Modulation of Phytochrome B Signalling by Phosphorylation

通过磷酸化调节光敏色素 B 信号传导

• 批准号: BB/K006975/1
• 负责人: Ferenc Nagy
• 金额: $ 46.7万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK006975%2F1
• 关键词: Modulation; Phytochrome; Signalling; Phosphorylation

Plants are sessile organisms and therefore have to adapt their growth to changes in the environment. Among the abiotic and biotic environmental factors that regulate plant growth, light plays a distinguished role. Light is not only used to drive photosynthesis but it is also an important developmental clue to ensure optimal adaptation to the changing environment. To monitor variations in the wavelength, intensity, direction and duration of light, plants evolved a battery of photoreceptors. The photoreceptors active in red/far-red light are called phytochromes. Phytochromes are dimeric chromoproteins with one covalently linked tetrapyrrol chromophore per molecule, and they cycle between their biologically inactive (Pr = red absorbing) and active (Pfr = far-red absorbing) forms. It is the Pfr conformer which is imported into the nuclei and whose interaction with specific cellular factors is required to launch the signaling cascade. It follows that phytochrome signaling is quantitatively determined (i) by the number of Pfr molecules available and (ii) by the kinetics of protein-protein interactions between Pfr molecules and signal transducers.Phytochromes are often also referred to as light-regulated enzymes as it was shown that phytochrome-A (phyA) can autophosphorylate and phosphorylate other proteins in vitro and is phosphorylated by unknown kinase(s) in planta. These data were interpreted to mean that autophosphorylation reduces the amount of phytochrome Pfr by increasing its degradation whereas phosphorylation by an unknown kinase at other amino acid residues decreases the capacity of the Pfr form to bind to its authentic signaling partners. Thereby it is generally accepted that phosphorylation negatively regulates phyA controlled signaling and physiological responses. However, the topic is surrounded by considerable controversy as fundamental biochemical evidences are still missing to validate this theory. In this proposal, we outline a research program to elucidate how reversible phosphorylation regulates phytochrome-B (phyB) controlled responses at the molecular level. We show that phyB, the major photoreceptor regulating photomorphogenic responses in adult plants, is (i) phosphorylated at multiple sites in planta, (ii) this post-translational modification increases the rate of dark-reversion, a light independent conversion of the thermodynamically unstable Pfr to Pr form and thereby (iii) results in decreased responsiveness to red light. To test if phosphorylation also affects interaction of phyB with its downstream signalling partners, we will perform in vitro experiments to characterise binding of the mutated phyB proteins to Phytochrome Interacting Factor (PIF) proteins. We also intend to identify those phosphatases which dephosphorylate phyB. We will therefore express candidate phosphatase proteins in bacterial cells and treat phosphorylated phyB purified from plants with recombinant phosphatases purified from E.coli. Finally we will determine whether phyB indeed functions as a kinase. To this end, we will define if phosphorylation detected in planta can be recapitulated at least partly in vitro by characterising autophosphorylation of phyB Pr and Pfr purified from insect cells. If phyB indeed autophosphorylates in vitro, we will use this approach to identify the catalytic domain and the ATP binding sites essential for kinase activity of the photoreceptor. Taken together, these experiments will help us deciphering whether phyB is phopshorylated by itself or other yet unknown kinases or if both of these mechanisms are involved in mediating post-translational modification of the photoreceptor.

植物是固着生物，因此必须使其生长适应环境的变化。在调节植物生长的非生物和生物环境因子中，光起着突出的作用。光不仅用于驱动光合作用，而且也是确保最佳适应不断变化的环境的重要发育线索。为了监测光的波长、强度、方向和持续时间的变化，植物进化出了一组光感受器。在红光/远红光中活跃的光感受器被称为光敏色素。光敏色素是每个分子具有一个共价连接的四吡咯发色团的二聚体色蛋白，并且它们在其生物非活性（Pr =红色吸收）和活性（Pfr =远红外吸收）形式之间循环。Pfr构象异构体被导入细胞核，其与特定细胞因子的相互作用是启动信号级联反应所必需的。光敏色素信号转导是通过Pfr分子的数量和Pfr分子与信号转导子之间蛋白质-蛋白质相互作用的动力学来定量确定的。光敏色素通常也被称为光调节酶，因为它表明光敏色素A（phyA）可以在体外自磷酸化和磷酸化其他蛋白质，并且在植物中被未知的激酶磷酸化。这些数据被解释为意味着自磷酸化通过增加其降解而减少光敏色素Pfr的量，而在其他氨基酸残基处通过未知激酶的磷酸化降低Pfr形式与其真实信号传导伙伴结合的能力。因此，普遍认为磷酸化负调节phyA控制的信号传导和生理反应。然而，这个话题被相当大的争议所包围，因为基本的生物化学证据仍然缺乏来验证这一理论。在这个建议中，我们概述了一个研究计划，以阐明可逆磷酸化如何调节光敏色素B（phyB）控制的反应在分子水平上。我们发现，phyB，主要的感光细胞调节光形态发生反应在成年植物，是（i）在植物中的多个位点磷酸化，（ii）这种翻译后修饰增加的黑暗逆转率，光不依赖转换的光不稳定的PFR到Pr的形式，从而（iii）结果在减少响应红光。为了测试磷酸化是否也影响phyB与其下游信号传导伙伴的相互作用，我们将进行体外实验以抑制突变的phyB蛋白与光敏色素相互作用因子（PIF）蛋白的结合。我们还打算鉴定那些使phyB去磷酸化的磷酸酶。因此，我们将在细菌细胞中表达候选磷酸酶蛋白，并用从大肠杆菌纯化的重组磷酸酶处理从植物中纯化的磷酸化phyB。最后，我们将确定phyB是否真的作为激酶发挥作用。为此，我们将定义，如果在植物中检测到的磷酸化可以重演至少部分在体外的特征phyB Pr和PFR从昆虫细胞纯化的自磷酸化。如果phyB确实在体外自磷酸化，我们将使用这种方法来确定的催化结构域和ATP结合位点的光感受器的激酶活性所必需的。两者合计，这些实验将帮助我们破译phyB是否是磷酸化本身或其他未知的激酶，或者如果这两种机制都参与介导的感光体的翻译后修饰。

项目成果

Molecular mechanisms for mediating light-dependent nucleo/cytoplasmic partitioning of phytochrome photoreceptors.

• DOI: 10.1111/nph.13207
• 发表时间: 2015-05
• 期刊: The New phytologist
• 作者: Klose C;Viczián A;Kircher S;Schäfer E;Nagy F
• 通讯作者: Nagy F

Article

• DOI: 10.1111/j.1944-8287.2000.tb00151.x
• 发表时间: 2000-10
• 期刊: Economic Geography
• 影响因子: 7
• 作者: Arifin Musthafa

New insights of red light-induced development.

• DOI: 10.1111/pce.12880
• 发表时间: 2017-02
• 期刊: Plant, cell & environment
• 作者: András Viczián;Cornelia Klose;É. Ádám;F. Nagy