Mutations in N-terminal Flanking Region of Blue Light-sensing Light-Oxygen and Voltage 2 (LOV2) Domain Disrupt Its Repressive Activity on Kinase Domain in the Chlamydomonas Phototropin
Mutations in N-terminal Flanking Region of Blue Light-sensing Light-Oxygen and Voltage 2 (LOV2) Domain Disrupt Its Repressive Activity on Kinase Domain in the Chlamydomonas Phototropin
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DOI：
10.1074/jbc.m111.324723
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发表时间：
2012-03-23
影响因子：
4.8
通讯作者：
Nagatani, Akira
中科院分区：
文献类型：
--
作者：
Aihara, Yusuke;Yamamoto, Takaharu;Nagatani, Akira
向光素是一种光调节激酶，在植物中介导向光性、叶绿体定位和气孔开放等多种光反应以提高光合效率。蓝光刺激首先诱导phototropin的发色团承载光氧和电压2（LOV 2）结构域中的局部构象变化，这进而激活C末端的丝氨酸/苏氨酸（Ser/Thr）激酶结构域。为了常规地检查全长趋光蛋白的激酶活性，我们采用了芽殖酵母酿酒酵母。在这种生物体中，存在与趋光蛋白的激酶结构域具有高度序列相似性的Ser/Thr激酶（Fpk 1 p和Fpk 2 p）。首先，我们证明了来自莱茵衣藻（CrPHOT）的趋光蛋白可以补充Fpk 1 p和Fpk 2 p的缺失，以允许细胞在酵母中生长。此外，该反应是蓝光依赖性的，表明CrPHOT在酵母细胞中确实是光激活的。我们将该系统应用于大规模筛选CrPHOT中的氨基酸取代，其在黑暗中提高激酶活性。因此，我们鉴定了位于LOV 2的N-末端侧翼区的突变簇（R199 C、L202 L、D203 N/G/V、L204 P、T207 I和R210 H）。体外磷酸化测定证实，这些突变大大降低了LOV 2在黑暗中对激酶结构域的抑制活性。此外，代表性的T207 I突变体的生化分析表明，该突变既不影响光谱也不影响CrPHOT的多聚化特性。因此，LOV 2的N-末端侧翼区，与C-末端侧翼J α区的情况一样，似乎在向光蛋白中激酶活性的调节中起关键作用。
Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking J alpha region, appears to play a crucial role in the regulation of kinase activity in phototropin.