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首页> 外文期刊>Biochemistry >Mapping of low- and high-fluence autophosphorylation sites in phototropin 1.
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Mapping of low- and high-fluence autophosphorylation sites in phototropin 1.

机译：在光蛋白1中的低和高通量自磷酸化位点的映射。

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Phototropins, originally detected by their blue light-dependent autophosphorylation, are plant photoreceptors involved in several blue light responses such as phototropism, chloroplast relocation, leaf expansion, rapid inhibition of hypocotyl growth, and stomatal opening. Three domains have been identified in phototropin sequences, two chromophore binding domains (LOV1 and LOV2) and a kinase domain. We describe here two additional domains, the N-terminus upstream of LOV1 and the hinge region between LOV1 and LOV2, as the regions for autophosphorylation; the phosphorylation sites were identified by site-directed mutagenesis as S27, S30, S274, S300, S317, S325, S332, and S349 of the PHOT1a sequence of Avena sativa. Investigation of the autophosphorylation in vivo revealed that serines close to the LOV1 domain are phosphorylated at lower fluence of blue light than the serines close to the LOV2 domain. Recovery of phosphorylation in vivo during a dark period after saturating irradiation is caused by dephosphorylation rather than by degradation of the phosphorylated form and new synthesis of nonphosphorylated phototropin. The results were obtained by a combination of autophosphorylation of phototropin with phosphorylation of recombinant domains by protein kinase A, which turned out to have the same site specificity as the phototropin kinase, followed by proteolysis and separation of phosphopeptides. With the knowledge of the phosphorylation sites, the physiological and biochemical consequences of autophosphorylation can now be approached by site-directed mutagenesis of phototropins.
机译：最初由它们的蓝光依赖性自磷酸化作用检测到的光蛋白是植物光感受器，参与几种蓝光响应，如光致性，叶绿体重新定位，叶片膨胀，下胚轴生长的快速抑制和气孔开放。已经在光蛋白序列中鉴定了三个结构域，两个生色团结合结构域（LOV1和LOV2）和一个激酶结构域。我们在这里描述两个额外的域，LOV1的N端上游和LOV1和LOV2之间的铰链区，作为自磷酸化的区域。磷酸化位点通过定点诱变鉴定为燕麦属植物的PHOT1a序列的S27，S30，S274，S300，S317，S325，S332和S349。体内自磷酸化的研究表明，与靠近LOV2域的丝氨酸相比，靠近LOV1域的丝氨酸在较低的蓝光通量下被磷酸化。在饱和辐射后的黑暗期中，体内磷酸化的恢复是由去磷酸化而不是由磷酸化形式的降解和非磷酸化光合蛋白的新合成引起的。通过结合蛋白激酶A使光蛋白的自身磷酸化与重组结构域的磷酸化相结合（结果证明具有与光养蛋白激酶相同的位点特异性），然后进行蛋白水解和分离磷酸肽，从而获得了结果。有了磷酸化位点的知识，现在就可以通过光敏蛋白的定点诱变来解决自磷酸化的生理和生化后果。

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