SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

控制生长和发育的信号通路

• 批准号: 7967848
• 负责人: ALAN R KIMMEL
• 金额: $ 104.68万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967848
• 关键词: Actins; Amino Acids; Amyloid beta-Protein Precursor; Avidity; Bacteria; Biochemical; Cell physiology; Cells; Chemotactic Factors; Chemotaxis; Complex; Conserved Sequence; Consultations; Cyclic AMP; Cyclic AMP Receptors; Cytoskeleton; Data; Development; Dictyostelium; Dictyostelium discoideum; Distant; Drosophila genus; Elements; Enzymes; Eukaryota; Eukaryotic Cell; Event; Family; Family member; Folate; GTP-Binding Proteins; Gene Proteins; Genes; Genome; Genomics; Growth; Growth Factor; Growth and Development function; Haploidy; Heterotrimeric GTP-Binding Proteins; Human; Individual; Insecta; Laboratories; Ligands; Lipids; Location; Mammalian Cell; Mediating; Membrane; Metabolic; Modeling; Molecular; Molecular Genetics; Movement; Mus; Natural Immunity; Neurotransmitters; Nomenclature; Nutrient; Organism; Orthologous Gene; PDPK1 gene; PH Domain; Pathway interactions; Peptides; Phagocytosis; Phosphorylation; Phosphotransferases; Physiological Processes; Plant Roots; Plants; Play; Population; Process; Protein Family; Protein Kinase; Proteins; Proto-Oncogene Proteins c-akt; RIPK3 gene; Radiation; Recruitment Activity; Regulation; Relative (related person); Research; Resources; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Source; Starvation; Stimulus; Surface; System; Techniques; Transmembrane Domain; Vertebrates; adenylyl cyclase A; amyloid precursor protein processing; cell fate specification; cell growth; cell type; chemokine; chemokine receptor; extracellular; familial Alzheimer disease; fungus; genetic manipulation; macrophage; member; morphogens; mutant; neutrophil; notch protein; perilipin; phosphoric diester hydrolase; presenilin; receptor; receptor coupling; research study; response; secretase; symposium

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.

正向和负向作用的细胞内途径都协调趋化运动。 对于许多真核细胞类型，化学引诱剂梯度由与异三聚体 G 蛋白偶联的七次跨膜受体 (7-TMR) 感知，以激活下游信号网络。除了化学引诱途径外，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传导。 盘基网柄菌 (Dictyostelium discoideum) 使用 7-TMR CAR1 来感知分泌的 cAMP，以协调运动以响应饥饿而形成聚集体。 聚集体形成的关键是细胞对 cAMP 刺激脱敏（适应）的过程。 此外，细胞必须通过细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 合成酶腺苷酸环化酶 A (ACA)，并启动自身磷酸化。 我们现在表明，在存在连续信号的情况下，ACA 活性在表达不可磷酸化 CAR1 突变体的细胞中持续存在，这表明至少需要受体磷酸化才能适应 ACA 途径。我们现在已经对这些事件进行了数学建模，并成功预测了振荡信号参数和发育反应。 AKT 和 PKBR1 是相关的 AGC 激酶，在盘基网柄菌生长和发育过程中的趋化作用中发挥关键作用。 AKT 具有 PH 结构域，通过与 PIP3 相互作用而短暂地募集到膜上，而 PKBR1 被肉豆蔻酰化并持续存在于膜上。 AKT 和 PKBR1 的不同位置表明不同激活机制和功能的潜力。尽管如此，AKT 和 PKBR1 都需要在其激酶 (PDK1) 结构域和 HM 基序内进行磷酸化。研究了生长过程中叶酸对 AKT 和 PKBR1 的趋化刺激作用以及发育过程中 cAMP 的趋化作用。在这两种情况下，AKT 激活都需要 PI3K，而 PKBR1 的激活不依赖于 PIP3。 TORC2 是 AKT 和 PKBR1 趋化性和调节的另一个关键模块。在缺乏 TORC2 成分 Pia、RIP3 或 lst8 的盘基网柄菌属中，PDK1 和 HM 位点上的 PKBR1 磷酸化被完全消除，但在叶酸刺激后，两个位点上的 AKT 磷酸化仍然存在。我们还表明，AKT 和 PKBR1 的 PDK1 磷酸化需要 HM 磷酸化，但是与之前的假设相反，TORC2 的磷酸化不足以激活 AKT 或 PKBR1。我们还研究了趋化剂刺激后 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，底物的磷酸化谱是不同的，具体取决于叶酸或 cAMP 的刺激。我们建议 AKT 和 PKBR1 优先磷酸化最佳底物。我们研究了不同趋化剂激活 AKT 和 PKBR1 的途径，发现这两种相关激酶受到上游因子的独特调节，并通过磷酸化特定底物发挥不同的功能。总的来说，这些数据为 AKT 和 PKBR1 调节的差异提供了功能证据，并为其他系统中多种 AGC 蛋白激酶的 PDK1 和 TORC2 调节的复杂性提供了背景。 早老素 (PS) 是 g-分泌酶复合物的催化部分。 PS/g 分泌酶成分在后生动物中非常保守，但它们在更遥远的物种中的存在/功能尚未解决。由于人类淀粉样前体蛋白 (APP) 的 g 分泌酶加工不当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元素对于功能研究至关重要。原始植物中已鉴定出不同的蛋白质，但实验未能证明 g 分泌酶活性。我们已经在缺乏 APP、Notch 和其他特征性 PS/g 分泌酶底物的古代真核生物盘基网柄菌中鉴定出每个 PS/g 分泌酶成分的高度分化的直系同源物。我们证明，WT 盘基网柄菌能够对异位表达的人 APP 进行淀粉样蛋白加工，生成 Ab40 和 Ab42 肽；缺乏g分泌酶的菌株不能产生Ab肽，但会积累加工后的APP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中间体共同迁移。我们进一步证明盘基网柄菌需要 PS/g 分泌酶成分来以细胞自主的方式进行吞噬作用和细胞命运规范；对于哺乳动物和果蝇细胞，有人提出 PS 信号传导对吞噬作用的调节，但尚未得到证实。我们的结果表明，PS 信号传导是一个古老的过程，出现在后生动物辐射之前，可能独立于 Notch。 PAT 蛋白家族已在脊椎动物、昆虫和变形虫等多种真核物种中得到鉴定。这些蛋白质具有高度保守的序列组织和对细胞内中性脂质存储液滴表面的亲合力。当前各成员的命名法缺乏一致性和精确性，更多地源自历史背景，而不是对进化关系和共享功能的认识。经与小鼠基因组命名委员会、HUGO 基因组命名委员会以及 2007 年 FASEB 脂滴会议：中性脂质储存的代谢后果的与会者协商，我们为基因和蛋白质家族成员建立了统一的命名法。每个成员都将包含根术语 PERILIPIN (PLIN)，即 PAT 家族的创始基因，不同的基因/蛋白质按顺序编号。