Phytochrome A: Structure/Function and Signaling Pathways

描述（由申请人提供）：感觉光感受器的五元光敏色素（phy）家族（phyA至phyE）对信息光信号的感知启动了细胞内转导过程，该过程最终导致核基因的表达改变，这些核基因指导生长和发育反应，称为光形态发生，适合于主要环境。该计划的长期目标是确定这一过程发生的细胞，分子和生化机制。目前的数据表明，转导过程涉及光激活的光感受器分子从细胞质到细胞核的快速易位，在细胞核中，它与bHLH转录因子家族的一部分成员（称为光敏色素相互作用因子（PIF））发生物理相互作用，诱导靶基因的转录反应。最近的证据表明，PIF家族的成员共同抑制在年轻的黑暗生长的幼苗的光形态建成，光激活的phy逆转这种抑制诱导快速降解的PIF分子在初始暴露于光。该过程涉及相互作用的bHLH蛋白的快速的、phy诱导的磷酸化，随后通过泛素蛋白酶体系统降解。尽管取得了这些进展，但仍存在一些核心问题，包括负责PIF磷酸化的蛋白激酶的身份以及phy-PIF信号通路的主要靶基因。我们建议在这里解决这些不足之处，拟南芥作为一个模型系统。（B）剖析在phy-PIF信号传导界面处发生的分子和生物化学交易，包括负责PIF蛋白的phy诱导的磷酸化的蛋白激酶组分的分子鉴定;和（c）确定与phy相互作用的bHLH因子与其靶基因之间的转录界面。实验方法将包括：（a）对pif突变组合进行遗传筛选和反向遗传分析，以剖析家族成员的差异和冗余功能，并确定其他因素;（B）对转基因植物中phy和PIF蛋白的靶向、位点特异性、错义突变体进行分子遗传学功能分析，以确定相互作用所需的残基，细胞内的磷酸化和泛素化;（c）蛋白质相互作用筛选，包括酵母“三杂交体”筛选和来自拟南芥属的亲和纯化复合物的质谱分析，目的在于鉴定与phy和/或PIF蛋白质结合的组分，在体内两种分子之间的光诱导信号传递期间和之后;和（d）整合的全基因组表达谱和染色质免疫沉淀（ChIP）分析，PIF-bHLH转录因子在phy调节的转录网络中的主要靶点。 公共卫生相关性：了解真核细胞感知和处理细胞外信息信号的分子和细胞机制的全谱仍然是生物医学研究的中心目标。这里提出的实验系统和策略有可能对这一目标做出重大贡献，通过定义感觉光感受器将感知到的光信号从环境转换到它控制的转录网络的基本机制。该计划的成功执行将提供对核定位信号中枢功能的机械洞察，其中多个相关的激活受体会聚以直接将其信号信息传递给多个相关的转录因子，这些转录因子负责调节反应途径中的初级转录网络。

DESCRIPTION (provided by applicant): The perception of informational light signals by the five-membered phytochrome (phy) family of sensory photoreceptors (phyA through phyE) initiates an intracellular transduction process that culminates in the altered expression of nuclear genes that direct growth and developmental responses, termed photomorphogenesis, appropriate to the prevailing environment. The long-term goal of this program is to define the cellular, molecular and biochemical mechanisms by which this process occurs. Current data indicate that the transduction process involves rapid translocation of the light-activated photoreceptor molecule from the cytoplasm to the nucleus, where it interacts physically with a subset of members of the bHLH transcription- factor family, termed phytochrome-interacting factors (PIFs), inducing transcriptional responses in target genes. Recent evidence shows that members of the PIF family collectively repress photomorphogenesis in young dark-grown seedlings, and that photoactivated phy reverses this repression by inducing rapid degradation of the PIF molecules upon initial exposure to light. This process involves rapid, phy-induced phosphorylation of the interacting bHLH protein, followed by degradation via the ubiquitin proteasome system. Despite these advances, several central questions remain, including the identity of the protein kinase responsible for PIF phosphorylation and the primary target genes of the phy-PIF signaling pathway. We propose to address these deficiencies here, using Arabidopsis as a model system. The specific objectives of this proposal are: (a) To define the functions of the different PIF-family members in controlling early, post- germinative seedling development; (b) To dissect the molecular and biochemical transactions occurring at the phy-PIF signaling interface, including molecular identification of the protein kinase component(s) responsible for phy-induced phosphorylation of the PIF proteins; and (c) To define the transcriptional interface between the phy-interacting bHLH factors and their target genes. The experimental approaches will include: (a) Genetic screens and reverse-genetic analyses of pif-mutant combinations to dissect out the differential and redundant functions of the family members and to identify additional factors; (b) Molecular-genetic functional analyses of targeted, site-specific, missense mutants of the phy and PIF proteins in transgenic plants to define residues necessary for interaction, phosphorylation and ubiquitylation in the cell; (c) Protein-interaction screens, including a yeast "tribrid" screen and mass-spectrometry of affinity-purified complexes from Arabidopsis, directed at identifying components that associate with either phy and/or PIF proteins before, during and after the light- induced signaling transaction between the two molecules in vivo; and (d) Integrated genome-wide expression profiling and chromatin-immunoprecipitation (ChIP) analyses aimed at identifying direct, primary targets of the PIF-bHLH transcription factors in the phy-regulated transcriptional network. PUBLIC HEALTH RELEVANCE: Understanding the full spectrum of molecular and cellular mechanisms by which eukaryotic cells perceive and transduce extracellular informational signals remains a central goal of biomedical research. The experimental system and strategies proposed here have the potential to contribute significantly to this goal, by defining the fundamental mechanism by which a sensory photoreceptor transduces perceived light signals from the environment to the transcriptional network that it controls. Successful execution of this program will provide mechanistic insight into the functioning of a nuclear-localized signaling hub where multiple, related, activated receptors converge to directly communicate their signaling information to multiple, related transcription factors that are responsible for regulating the primary transcriptional network in the response pathway.