-κ B的活化是必需的。已知IKK β激活环中的Cys-179是IKK抑制剂（如环戊烯酮类化合物、亚砷酸盐和抗风湿金化合物）的靶位点。在这里，我们表明，突变体IKK β，其中Cys-179被丙氨酸取代，当它在HEK-293细胞中表达时，活性降低，TNF刺激没有恢复活性。IKKO激活所需的激活环丝氨酸（Ser-177和Ser-181）的磷酸化在IKK β（C179 A）突变体中减少。当IKK β（C179 A）与丝裂原活化蛋白激酶（MAPKKK）如NF-κ B诱导激酶（NIK）和MAPK/细胞外信号调节激酶1（MEKK 1）共表达或当丝氨酸残基被磷酸模拟谷氨酸取代时，其磷酸化可部分恢复。IKKO（C179 A）突变体在二聚体形成方面是正常的，而其活性异常地响应于反应混合物中底物ATP浓度的变化。我们的研究结果表明，IKKO的Cys-179通过促进激活环丝氨酸的磷酸化和与ATP的相互作用在酶激活中起着关键作用。
I kappa B kinase beta (IKKO) subunit of IKK beta complex is essential for the activation of NF-kappa B in response to various proinflammatory signals. Cys-179 in the activation loop of IKK beta is known to be the target site for IKK inhibitors such as cyclopentenone prostaglandins, arsenite, and antirheumatic gold compounds. Here we show that a mutant IKK beta in which Cys-179 is substituted with alanine had decreased activity when it was expressed in HEK-293 cells, and TNF stimulation did not restore the activity. Phosphorylation of activation loop serines (Ser-177 and Ser-181) which is required for IKKO activation was reduced in the IKK beta(C179A) mutant. The activity of IKK beta (C179A) was partially recovered when its phosphorylation was enforced by coexpression with mitogen-activated protein kinase kinase kinases (MAPKKK) such as NF-kappa B inducing kinase (NIK) and MAPK/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) or when the serine residues were replaced with phospho-mimetic glutamate. The IKKO (C179A) mutant was normal in dimer formation, while its activity abnormally responded to the change in the concentration of substrate ATP in reaction mixture. Our results suggest that Cys-179 of IKKO plays a critical role in enzyme activation by promoting phosphorylation of activation-loop serines and interaction with ATP.