Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1.
Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1.
复制标题
拟南芥光蛋白1的组成型活性激酶变体的功能表征。
DOI：
10.1074/jbc.m117.799643
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发表时间：
2017-08-18
期刊：
影响因子：
--
通讯作者：
Christie JM
中科院分区：
文献类型：
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作者：
Petersen J;Inoue SI;Kelly SM;Sullivan S;Kinoshita T;Christie JM
趋光性激素(PHTS)是一种质膜相关的丝氨酸/苏氨酸激酶，它协调一系列与优化植物光合作用效率相关的过程。这些光感受器在其N端含有两个光、氧或电压敏感(LOV)结构域，每个结构域都结合一个黄素单核苷酸分子作为吸收紫外光/蓝光的发色团。尽管PHITs含有两个LOV结构域，但光诱导的C末端激酶结构域的激活和随后的受体自动磷酸化主要由A‘α-LOV2-Jα光敏模块控制。破坏LOV2核心及其侧翼螺旋片段之间相互作用的突变可以解偶联这种光调节模式。然而，这些突变对拟南芥phot功能的影响还没有被探索。在此，我们报道了位于拟南芥PHIT1 A‘α-螺旋内的Arg-472的组氨酸取代在体外结构性地激活了PHIK活性，而不影响LOV2光化学。对该缺光突变体中的Pho1 R472H的表达分析证实，它在体内的黑暗中是自动磷酸化的，但在没有光的情况下不能启动Pho1信号。相反，我们发现Phot1R472H在弱光条件下功能不佳，但在较高的光强下可以恢复向光性、叶绿体积累、气孔开放以及叶片定位和扩张。我们的发现表明，拟南芥可以通过降低其稳定性来部分适应Pho1 R472H突变体的磷酸化状态，而突变体在强光条件下的活性可以归因于LOV2介导的光感受器自动磷酸化的额外增加。
Phototropins (phots) are plasma membrane–associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen-, or voltage-sensing (LOV) domains within their N terminus, with each binding one molecule of flavin mononucleotide as a UV/blue light–absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A′α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2 core and its flanking helical segments can uncouple this mode of light regulation. However, the impact of these mutations on phot function in Arabidopsis has not been explored. Here we report that histidine substitution of Arg-472 located within the A′α-helix of Arabidopsis phot1 constitutively activates phot1 kinase activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo but unable to initiate phot1 signaling in the absence of light. Instead, we found that phot1 R472H is poorly functional under low-light conditions but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant in part by reducing its stability, whereas the activity of the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation.