REGULATION OF DEVELOPMENT BY PHOSPHORELAY SIGNALING

通过磷传递信号调节发育

• 批准号: 6181472
• 负责人: CHARLES K SINGLETON
• 金额: $ 21.05万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6181472
• 关键词: Dictyostelium; biological signal transduction; cell growth regulation; cyclic AMP; enzyme activity; eukaryote; gene expression; gene mutation; growth inhibitors; molecular cloning; phosphomonoesterases; phosphorylation; polymerase chain reaction; protein kinase A

During development, cells must make choices between alternative pathways, and they must maintain appropriate developmental timing so that differentiation and morphogenesis are coordinated. This is accomplished by an assessment of the cell's environment through sensory signal transduction mechanisms and integration of the relevant extracellular and intracellular information to allow the appropriate decision to be made and timing to be maintained. Recent genetic evidence suggests that the major signaling mechanism of prokaryotes couples to a major eukaryotic effector to regulate the timing of multicellular development and choices between alternative developmental pathways and cell fates in Dictyostelium. We hypothesize that a two-component phosphorelay, controlled by sensory histidine kinases DHKC and DHKB, regulate these important aspects of development by modulating the activity of cAMP-dependent protein kinase. Molecular genetic and biochemical approaches are proposed that will establish the molecular mechanisms that result in the regulation and control of multicellular development. Specifically, experiments in vivo and in vitro will examine the biochemical details of the phosphorelay by characterizing the autophosphorylation, phosphatase, and phosphotransfer activities of the various components and by determining the effects of one component on the activities of the others. Ectopic expression of stimulus-independent activators or inhibitors of the pathway, using cell type-specific promoters, will reveal in which cell type(s) the pathway functions and if the pathway results in cell to cell signaling to couple differentiation and morphogenesis. Finally genetic screens for suppressers or synthetic phenotypes will identify new and perhaps novel components of the regulatory pathway, including upstream inputs, phosphorelay regulators, and downstream effectors.

在发育过程中，细胞必须在不同的途径之间做出选择，并且必须保持适当的发育时间，以协调分化和形态发生。这是通过感觉信号转导机制对细胞环境的评估以及相关的细胞外和细胞内信息的整合来完成的，从而做出适当的决定并保持时间。最近的遗传证据表明，Dictyostelium中原核生物的主要信号机制与真核生物的主要效应物偶联，以调节多细胞发育的时间和选择不同的发育途径和细胞命运。我们假设，由感觉组氨酸激酶DHKC和DHKB控制的双组分磷接力通过调节camp依赖性蛋白激酶的活性来调节这些重要的发育方面。提出了分子遗传学和生物化学方法，将建立导致多细胞发育调控的分子机制。具体来说，体内和体外实验将通过表征各种成分的自磷酸化、磷酸酶和磷转移活性，并通过确定一种成分对其他成分活性的影响，来检查磷接力的生化细节。使用细胞类型特异性启动子，异位表达刺激非依赖性通路激活因子或抑制剂，将揭示该通路在哪种细胞类型中起作用，以及该通路是否导致细胞间信号传导以偶联分化和形态发生。最后，抑制因子或合成表型的遗传筛选将确定调控途径的新成分，包括上游输入、磷接力调节因子和下游效应因子。