MOLECULAR NETWORKS THAT COORDINATE DIFFERENTIATION

我们计划重点研究一组在盘基骨柱终末分化中起重要作用的基因。从我们正在进行的近饱和诱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协调形态发生和封装的网络中起作用。cAMP依赖性蛋白激酶（PKA）在盘基骨门菌的发育过程中起着关键作用，并在顶生过程中触发孢子的末端分化。由于PKA的活性是由cAMP的内部水平决定的，因此这种环核苷酸的合成和降解的相对速率是其调控的核心。我们建议深入研究的基因之一acrA编码一种新的腺苷酸环化酶，该酶在发育后期起重要作用。我们将对PKA催化亚基过表达和编码发育腺苷酸环化酶acaA和acrA基因失活的菌株进行表征，以确定在没有cAMP的情况下Dictyostelium可以发育到何种程度。acrA仅在两种主要细胞类型（前孢子或前柄细胞）中的一种中表达，将被分析，以测试在终末分化过程中是否通过cAMP进行细胞间通信。在顶点期间观察到的cAMP的增加不仅取决于ACR，而且取决于胞质磷酸二酯酶RegA的抑制。RegA的羧基末端区域有几个位点被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。我们拟深入研究的另外两个基因splA和mkcB编码的蛋白与激活MAP激酶级联反应的raf样蛋白激酶有显著的相似性。这些基因中任何一个的零突变都显著降低了包封的程度。我们将确定这些蛋白激酶的显性激活形式是否可以挽救splA-或mkcB-菌株中孢子形成的阻滞，并使野生型细胞的孢子形成不依赖于前茎衍生的信号。结果将验证一个假设，即分泌的信号肽SDF-2与组氨酸激酶受体DhkA结合，可能通过直接与Sp1A相互作用激活MAP激酶级联。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.