MOLECULAR NETWORKS THAT COORDINATE DIFFERENTIATION

我们计划集中在一组基因上，这些基因在网柄网柄藻的末端分化中起着重要的作用。从我们正在进行的近饱和突变筛选中发现的基因中选择了三个基因。这些基因预测产物中的保守结构域表明，它们可能在一个协调形态发生和封装的网络中发挥作用。CAMP依赖的蛋白激酶PKA在网柄苔藓的整个发育过程中起着至关重要的作用，并在终末分化过程中触发孢子的终末分化。由于PKA的活性是由cAMP的内部水平决定的，所以这种环核苷酸的合成和降解的相对速度是其调控的核心。我们打算深入研究的基因之一acrA编码一种新的腺酰环化酶，它在发育后期起着至关重要的作用。我们将对PKA催化亚单位过度表达以及编码发育性腺酰环化酶的两个基因ACAA和AcrA失活的菌株进行鉴定，以确定Dictyostelials在没有cAMP的情况下可以发育到什么程度。将分析AcrA在两种主要细胞类型中的一种，即前孢子或前柄细胞中的表达，以测试在末端分化过程中是否存在通过cAMP进行的细胞间通讯。在顶峰过程中观察到的cAMP的增加不仅依赖于ACR，而且还依赖于细胞质磷酸二酯酶RegA的抑制。在RegA的羧基末端结构域中，有几个位点被MAP激酶磷酸化。MAP激酶ERK2对这些位点的磷酸化导致cAMP的增加。我们建议深入研究的另外两个基因，splA和mkcB，编码的蛋白质与激活MAP激酶级联的Raf样蛋白激酶有很大的相似性。这两个基因中的任何一个的零突变都会极大地减少包裹体的程度。我们将确定这些蛋白激酶的主要激活形式是否可以挽救Spla或MkcB菌株的孢子形成障碍，并使野生型细胞的孢子形成独立于前柄来源的信号。这一结果将检验这样一个假设，即与组氨酸激酶受体DhkA结合的分泌型信号肽SDF-2可能通过直接与Sp1a相互作用激活MAP激活级联反应。总之，这些研究将有助于建立一个信号转导途径，可以作为其他系统研究的模型。

We plan to focus on a set of genes that play essential roles in terminal differentiation of Dictyostelium. Three genes were chosen from among those discovered in our on-going near- saturation mutagenesis screen. Conserved domains in the predicted products of these genes suggest that they may function in a network that coordinates morphogenesis and encapsulation. The cAMP dependent protein kinase, PKA, plays a critical role throughout development in Dictyostelium and triggers terminal differentiation of spores during culmination. Since the activity of PKA is determined by the internal levels of cAMP, the relative rates of synthesis and degradation of this cyclic nucleotide are central to its regulation. One of the genes we propose to study in depth, acrA, encodes a novel adenylyl cyclase that plays an essential role during late development. We will characterize strains in which the catalytic subunit of PKA is overexpressed and both of the genes encoding developmental adenylyl cyclases, acaA and acrA, are inactivated to establish the extent to which Dictyostelium can develop without cAMP. Expression of acrA in only one of the two major cell types, prespore or prestalk cells, will be analyzed to test whether there is intercellular communication through cAMP during terminal differentiation. The increase in cAMP that is observed during culmination not only depends on ACR but also on the inhibition of the cytoplasmic phosphodiesterase RegA. There are several sites in the carboxy- terminal domain of RegA that are phosphorylated by a MAP kinase. Phosphorylation of these sites by the MAP kinase ERK2 results in an increase in cAMP. The other two genes that we propose to study in depth, splA and mkcB, encode proteins that show significant similarity to Raf-like protein kinases that activate MAP kinase cascades. Null mutations in either of these genes reduce the extent of encapsulation dramatically. We will determine whether dominant activated forms of these protein kinases can rescue the block in spore formation in either splA- or mkcB- strain as well as make sporulation of wild type cells independent of prestalk derived signals. The results will test the hypothesis that a secreted signal peptide, SDF-2, that binds to the histidine kinase receptor, DhkA, activates the MAP kinase cascade, possibly by directly interacting with Sp1A. Together these studies will help to establish a signal transduction pathway that can serve as a model for studies in other systems.