Direct activation of mitogen-activated protein kinase kinase kinase MEKK1 by the Ste20p homologue GCK and the adapter protein TRAF2
Direct activation of mitogen-activated protein kinase kinase kinase MEKK1 by the Ste20p homologue GCK and the adapter protein TRAF2
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DOI：
10.1128/mcb.22.3.737-749.2002
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发表时间：
2002-02-01
影响因子：
5.3
通讯作者：
Kyriakis, JM
中科院分区：
文献类型：
--
作者：
Chadee, DN;Yuasa, T;Kyriakis, JM
丝裂原活化蛋白激酶（MAPK）途径协调对丝裂原、应激和发育线索的关键细胞反应。MAPK激酶激酶(MAP3K) -> MAPK激酶(MEK) -> MAPK核心途径与细胞表面受体的偶联仍然知之甚少。重组形式的MAP3K MEK激酶I （MEKK1）在体内和体外与STE20蛋白同源生发中心激酶（GCK）相互作用，GCK和MEKK1在体内都与适配蛋白肿瘤坏死因子（TNF）受体相关因子2 （TRAF2）相关联。这些相互作用可能将TNF受体与MAPKs的SAPK/JNK家族偶联；然而，这些蛋白协同募集SAPKs/JNKs的分子机制仍然难以捉摸。我们发现内源性GCK和MEKK1在体内相互关联。此外，我们还开发了一种体外检测系统，通过该系统，我们证明纯化的、活性的GCK和TRAF2可以激活MEKK1。TRAF2的RING结构域是体外激活MEKK1的必要条件，而GCK的激酶结构域则不是。MEKK1激酶结构域激活环内的自磷酸化是激活所必需的，强制寡聚化也激活了MEKK1， GCK在体内诱导了共表达的MEKK1的增强寡聚化。这些结果代表了MEKK1首次在体外使用纯化蛋白激活，并提出了MEKK1激活的机制，包括诱导寡聚化和随后由上游蛋白介导的自磷酸化。
Mitogen-activated protein kinase (MAPK) pathways coordinate critical cellular responses to mitogens, stresses, and developmental cues. The coupling of MAPK kinase kinase (MAP3K) --> MAPK kinase (MEK) --> MAPK core pathways to cell surface receptors remains poorly understood. Recombinant forms of MAP3K MEK kinase I (MEKK1) interact in vivo and in vitro with the STE20 protein homologue germinal center kinase (GCK), and both GCK and MEKK1 associate in vivo with the adapter protein tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2). These interactions may couple TNF receptors to the SAPK/JNK family of MAPKs; however, a molecular mechanism by which these proteins might collaborate to recruit the SAPKs/JNKs has remained elusive. Here we show that endogenous GCK and MEKK1 associate in vivo. In addition, we have developed an in vitro assay system with which we demonstrate that purified, active GCK and TRAF2 activate MEKK1. The RING domain of TRAF2 is necessary for optimal in vitro activation of MEKK1, but the kinase domain of GCK is not. Autophosphorylation within the MEKK1 kinase domain activation loop is required for activation, Forced oligomerization also activates MEKK1, and GCK elicits enhanced oligomerization of coexpressed MEKK1 in vivo. These results represent the first activation of MEKK1 in vitro using purified proteins and suggest a mechanism for MEKK1 activation involving induced oligomerization and consequent auto phosphorylation mediated by upstream proteins.