Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis
Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis
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DOI：
10.1046/j.1365-313x.2003.01620.x
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发表时间：
2003-01-01
期刊：
影响因子：
7.2
通讯作者：
Kieber, JJ
中科院分区：
文献类型：
--
作者：
Huang, YF;Li, H;Kieber, JJ
CTR 1编码拟南芥乙烯反应途径的负调控因子。CTR 1的C-末端结构域类似于Raf蛋白激酶家族，但其前三分之二编码一个新的蛋白质结构域。我们使用了多种方法来研究这两个CTR 1结构域的功能。重组CTR 1蛋白从杆状病毒表达系统中纯化，并显示具有内在的Ser/Thr蛋白激酶活性，具有与Raf-1相似的酶性质。N-末端结构域的缺失并没有提高CTR 1的激酶活性，这表明，至少在体外，该结构域不自动抑制激酶功能。对功能丧失的ctr 1等位基因的分子分析表明，几个突变破坏了激酶催化结构域，体外研究证实，这些突变中至少有一个消除了激酶活性，这表明CTR 1功能需要激酶活性。一个错义突变，ctr 1 -8，被发现是由于一个新的保守基序内的N-末端结构域的氨基酸取代。Ctr 1 -8在体外对CTR 1的激酶活性没有可检测的影响，而是破坏了与乙烯受体ETR 1的相互作用。这种突变也破坏了转基因拟南芥中CTR 1氨基末端结构域过表达所产生的显性负效应。这些结果表明，CTR 1与ETR 1在体内相互作用，这种关联是关闭乙烯信号通路所必需的。
CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana . The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro , this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8 , was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis . These results suggest that CTR1 interacts with ETR1 in vivo , and that this association is required to turn off the ethylene-signaling pathway.