Phosphoinositide-calcium Signaling In Cell Regulation

细胞调节中的磷酸肌醇-钙信号转导

• 批准号: 7594120
• 负责人: TAMAS BALLA
• 金额: $ 96.96万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/7594120
• 关键词: 1,2-diacylglycerol; 1-Phosphatidylinositol 3-Kinase; 1-Phosphatidylinositol 4-Kinase; Actin-Binding Protein; Actins; Adaptor Signaling Protein; Affect; Apoptosis; Apoptotic; Binding; Biochemical Reaction; Branchial arch structure; Calcium; Calcium Channel; Calcium Signaling; Capsaicin; Catalytic Domain; Cell membrane; Cell physiology; Cell surface; Cells; Cellular Membrane; Class; Clathrin; Clathrin Adaptors; Code; Collaborations; Commit; Complex; Connexin 43; Data; Defect; Development; Developmental Process; Diglycerides; Dissociation; Dose; Down-Regulation; Dynamin; Embryo; Embryonic Development; Employee Strikes; Enzymes; Equilibrium; Erinaceidae; Esthesia; Event; Exons; FGF8 gene; Family; Fibroblast Growth Factor; Fibroblast Growth Factor 8; Fibroblast Growth Factor Receptors; Fluorescence; Focus Groups; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Silencing; Genes; Growth Factor; Heterodimerization; Hormones; Hydrolysis; Individual; Injection of therapeutic agent; Inositol; Investigation; Ion Channel; LY294002; Laboratories; Life; Lipids; MAP Kinase Gene; Mammalian Cell; Mediating; Membrane; Membrane Transport Proteins; Metabolism; Mitogen-Activated Protein Kinases; Molecular; Netherlands; Neurotransmitters; Numbers; Pathway interactions; Pattern; Pectoral; Pharmaceutical Preparations; Phenotype; Phosphatidylinositols; Phospholipase C; Phospholipids; Phosphoric Monoester Hydrolases; Process; Protein Isoforms; Protein Kinase; Proteins; RNA Interference; RNA Splicing; Receptor Activation; Receptor Protein-Tyrosine Kinases; Regulation; Reporting; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Sirolimus; Specificity; Spottings; Staging; Surface; Tacrolimus Binding Protein 1A; Time; Transferrin Receptor; Yeasts; Zebrafish; base; cell motility; coated pit; follow-up; genetic regulatory protein; hindbrain; human FRAP1 protein; inhibitor/antagonist; intercellular communication; interest; kinase inhibitor; phosphatidylinositol 3,4,5-triphosphate; phosphatidylinositol phosphate, PtdIns(4,5)P2; platelet protein P47; receptor internalization; research study; response; second messenger; small molecule; tool

We have previously identified four isoforms of the phosphatidylinositol 4-kinase enzymes (PI4Ks) that are expressed in Zebrafish and characterized their expression patterns during embryonic development. Although these enzymes catalyze the same biochemical reaction, their intracellular localization and presumably their regulation are different. Based on studies in yeast, these enzymes assume non-redundant functions. To identify developmental processes and signal transduction pathways in which specific phosphatidylinositol 4-kinases (PI4Ks) have pivotal roles, the expression of these enzymes were down-regulated by injection of morpholinos targeting the splicing of exons coding for the catalytic domains of the individual enzymes. In the first set of studies the role of PI4KIIIalpha was investigated. Down-regulation of PI4KIIIalpha resulted in multiple developmental abnormalities most severely affecting the hindbrain and the branchial arches. These changes were associated with highly increased apoptotic activity and were partially mimicked by treatment of the embryos with the PI 3-kinase inhibitor, LY294002. The most striking phenotype of PI4KIIIalpha down-regulation was the lack of pectoral fin development. Downstream targets of the FGF8 signaling pathway, such as MKP3 (a MAP kinase phosphatase) and Sef were strongly inhibited in the morphant embryos especially in the branchial arches and pectoral finbuds, whereas genes mediating hedgehog (Hh) signaling were only marginally affected. In HEK293T cells downregulation of PI4KIIIalpha by RNAi mediated gene silencing, inhibited constitutive Akt activation and potentiated the FGF- but not EGF-stimulated MAPK response. These data suggest that PI4KIIIalpha is critically important in supplying phosphoinositides for PI 3-kinases to activate anti-apoptotic pathways and that its down-regulation changes the balance between the MAPK and PI3K signaling pathways in FGF signaling causing a defect in pectoral fin development. Further studies are in progress to define the molecular details of how this PI4K isoform contributes to signaling from FGF receptors. Phosphatidylinositol 4,5-bisphosphate PtdIns(4,5)P2 is the major phosphoinositide species in mammalian cells that has been associated with numerous molecular events critical for cellular signaling. PtdIns(4,5)P2 is hydrolyzed by phospholipase C enzymes to generate diacylglycerol and Ins(1,4,5)P3, two pivotal second messengers. PtdIns(4,5)P2 is also converted by Class I PI 3-kinases to PtdIns(3,4,5)P3, another important membrane-bound messenger molecule. PtdIns(4,5)P2 directly interacts with several ion channels, transporters, actin binding proteins, and regulates enzymes such as PLC and PLD. A number of molecules that are part of the receptor internalization machinery have also been shown to contain inositide binding domains but the exact lipid species that regulates them in the cell has not been firmly established. It is a major challenge to understand how a single type of molecule is able to regulate so many processes simultaneously and perhaps independently within the plasma membrane (PM). Last year we reported on the development of a new strategy to promptly regulate membrane PtdIns(4,5)P2 levels by a drug-inducible membrane targeting strategy based on the heterodimerization of the FRB domain of mTOR and FKBP12. In this approach the enzyme of interest, in this case a type-IV phosphoinositide 5-phosphatase (5-ptase,) was fused to the FKBP12 protein and upon addition of rapamycin (Rapa) the enzyme rapidly translocates to the membrane where its binding partner, the FRB domain, is targeted. We have shown that this manipulation rapidly eliminates PtdIns(4,5)P2 from the plasma membrane. This approach has been applied to study the PtdIns(4,5)P2 regulation of a number of ion channels and transporters. In collaboration with Dr. Rohacs, the phosphoinositide regulation of the TrpV1 and TrpM8 channels (the calcium channels responsible for hot and cold sensations, respectively) was compared. Intriguingly, while both of these channels are regulated by phosphoinositides, the regulation of TrpM8 is relatively simple, as it requires PtdIns(4,5)P2 for activity, and PtdIns4P cannot substitute for the former lipid. In contrast, TrpV1 channels are inhibited as well as stimulated by PtdIns(4,5)P2 depending on the dose of capsaicin, and PtdIns4P is also able to support its calcium channel activity. The molecular basis for this regulatory difference between these channels is currently under further investigation in the laboratory of Dr. Rohacs. In similar experiments we also showed that the Orai1 channel that supports capacitative calcium entry does not require PtdIns(4,5)P2 for its activity. In another collaboration with the group of Dr. Moolenaar in the Netherlands, the regulation of the GAP junction protein, connexin43, by PtdIns(4,5)P2 has been revealed. These studies showed that transport of small molecules from cell-to-cell via GAP junctions formed by the molecule, connexin43, requires PtdIns(4,5)P2 in the plasma membrane, and that decreasing the level of this lipid rapidly shuts down this form of intercellular communication. The Moolenaar group also showed that connexin43 regulation by PtdIns(4,5)P2 occurs via receptor activation of PLCbeta3, which associates with the connexin43 protein indirectly. Last year we also reported that PtdIns(4,5)P2 was required for the internalization of transferrin receptors. This observation was followed up in a collaboration with the group of Dr. De Camilli in which the role of PtdIns(4,5)P2 in the plasma membrane recruitment of several clathrin adaptor proteins was investigated. PtdIns(4,5)P2 depletion resulted in a rapid loss of clathrin puncta from the plasma membrane, which correlated with a massive dissociation of endocytic adaptors. The remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which are concentrated at late-stage clathrin-coated pits and in lamellipodia, also dissociated from the plasma membrane, and these changes correlated with an arrest of motility at the cell edge. These findings demonstrate the critical importance of PtdIns(4,5)P2 in clathrin coat dynamics and Arp2/3-dependent actin regulation.

我们先前已经鉴定出四种在斑马鱼中表达的磷脂酰肌醇4-激酶（PI4K）的四种同工型，并在胚胎发育过程中表征了它们的表达模式。尽管这些酶催化了相同的生化反应，但它们的细胞内定位和大概它们的调节是不同的。 根据酵母的研究，这些酶采用非冗余功能。为了鉴定发育过程和信号转导途径，在这种过程中，特定的磷脂酰肌醇4-激酶（PI4K）具有关键作用，这些酶的表达通过针对编码单个enzymes催化域的外显子的拼接的形态片的注射来降低这些酶的表达。 在第一组研究中，研究了PI4Kiiialpha的作用。 PI4KIIIALPHA的下调导致多种发育异常最严重影响后脑和分支拱形。这些变化与高度增加的凋亡活性有关，并通过用Pi 3-激酶抑制剂LY294002处理胚胎来部分模仿。 PI4Kiiialpha下调的最引人注目的表型是缺乏胸鳍发育。 FGF8信号通路的下游靶标，例如MKP3（MAP激酶磷酸酶）和SEF，在形态胚胎中尤其是在分支拱门和胸骨鳍中受到强烈抑制，而介导HedgeHog（HH）信号传导的基因仅受到质量影响。在HEK293T细胞中，通过RNAi介导的基因沉默对PI4Kiiialpha的下调，抑制了组成型AKT激活，并增强了FGF-但不增强EGF刺激的MAPK响应。这些数据表明，PI4Kiiialpha对于为PI 3-激酶提供磷酸肌醇至关重要，以激活抗凋亡途径，并且其下调改变了MAPK和PI3K信号传导途径之间在FGF中引起pectoral Fin fin发育不足的FGF信号之间的平衡。 进一步的研究正在进行中，以定义该PI4K同工型如何促进FGF受体信号传导的分子细节。 磷脂酰肌醇4,5-双磷酸PTDINS（4,5）P2是哺乳动物细胞中主要的磷酸肌醇物种，与许多分子事件有关，对细胞信号传导至关重要。 PTDINS（4,5）P2被磷脂酶C酶水解，以生成二酰基甘油和INS（1,4,5）P3，两个关键的第二章voters。 PTDINS（4,5）P2也通过I类PI 3-激酶转换为PTDINS（3,4,5）P3，这是另一个重要的膜结合的质量分子。 PTDINS（4,5）P2直接与多种离子通道，转运蛋白，肌动蛋白结合蛋白相互作用，并调节PLC和PLD等酶。 许多是受体内在化机制的一部分的许多分子也已显示包含inositide结合结构域，但是调节它们在细胞中的确切脂质物种尚未牢固确定。了解单一类型的分子如何能够同时和可能独立于质膜（PM）独立地调节这么多过程是一个主要的挑战。 去年，我们报告了一种新策略，该战略通过基于MTOR和FKBP12的FRB域的异二映基于药物诱导的膜靶向策略来迅速调节膜PTDIN（4,5）P2水平。 在这种方法中，感兴趣的酶在这种情况下，将IV型磷酸肌醇5-磷酸酶（5-PTase，，）融合到FKBP12蛋白上，并在添加雷帕霉素（Rapa）后，酶迅速易位到膜的膜，其粘合伙伴，FRB Domain，目标是目标。我们已经表明，这种操作迅速消除了质膜中的PTDIN（4,5）P2。 该方法已应用于研究许多离子通道和转运蛋白的PTDINS（4,5）P2调节。与Rohacs博士合作，比较了TRPV1和TRPM8通道的磷酸肌醇调节（分别对负责热和冷感觉的钙通道）进行了合作。 有趣的是，尽管这两个通道都受磷酸肌醇调节，但TRPM8的调节相对简单，因为它需要PTDINS（4,5）P2才能进行活性，而PTDINS4P无法代替以前的脂质。相反，根据辣椒素的剂量，PTDINS（4,5）P2抑制了TRPV1通道，并且PTDINS4P也能够支持其钙通道活性。这些通道之间这种调节差异的分子基础目前正在Rohacs博士的实验室进行进一步研究。在类似的实验中，我们还表明，支持电容性钙进入的ORAI1通道不需要PTDINS（4,5）P2才能进行活性。 在与荷兰的Moolenaar博士的另一次合作中，PTDINS（4,5）P2对GAP连接蛋白Connexin43的调节。 这些研究表明，小分子通过该分子形成的connexin43形成的小分子从细胞到细胞的间隙连接，需要质膜中的PTDINS（4,5）P2，这会迅速降低这种脂质的水平。 Moolenaar组还表明，PTDINS（4,5）P2通过PLCBETA3的受体激活发生了Connexin43（4,5）P2，该受体激活与Connexin43蛋白间接相关。 去年，我们还报道了转铁蛋白受体的内在化需要PTDINS（4,5）P2。 在与Camilli博士的一组合作中进行了观察，其中PTDINS（4,5）P2在几种网状蛋白适配蛋白的质膜募集中的作用进行了研究。 PTDINS（4,5）P2耗竭导致质膜迅速损失网状蛋白，这与内吞转移器的大量分离有关。细胞表面的剩余网状蛋白斑点只有弱荧光，并且随着时间的流逝而静态。 ARP2/3肌动蛋白调节络合物的Dynamin和P20亚基，它们集中在晚期网状蛋白涂层的凹坑和Lamellipodia中，也与质膜分离，这些变化与细胞边缘的运动性的禁止相关。这些发现证明了PTDINS（4,5）P2在网格蛋白外套动力学和ARP2/3依赖性肌动蛋白调节中的重要性。