Probing kinase activation and substrate specificity with an engineered monomeric IKK2.
Probing kinase activation and substrate specificity with an engineered monomeric IKK2.
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DOI：
10.1021/bi401551r
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发表时间：
2014-04-01
期刊：
影响因子：
2.9
通讯作者：
Huxford T
中科院分区：
文献类型：
--
作者：
Hauenstein AV;Rogers WE;Shaul JD;Huang DB;Ghosh G;Huxford T
IκB激酶（IKK）的催化亚基IKK1/IKKα和IKK2/IKKβ在体内作为二聚体发挥作用，并与必需的支架亚基NEMO/IKKγ相关联。近期IKK2的X射线晶体结构表明，二聚化可能是由比之前认为的更小的蛋白质 - 蛋白质相互作用所介导。在此，我们报道去除人IKK2的一部分支架二聚化结构域（SDD）会产生一种在溶液中保持单体形式的激酶亚基。在杆状病毒感染的Sf9昆虫细胞中表达并纯化这种经过改造的单体人IKK2酶，能够对其底物特异性和激活机制进行体外分析。我们发现，这种单体酶包含所有氨基末端激酶和泛素样结构域以及SDD较近端部分，在体外仅能将其底物IκBα的S32和S36残基磷酸化。因此，IKK2诱导NF - κB的潜能在改造后的单体中得以保留。此外，我们观察到我们改造的IKK2单体容易反式自磷酸化激活环的丝氨酸177和181。然而，当在单体背景下对先前观察到的在转染细胞中干扰IKK2反式自磷酸化的残基进行突变时，所得到的在Sf9细胞中表达并纯化的蛋白质在体外的反式自磷酸化活性显著受损。这项研究进一步明确了底物特异性的决定因素，并为一种模型提供了额外证据，该模型认为IKK2中激活环丝氨酸通过反式自磷酸化激活需要高阶寡聚体的瞬时组装。
Catalytic subunits of the IκB kinase (IKK), IKK1/IKKα, and IKK2/IKKβ function in vivo as dimers in association with the necessary scaffolding subunit NEMO/IKKγ. Recent X-ray crystal structures of IKK2 suggested that dimerization might be mediated by a smaller protein–protein interaction than previously thought. Here, we report that removal of a portion of the scaffold dimerization domain (SDD) of human IKK2 yields a kinase subunit that remains monomeric in solution. Expression in baculovirus-infected Sf9 insect cells and purification of this engineered monomeric human IKK2 enzyme allows for in vitro analysis of its substrate specificity and mechanism of activation. We find that the monomeric enzyme, which contains all of the amino-terminal kinase and ubiquitin-like domains as well as the more proximal portions of the SDD, functions in vitro to direct phosphorylation exclusively to residues S32 and S36 of its IκBα substrate. Thus, the NF-κB-inducing potential of IKK2 is preserved in the engineered monomer. Furthermore, we observe that our engineered IKK2 monomer readily autophosphorylates activation loop serines 177 and 181 in trans. However, when residues that were previously observed to interfere with IKK2 trans autophosphorylation in transfected cells are mutated within the context of the monomer, the resulting Sf9 cell expressed and purified proteins were significantly impaired in their trans autophosphorylation activity in vitro. This study further defines the determinants of substrate specificity and provides additional evidence in support of a model in which activation via trans autophosphorylation of activation loop serines in IKK2 requires transient assembly of higher-order oligomers.
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