Mutational analysis of phototropin 1 provides insights into the mechanism underlying LOV2 signal transmission
Mutational analysis of phototropin 1 provides insights into the mechanism underlying LOV2 signal transmission
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DOI：
10.1074/jbc.m605969200
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发表时间：
2007-03-02
影响因子：
4.8
通讯作者：
Christie, John M.
中科院分区：
文献类型：
--
作者：
Jones, Matthew A.;Feeney, Kevin A.;Christie, John M.
向光素（phot1 和 phot2）是蓝光激活的丝氨酸/苏氨酸蛋白激酶，可在植物中引发多种光响应。向光素的光感应是由位于蛋白质 N 末端区域的两个黄素单核苷酸 (FMN) 结合域（称为 LOV1 和 LOV2）介导的。暴露在光下会导致 FMN 发色团与 LOV 结构域内的保守半胱氨酸残基之间形成共价加合物。 LOV2 光激发对于拟南芥中的 phot1 功能至关重要，并且是通过光诱导位于 LOV2 C 末端的保守 a 螺旋内的结构变化来激活 photl 激酶活性所必需的。在这里，我们使用定点诱变来进一步鉴定对于光激活 photl 很重要的氨基酸残基。细菌表达的 LOV2 和昆虫细胞中表达的全长 phot1 的诱变表明，LOV2 表面保守盐桥的扰动在受体激活中不起作用。然而，LOV2 内保守谷氨酰胺残基 (Gln 575) 的突变（先前报道需要在 LOV2 表面传播结构变化）会减弱昆虫细胞中表达的 photl 的光诱导自磷酸化，而不影响 FMN 结合。这些发现与双突变体分析相结合，表明 Gln'' 在 LOV2 内部光驱动的半胱氨酰加合物形成与 LOV2 表面结构变化（导致 C 端激酶结构域激活）的耦合中发挥着重要作用。
Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that elicit a variety of photoresponses in plants. Light sensing by the phototropins is mediated by two flavin mononucleotide (FMN)-binding domains, designated LOV1 and LOV2, located in the N-terminal region of the protein. Exposure to light results in the formation of a covalent adduct between the FMN chromophore and a conserved cysteine residue within the LOV domain. LOV2 photoexcitation is essential for phot1 function in Arabidopsis and is necessary to activate photl kinase activity through light-induced structural changes within a conserved a-helix situated C-terminal to LOV2. Here we have used site-directed mutagenesis to identify further amino acid residues that are important for photl activation by light. Mutagenesis of bacterially expressed LOV2 and full-length phot1 expressed in insect cells indicates that perturbation of the conserved salt bridge on the surface of LOV2 does not play a role in receptor activation. However, mutation of a conserved glutamine residue (Gln 575) within LOV2, reported previously to be required to propagate structural changes at the LOV2 surface, attenuates light-induced autophosphorylation of photl expressed in insect cells without compromising FMN binding. These findings, in combination with double mutant analyses, indicate that Gln"' plays an important role in coupling light-driven cysteinyl adduct formation from within LOV2 to structural changes at the LOV2 surface that lead to activation of the C-terminal kinase domain.