IKK-i and TBK-1 are enzymatically distinct from the homologous enzyme IKK-2 - Comparative analysis of recombinant human IKK-i, TBK-1, and IKK-2
IKK-i and TBK-1 are enzymatically distinct from the homologous enzyme IKK-2 - Comparative analysis of recombinant human IKK-i, TBK-1, and IKK-2
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DOI：
10.1074/jbc.m110474200
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发表时间：
2002-04-19
影响因子：
4.8
通讯作者：
Tripp, CS
中科院分区：
文献类型：
--
作者：
Kishore, N;Huynh, QK;Tripp, CS
NF-κ B被抑制性IkappaB蛋白隔离在细胞质中。激动剂对细胞的刺激导致IkappaB的快速磷酸化，导致其降解，导致NT-kappaB活化。IKK-1和IKK-2是两种直接的licB激酶。最近鉴定的两种新型IKK是IKK-1和TBK-1。我们克隆、表达并纯化了同源重组人（rh）IKK-1和rhTBK-1，并将它们的酶性质与rhIKK-2的酶性质进行了比较。我们发现rhIKK-1和rhTBK-1在酶促方面彼此相似。我们通过磷酸肽作图和位点特异性诱变证明，rhIKK-1和rhTBK-1在丝裂原活化蛋白激酶激酶激活环中的丝氨酸172上被磷酸化，并且这种磷酸化是激酶活性所必需的。此外，与rhIKK-2相比，rhIKK-i和rhTBK-1具有不同的肽底物特异性，IKK-2的丝裂原活化蛋白激酶激酶活化环是比IkappaB α肽更有利的底物。最后，使用ATP类似物，我们证明了rbIKK-1，rhTBK-1和rhIKK-2的ATP结合位点的独特差异。因此，尽管这些IKK在结构上相似，但它们的酶性质可以提供对其独特功能的见解。
NF-kappaB is sequestered in the cytoplasm by the inhibitory IkappaB proteins. Stimulation of cells by agonists leads to the rapid phosphorylation of IkappaBs leading to their degradation that results in NT-kappaB activation. IKK-1 and IKK-2 are two direct licB kinases. Two recently identified novel IKKs are IKK-i and TBK-1. We have cloned, expressed, and purified to homogeneity recombinant human (rh)IKK-i and rhTBK-1 and compared their enzymatic properties with those of rhIKK-2. We show that rhIKK-i and rhTBK-1 are enzymatically similar to each other. We demonstrate by phosphopeptide mapping and site-specific mutagenesis that rhIKK-i and rhTBK-1 are phosphorylated on serine 172 in the mitogen-activated protein kinase kinase activation loop and that this phosphorylation is necessary for kinase activity. Also, rhIKK-i and rhTBK-1 have differential peptide substrate specificities compared with rhIKK-2, the mitogen-activated protein kinase kinase activation loop of IKK-2 being a more favorable substrate than the IkappaBalpha peptide. Finally, using analogs of ATP, we demonstrate unique differences in the ATP-binding sites of rbIKK-i, rhTBK-1, and rhIKK-2. Thus, although these IKKs are structurally similar, their enzymatic properties may provide insights into their unique functions.