Modulation of Phytochrome B Signalling by Phosphorylation

植物是无根的生物，因此必须适应环境的变化而生长。在调节植物生长的非生物和生物环境因子中，光起着重要的作用。光不仅用于驱动光合作用，也是确保对不断变化的环境进行最佳适应的重要发育线索。为了监测光的波长、强度、方向和持续时间的变化，植物进化出一组光感受器。在红光/远红光下活跃的光感受器被称为光敏色素。光敏色素是二聚体色蛋白，每个分子有一个共价连接的四吡啶色团，它们在生物无活性（Pr =红色吸收）和活性（Pfr =远红色吸收）形式之间循环。导入细胞核的是Pfr构象，它需要与特定的细胞因子相互作用才能启动信号级联。由此可见，光敏色素信号的定量决定于(i)可利用的Pfr分子数量和（ii） Pfr分子与信号转导器之间蛋白-蛋白相互作用的动力学。光敏色素通常也被称为光调节酶，因为研究表明光敏色素a （phyA）可以在体外自磷酸化和磷酸化其他蛋白质，并在植物中被未知的激酶磷酸化。这些数据被解释为自磷酸化通过增加其降解来减少光敏色素Pfr的数量，而未知激酶在其他氨基酸残基上的磷酸化则降低了Pfr形式与其真实信号伙伴结合的能力。因此，人们普遍认为磷酸化负调控phyA控制的信号和生理反应。然而，由于缺乏基本的生物化学证据来验证这一理论，这一主题仍然存在相当大的争议。在本提案中，我们概述了一项研究计划，以阐明可逆磷酸化如何在分子水平上调节光敏色素b （phyB）控制的反应。我们发现phyB，在成年植物中调节光形态形成反应的主要光受体，在植物中被(i)在多个位点磷酸化，（ii）这种翻译后修饰增加了暗还原的速率，这是一种不依赖于光的热不稳定的Pfr到Pr形式的转化，因此（iii）导致对红光的响应性降低。为了测试磷酸化是否也会影响phyB与其下游信号伙伴的相互作用，我们将进行体外实验，以表征突变的phyB蛋白与光敏色素相互作用因子（PIF）蛋白的结合。我们还打算确定那些磷酸酶去磷酸化phyB。因此，我们将在细菌细胞中表达候选磷酸酶蛋白，并用从大肠杆菌中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是否确实作为激酶起作用。为此，我们将通过表征从昆虫细胞中纯化的phyB Pr和Pfr的自磷酸化，来确定在植物中检测到的磷酸化是否可以至少部分地在体外重现。如果phyB确实在体外自磷酸化，我们将使用这种方法来鉴定光感受器激酶活性所必需的催化结构域和ATP结合位点。综上所述，这些实验将帮助我们破译phyB是否被自身或其他未知的激酶磷酸化，或者是否这两种机制都参与了介导光受体的翻译后修饰。

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.