SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

网柄苔藓的蛋白激酶AKT和PKBR1在趋化作用中起着至关重要的作用，它们被PDK1和TORC2激酶磷酸化并激活，但具有不同的细胞定位区域19、20。AKT具有PI3K/PIP3调节的PH结构域，而PKBR1是肉豆蔻酰化的，并持续存在于膜上。我们使用PI3K/PIP3-、PDK1-和TORC2-信号缺陷或表达AKT和PKBR1磷酸化位点突变的菌株来研究它们的调控。尽管有某些相似之处，AKT和PKBR1具有影响激活和效应器靶向的不同调控路径，其中PDK1发挥着新的核心作用。AKT/PKBR1的激活需要PDK1的磷酸化。虽然TORC2不能激活AKT/PKBR1，但PDK1对AKT/PKBR1的磷酸化需要TORC2的磷酸化，这表明PDK1/TORC2之间存在调节作用。我们还发现，与其他一些系统不同，DictyostelialPDK1不需要PI3K/PIP3，并且，在缺乏PI3K的细胞中，PKBR1，而不是AKT，可以被TORC2/PDK1激活。最后，我们发现AKT和PKBR1具有底物选择性，并鉴定出两种新的脂类相互作用蛋白，它们优先被AKT磷酸化。这些数据揭示了AKT家族激酶的上游和下游的新的调控途径。 早老素(PS)是G-分泌酶复合体13、14的催化部分。人类淀粉样前体蛋白(APP)不适当的G-分泌酶处理与家族性阿尔茨海默病有关。因此，了解每个PS/g-分泌酶成分中的基本成分是至关重要的。我们鉴定了网柄柄菌中每个PS/g-分泌酶组分的高度分化的同源基因，它缺乏内源APP、Notch和其他特征的PS/g-分泌酶底物22。尽管如此，WT Dictyostelials可以准确地处理人类APP，而缺乏分泌酶成分的菌株则不能。我们进一步证明，网柄菌需要PS/g-分泌酶成分来进行吞噬作用和细胞命运的指定，而吞噬作用的调节需要活跃的g-分泌酶，这是哺乳动物巨噬细胞的一条途径，但尚未得到证实。因此，网柄菌可用于识别新的PS/-分泌信号靶点，并为高通量筛选新疗法的小分子文库提供一个独特的系统。 刺激DictyostelialcAMP受体调节GSK3的激活/失活，GSK3介导发育细胞模式6，7。虽然Dictyostelial极化到细胞外cAMP，但GSK3在这一途径中的潜在作用尚未被研究。我们已经证明ZAK1是GSK3的激活酪氨酸激酶，现在已经在GSK323的cAMP激活途径中发现了另一种酪氨酸激酶ZAK2。我们发现，ZAK2和ZAK1分别调节不同分化细胞群中GSK3的酪氨酸磷酸化/激活，ZAK2通过自主和非自主途径调节这些细胞类型的分化。最后，我们证明了Dictyostelius对cAMP的有效极化依赖于ZAK1介导的GSK3酪氨酸磷酸化。DictyostelialZak激酶对GSK3的组合调控指导细胞极性、定向细胞迁移和细胞分化，这些途径使我们对GSK3信号的理解贯穿整个发育过程。 我们开发了小型化的高通量小分子筛选，以确定趋化和发育的新调控途径。通过对有毒化合物的计数器筛选和使用已知抑制剂的剂量-反应分析，这些分析以1536孔板格式进行了验证。我们筛选了4000个小分子，寻找趋化作用的抑制剂和发育的激活剂和抑制剂。已确定的几个化合物是已知的Dictyostelius发育调节因子；其他化合物在哺乳动物细胞中具有假定的靶标，这将有助于它们在Dictyostelius中的生化评估。这些大规模的应用将系统地发现网柄网柄动物中的新因子/途径，并进一步探索复杂后生动物之间共享机制的可能性。

Protein kinases AKT and PKBR1 of Dictyostelium play essential roles for chemotaxis, are phosphorylated and activated by PDK1 and TORC2 kinases, but have different cellular localizing domains19,20. AKT has a PI3K/PIP3-regulated PH domain while PKBR1 is myristoylated and persistently on membranes. We used strains defective for PI3K/PIP3-, PDK1-, and TORC2-signaling or that express phospho-site mutants of AKT and PKBR1 to study their regulations21. Despite certain similarities, AKT and PKBR1 have distinct regulatory paths that impact activation and effector targeting, with PDK1 serving a novel, central role. Activation of AKT/PKBR1 requires phosphorylation by PDK1. Although TORC2 cannot activate AKT/PKBR1, phosphorylation of AKT/PKBR1 by PDK1 requires phosphorylation by TORC2, suggesting regulatory interaction between PDK1/TORC2. We also showed that Dictyostelium PDK1, unlike some other systems, does not require PI3K/PIP3, and, that PKBR1, but not AKT, can be TORC2/PDK1 activated in cells lacking PI3K. Finally, we showed that AKT and PKBR1 exhibit substrate selectivity and identified 2 novel lipid-interacting proteins, preferentially phosphorylated by AKT. These data reveal new regulatory paths upstream and downstream of AKT family kinases. Presenilin (PS) is the catalytic moiety of the g-secretase complex13,14. Inappropriate g-secretase processing of amyloid precursor protein (APP) in humans is associated with familial Alzheimers disease12. Thus, understanding essential elements within each PS/g-secretase component is critical. We identified highly diverged orthologs for each PS/g-secretase component in Dictyostelium, which lacks endogenous APP, Notch, and other characterized PS/-secretase substrates22. Nonetheless, WT Dictyostelium accurately processed human APP, while strains deficient in -secretase components did not22. We further demonstrated that Dictyostelium require PS/g-secretase components for phagocytosis and cell-fate specification and that regulation of phagocytosis required an active g-secretase, a pathway suggested, but not proven, for mammalian macrophages. Dictyostelium may, therefore, serve to identify novel PS/-secretase signaling targets and provide a unique system for high-throughput screening of small molecule libraries for new therapeutics. Stimulation of cAMP receptors in Dictyostelium regulates the activation/de-activation of GSK3, which mediates developmental cell patterning6,7. While Dictyostelium polarize to extracellular cAMP, a potential role for GSK3 in this pathway had not been investigated. We had shown that ZAK1 was an activating tyrosine kinase for GSK3 and have now identified another tyrosine kinase, ZAK2, in the cAMP-activation pathway for GSK323. We found that ZAK2 and ZAK1 separately regulate tyrosine phosphorylation/activation of GSK3 in distinct differentiated cell populations and that ZAK2 acts in both autonomous and non-autonomous pathways to regulate these cell-type differentiations. Finally, we demonstrated that efficient polarization of Dictyostelium toward cAMP depends on ZAK1-mediated tyrosine phosphorylation of GSK3. Combinatorial regulation of GSK3 by ZAK kinases in Dictyostelium guides cell polarity, directional cell migration, and cell differentiation, pathways that extend our understanding of GSK3 signaling throughout the development. We developed miniaturized high-throughput small molecule screens to identify novel regulatory pathways for chemotaxis and development. The assays were validated in 1536-well plate formats by counter screening for toxic compounds and by dose-response analyses using known inhibitors. We screened 4,000 small molecules for inhibitors of chemotaxis and activators and inhibitors of development. Several of the identified compounds are known regulators of Dictyostelium development; other compounds have presumptive targets in mammalian cells, which will assist their biochemical evaluation in Dictyostelium. These large-scale applications will systematically uncover new factors/pathways in Dictyostelium and further explore the potential for shared mechanisms among complex metazoa.