SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

正向和负向作用的细胞内通路都协调趋化运动。对于许多真核细胞类型，七个跨膜受体(7-TMR)与异源三聚体G蛋白偶联来激活下游信号网络，从而感受到化学吸引的梯度。除趋化通路外，7-TMR还可以激活多种信号网络，在这些系统中，需要受体磷酸化来关闭下游信号。盘基网柄菌，利用7-TMR-CAR1来感应分泌的cAMP，以协调运动到聚集体中，以响应饥饿。聚集形成的关键是细胞对cAMP刺激不敏感(适应)的过程。此外，细胞必须通过胞外磷酸二酯酶破坏分泌的cAMP配体，才能恢复敏感性。CAR1激活多个网络，包括cAMP合成酶腺苷环化酶A(ACA)，并启动其自身的磷酸化。我们现在证明，在连续信号存在的情况下，表达非磷酸化CAR1突变体的细胞中ACA活性持续存在，这表明至少ACA途径的适应需要受体磷酸化。我们现在已经对这些事件进行了数学建模，并成功地预测了振荡信号参数和发育反应。 AKT和PKBR1是与AGC相关的蛋白激酶，在网柄苔藓的生长发育过程中发挥着重要的趋化作用。AKT有一个PH结构域，通过与PIP3的相互作用被瞬时招募到膜上，而PKBR1是肉豆蔻酰化的，并持续在膜上。AKT和PKBR1的不同位置表明可能存在不同的激活机制和功能。尽管如此，AKT和PKBR1都需要在其激酶(PDK1)结构域和HM基序中进行磷酸化。在生长、叶酸和发育过程中，用cAMP研究了AKT和PKBR1的化学吸引刺激。在这两种情况下，AKT的激活都需要PI3K，而PKBR1的激活不依赖于PIP3。TORC2是AKT和PKBR1趋化和调控的另一个关键模块。在缺乏TORC2组分Pia、RIP3或Lst8的网茎中，PKBR1在PDK1和Hm位点的磷酸化被完全消除，但在叶酸刺激后，AKT在这两个位点上的磷酸化仍然存在。我们还表明，PDK1对AKT和PKBR1的磷酸化需要HM的磷酸化，但与先前的假设相反，TORC2的磷酸化不足以激活AKT或PKBR1。我们还研究了在化学诱导剂刺激后被AKT和PKBR1瞬时磷酸化的蛋白质。有趣的是，在AKT或pkbr1缺失菌株中，底物的磷酸化情况不同，这取决于叶酸或cAMP的刺激。我们建议AKT和PKBR1优先磷酸化最佳底物。我们研究了不同的化学诱导剂激活AKT和PKBR1的途径，发现这两个相关的激酶受到上游因素的不同调控，并通过磷酸化特定的底物发挥不同的功能。总之，这些数据为AKT和PKBR1调控的差异以及PDK1和TORC2对其他系统中多种AGC蛋白激酶调控的复杂性提供了功能证据。 早老素(PS)是G-分泌酶复合体的催化部分。PS/g-分泌酶组分在后生动物中保存良好，但它们在更远的物种中的存在/功能尚未得到解决。由于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对于功能研究至关重要。已经在原始植物中发现了分化蛋白，但实验未能证明g-分泌酶的活性。我们已经在缺乏APP、Notch和其他特征PS/g-分泌酶底物的古代真核生物Dictyostelial中确定了每个PS/g-分泌酶成分的高度分化的同源物。我们发现WT Dictyostelials能够对异位表达的人APP进行淀粉样变性处理，产生Ab40和AB42肽；g-分泌酶缺陷菌株不能产生Ab多肽，但积累APP的加工中间体，这些中间体与哺乳动物细胞中发现的APP的a-和b-CTF中间体共迁移。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，并以细胞自主的方式指定细胞命运；已经有人建议通过PS信号来调节哺乳动物和果蝇细胞的吞噬作用，但尚未得到证实。我们的结果表明，PS信号是一个古老的过程，在后生动物辐射之前就出现了，可能独立于Notch。 PAT蛋白家族在真核生物中已被鉴定，包括脊椎动物、昆虫和阿米巴虫。这些蛋白质具有高度保守的序列组织和细胞内中性脂滴表面的亲和力。目前对各成员的命名缺乏一致性和精确性，更多地源于历史背景，而不是对进化关系和共同功能的认识。在与小鼠基因组命名委员会、雨果基因组命名委员会以及2007年FASEB脂滴会议：中性脂类储存的代谢后果会议的与会者协商后，我们为基因和蛋白质家族成员建立了一个统一的命名法。每个成员将结合根术语Perilipin(Plin)，这是PAT家族的创始基因，不同的基因/蛋白质按顺序编号。

Both positively and negatively acting intracellular pathways coordinate chemotactic movement. For numerous eukaryotic cell-types, chemoattractant gradients are perceived by seven-transmembrane receptors (7-TMRs) coupled to heterotrimeric G proteins to activate downstream signaling networks. 7-TMRs can activate a variety of signaling networks in addition to chemoattractant pathways, and in these systems, receptor phosphorylation is required to turn off downstream signaling. Dictyostelium discoideum, uses the 7-TMR CAR1 to sense secreted cAMP to coordinate movement into aggregates in response to starvation. Essential to aggregate formation is the process where cells desensitize (adapt) to cAMP stimulation. In addition, cells must destroy the secreted cAMP ligand via an extracellular phosphodiesterase to regain sensitivity. CAR1 activates multiple networks including the cAMP-synthesizing enzyme adenylyl cyclase A (ACA) and it initiates its own phosphorylation. We now show that ACA activity persists in cells expressing a non-phosphorylatable CAR1 mutant in the presence of continuous signal, demonstrating that receptor phosphorylation is required, at least, for adaptation of the ACA pathway. We have now mathematically modeled these events and successfully predict oscillatory signaling parameters and developmental response. AKT and PKBR1 are related AGC kinases that play pivotal roles in Dictyostelium chemotaxis during growth and development. AKT has a PH domain and is transiently recruited to the membrane by interaction with PIP3, whereas PKBR1 is myristoylated and is persistently at the membrane. The disparate locations of AKT and PKBR1 indicate the potential for different activation mechanisms and function. Nonetheless, AKT and PKBR1 both require phosphorylation within their kinase (PDK1) domains and within their HM motifs. Chemoattractant stimulation of AKT and PKBR1 was studied during growth, by folate, and during development, by cAMP. Under both situations, AKT activation requires PI3K, while activation of PKBR1 is PIP3-independent. TORC2 is another key module for chemotaxis and regulation for AKT and PKBR1. Phosphorylation of PKBR1 at both the PDK1 and HM sites is completely eliminated in Dictyostelium that lack TORC2 components Pia, RIP3, or lst8, but, following folate stimulation, AKT phosphorylations persist at both sites. We also show that PDK1 phosphorylation of AKT and PKBR1 requires HM phosphorylation, but, in contrast to what has been previously assumed, phosphorylation by TORC2 is insufficient to activate either AKT or PKBR1. We also investigated the proteins transiently phosphorylated by AKT and PKBR1 following chemoattractant stimulation. Interestingly, the phosphorylation profiles of substrates were different in akt- or pkbr1- null strains, depending upon stimulation with either folate or cAMP. We suggest that AKT and PKBR1 preferentially phosphorylate optimal substrates. We have investigated the pathways of activation of AKT and PKBR1 by different chemoattractants, and discovered that these two related kinases are distinctively regulated by upstream factors and function differently through phosphorylating specific substrates. Collectively, these data provide functional proof for differences in the regulation of AKT and PKBR1 and context for complexity of PDK1 and TORC2 regulation of multiple AGC protein kinases in other systems. Presenilin (PS) is the catalytic moiety of the g-secretase complex. PS/g-secretase components are well-conserved among metazoa, but their presence/function in more distant species is not resolved. Since inappropriate g-secretase processing of amyloid precursor protein (APP) in humans is associated with familial Alzheimer's disease, understanding essential elements within each PS/g-secretase component is critical to functional studies. Diverged proteins have been identified in primitive plants but experiments have failed to demonstrate g-secretase activity. We have identified highly diverged orthologs for each PS/g-secretase component in the ancient eukaryote Dictyostelium that lacks APP, Notch, and other characterized PS/ -secretase substrates. We show that WT Dictyostelium is capable of amyloidogenic processing of ectopically expressed human APP to generate Ab40 and Ab42 peptides; strains deficient in g-secretase cannot produce Ab peptides but accumulate processed intermediates of APP that co-migrate with the a- and b-CTF intermediates of APP that are found in mammalian cells. We further demonstrate that Dictyostelium require PS/g-secretase components for phagocytosis and cell-fate specification in a cell-autonomous manner; regulation of phagocytosis by PS-signaling had been suggested, but not proven, for mammalian and Drosophila cells. Our results indicate that PS-signaling is an ancient process that arose prior to metazoan radiation, perhaps independently of Notch. The PAT family of proteins has been identified in eukaryotic species as diverse as vertebrates, insects, and amebazoa. These proteins share a highly conserved sequence organization and avidity for the surfaces of intracellular, neutral lipid storage droplets. The current nomenclature of the various members lacks consistency and precision, deriving more from historic context than from recognition of evolutionary relationship and shared function. In consultation with the Mouse Genomic Nomenclature Committee, the HUGO Genomic Nomenclature Committee, and conferees at the 2007 FASEB Conference on Lipid Droplets: Metabolic Consequences of the Storage of Neutral Lipids, we have established a unifying nomenclature for the gene and protein family members. Each member will incorporate the root term PERILIPIN (PLIN), the founding gene of the PAT family, with the different genes/proteins numbered sequentially.