O'Mahony A, Lin X, Geleziunas R, Greene W C
Gladstone Institute of Virology and Immunology, Microbiology and Immunology, University of California, San Francisco, California 94141, USA.
Mol Cell Biol. 2000 Feb;20(4):1170-8. doi: 10.1128/MCB.20.4.1170-1178.2000.
Signal-induced nuclear expression of the eukaryotic NF-kappaB transcription factor involves the stimulatory action of select mitogen-activated protein kinase kinase kinases on the IkappaB kinases (IKKalpha and IKKbeta) which reside in a macromolecular signaling complex termed the signalsome. While genetic studies indicate that IKKbeta is the principal kinase involved in proinflammatory cytokine-induced IkappaB phosphorylation, the function of the equivalently expressed IKKalpha is less clear. Here we demonstrate that assembly of IKKalpha with IKKbeta in the heterodimeric signalsome serves two important functions: (i) in unstimulated cells, IKKalpha inhibits the constitutive IkappaB kinase activity of IKKbeta; (ii) in activated cells, IKKalpha kinase activity is required for the induction of IKKbeta. The introduction of kinase-inactive IKKalpha, activation loop mutants of IKKalpha, or IKKalpha antisense RNA into 293 or HeLa cells blocks NIK (NF-kappaB-inducing kinase)-induced phosphorylation of the IKKbeta activation loop occurring in functional signalsomes. In contrast, catalytically inactive mutants of IKKbeta do not block NIK-mediated phosphorylation of IKKalpha in these macromolecular signaling complexes. This requirement for kinase-proficient IKKalpha to activate IKKbeta in heterodimeric IKK signalsomes is also observed with other NF-kappaB inducers, including tumor necrosis factor alpha, human T-cell leukemia virus type 1 Tax, Cot, and MEKK1. Conversely, the theta isoform of protein kinase C, which also induces NF-kappaB/Rel, directly targets IKKbeta for phosphorylation and activation, possibly acting through homodimeric IKKbeta complexes. Together, our findings indicate that activation of the heterodimeric IKK complex by a variety of different inducers proceeds in a directional manner and is dependent on the kinase activity of IKKalpha to activate IKKbeta.
真核生物NF-κB转录因子的信号诱导核表达涉及特定丝裂原活化蛋白激酶激酶激酶对IkappaB激酶（IKKα和IKKβ）的刺激作用，这些激酶存在于一种称为信号体的大分子信号复合物中。虽然遗传学研究表明IKKβ是参与促炎细胞因子诱导的IkappaB磷酸化的主要激酶，但等量表达的IKKα的功能尚不清楚。在这里，我们证明IKKα与IKKβ在异二聚体信号体中的组装具有两个重要功能：（i）在未受刺激的细胞中，IKKα抑制IKKβ的组成型IkappaB激酶活性；（ii）在活化的细胞中，IKKα激酶活性是诱导IKKβ所必需的。将激酶失活的IKKα、IKKα的激活环突变体或IKKα反义RNA引入293或HeLa细胞中，可阻断在功能性信号体中发生的NIK（NF-κB诱导激酶）诱导的IKKβ激活环磷酸化。相反，IKKβ的催化失活突变体不会阻断这些大分子信号复合物中NIK介导的IKKα磷酸化。在其他NF-κB诱导剂中也观察到，在异二聚体IKK信号体中，需要激酶功能正常的IKKα来激活IKKβ，这些诱导剂包括肿瘤坏死因子α、人类T细胞白血病病毒1型Tax、Cot和MEKK1。相反，蛋白激酶C的θ亚型也诱导NF-κB/Rel，它直接靶向IKKβ进行磷酸化和激活，可能通过同二聚体IKKβ复合物起作用。总之，我们的研究结果表明，多种不同诱导剂对异二聚体IKK复合物的激活是以定向方式进行的，并且依赖于IKKα的激酶活性来激活IKKβ。
