Phytochrome A: Structure/Function and Signaling Pathways

光敏色素 A：结构/功能和信号传导途径

• 批准号: 8004994
• 负责人: Peter H. Quail
• 金额: $ 36.98万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8004994
• 关键词: Address; Affinity; Arabidopsis; Biochemical; Biological Assay; Biological Models; Biological Process; Biomedical Research; Cell Nucleus; Cells; Complex; Cytoplasm; Data; Development; ERG gene; Environment; Eukaryotic Cell; Exposure to; Family; Family member; Funding; Gene Expression; Gene Targeting; Genes; Genetic Screening; Goals; Growth; Light; Mass Spectrum Analysis; Molecular; Molecular Genetics; Molecular Profiling; Nuclear; Pathway interactions; Perception; Phosphorylation; Photoreceptors; Phytochrome; Procedures; Process; Protein Kinase; Proteins; Repression; Research; Seedling; Sensory; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Structure; System; Transgenic Plants; Ubiquitin; Yeasts; chromatin immunoprecipitation; combinatorial; extracellular; genetic analysis; genome-wide; in vivo; insight; member; multicatalytic endopeptidase complex; mutant; phyA phytochrome; phyE; positional cloning; programs; promoter; protein complex; public health relevance; receptor; response; transcription factor

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.

描述（由申请人提供）：五成员光敏色素（phy）家族的感觉光感受器（phyA至phyE）对信息光信号的感知启动细胞内转导过程，最终导致核基因表达改变，从而指导生长和发育反应，称为光形态发生，适合于当前环境。这个程序的长期目标是定义细胞，分子和生化机制，通过这个过程发生。目前的数据表明，转导过程涉及光激活光受体分子从细胞质到细胞核的快速易位，在那里它与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））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暗生长幼苗的光形态发生，而光激活物理通过诱导PIF分子在初始暴露于光下的快速降解来逆转这种抑制。这个过程包括快速的，物理诱导的相互作用bHLH蛋白磷酸化，随后通过泛素蛋白酶体系统降解。尽管取得了这些进展，但仍存在几个核心问题，包括负责PIF磷酸化的蛋白激酶的身份以及物理-PIF信号通路的主要靶基因。我们建议在这里解决这些不足，以拟南芥为模型系统。本提案的具体目标是：(a)确定不同pif家族成员在控制早期发芽后幼苗发育方面的功能；(b)剖析发生在物理-PIF信号界面的分子和生化交易，包括对负责物理诱导的PIF蛋白磷酸化的蛋白激酶成分的分子鉴定；(c)确定bHLH因子与其靶基因之间的转录界面。实验方法将包括：(a)基因筛选和pif突变组合的反向遗传分析，以剖析家庭成员的差异和冗余功能，并查明其他因素；(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，以确定细胞中相互作用、磷酸化和泛素化所必需的残基；(c)蛋白质相互作用筛选，包括酵母“三杂交”筛选和拟南芥亲和纯化复合物的质谱分析，旨在确定在体内两种分子之间光诱导信号交易之前、期间和之后与phy和/或PIF蛋白质相关的成分；(d)整合全基因组表达谱和染色质免疫沉淀（ChIP）分析，旨在确定PIF-bHLH转录因子在生理调节转录网络中的直接、主要靶点。