REGULATION OF DEVELOPMENT BY PHOSPHORELAY SIGNALING

在发育过程中，细胞必须在不同的途径之间做出选择，它们必须保持适当的发育时机，以便分化和形态发生协调。这是通过感觉信号转导机制对细胞环境进行评估，并整合相关的细胞外和细胞内信息，以便做出适当的决定和保持时机。最近的遗传学证据表明，原核生物的主要信号机制是与一个主要的真核效应器偶联，以调节多细胞发育的时间，以及在网柄藻不同的发育途径和细胞命运之间的选择。我们假设，由感觉组氨酸激酶DHKC和DHKB控制的双组分磷酸继电器通过调节cAMP依赖的蛋白激酶的活性来调节这些重要的发育方面。分子遗传学和生物化学方法被提出，将建立导致多细胞发育的调控的分子机制。具体地说，体内和体外实验将通过表征各组分的自磷酸化、磷酸酶和磷转移活性以及通过确定一种组分对其他组分的活性的影响来检查磷继电器的生化细节。利用细胞类型特异性启动子异位表达刺激非依赖性激活因子或抑制因子，将揭示该通路在哪种细胞类型(S)中发挥作用，以及该通路是否导致细胞间信号耦合分化和形态发生。最后，对抑制物或合成表型的遗传筛选将识别调控途径的新的、甚至可能是新的组成部分，包括上游输入、磷继电调节因子和下游效应器。

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.