Phosphoinositide-calcium Signaling In Cell Regulation

细胞调节中的磷酸肌醇-钙信号转导

• 批准号: 6541099
• 负责人: TAMAS BALLA
• 金额: --
• 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/6541099
• 关键词: alcohol phosphotransferase; androstane compound; biological signal transduction; calcium flux; cell growth regulation; cell line; diacylglycerols; enzyme activity; enzyme mechanism; enzyme structure; green fluorescent proteins; guanine nucleotide binding protein; hormone regulation /control mechanism; inositol phosphates; intermolecular interaction; lipids; phosphatidylinositols; phosphorylation; posttranslational modifications; protein binding; protein kinase; protein localization; protein purification; protein structure function; second messengers

The Unit of Molecular Signal Transduction directed by Tamas Balla investigates signal transduction pathways that mediate the actions of hormones and growth factors in mammalian cells, with special emphasis on the role of phosphoinositide-derived messengers. Current studies are aimed at (1) understanding the function and regulation of several phosphatidylinositol (PI) 4-kinases in the control of the synthesis of hormone-sensitive phosphoinositide pools; (2) characterizing the structural features that determine the catalytic specificity and inhibitor sensitivity of PI 3- and PI 4-kinases; (3) defining the molecular basis of protein-phosphoinositide interactions via the pleckstrin homology and other domains of selected regulatory proteins; (4) developing tools to analyze inositol lipid dynamics in live cells; (5) determining the importance of the lipid-protein interactions in the activation of cellular responses by G protein-coupled receptors and receptor tyrosine kinases. Neuronal calcium sensor-1 regulates phosphatidylinositol 4-kinase beta in mammalian cells - Inositol lipid kinases are increasingly recognized as regulators of membrane remodeling events including Golgi-to-plasma membrane transport, exocytosis or endocytosis. PI 4-kinases (PI4Ks) are the enzymes that catalyze the formation of PI(4)P, the main precursor of several other polyphosphoinositides with important regulatory functions. Investigators in this unit have recently purified and cloned two mammalian PI4Ks, a larger (~200 kDa) alpha , and a smaller (~100 kDa) beta form from bovine adrenal and brain. These enzymes are mammalian homologues of the yeast STT4 and PIK1 gene products, respectively, and are greatly conserved in all eukaryotes, including plants. Recently, it has been reported that the yeast homologue of the Ca2+-dependent regulatory protein, NCS-1, is able to stimulate PI 4-kinase activity of yeast homogenates apparently through interaction with the Pik1 protein. NCS-1 was first identified in Drososphila (where it was named frequenin) as an important determinant of synaptic plasticity and a regulator of synaptic development. In a series of studies it was investigated whether mammalian NCS-1 is able to interact and regulate PI4Kbeta in mammalian cells. Recombinant PI4Kbeta, but not its GST-fused form showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro-translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro-translated PI4Kbeta in PI4Kbeta-immunoprecipitates. When expressed in COS-7 cells, PI4Kbeta and NCS-1 formed a complex that could be immuno-precipitated with antibodies against either proteins and PI 4-kinase activity was present in anti-NCS-1 immuno-precipitates. Confocal analysis of the distribution of expressed NCS-1-YFP showed that NCS-1 is co-localized with endogenous PI4Kbeta primarily in the Golgi but is also present in the plasma membrane and the walls of numerous large perinuclear vesicles that are not observed in untransfected COS-7 cells. Co-expression of a catalytically inactive PI4Kbeta inhibited the development of the vesicular phenotype, suggesting that formation of these vesicles are the consequence of NCS-1 activating PI4Kbeta. Transfection of PI4Kbeta and NCS-1 had no effect on basal PIP synthesis in permeabilized COS-7 cells, but increased wortmannin-sensitive 32P-phosphate incorporation into phosphatidylinositol 4-phosphate during Ca2+-induced phospholipase C activation. These results together indicate that NCS-1 is able to interact with PI4Kbeta in mammalian cells and may play a role in the regulation of this enzyme in specific cellular compartments affecting vesicular trafficking. Analysis of inositol phospholipid changes in relation to activation of small GTP binding proteins - Activation of small GTP binding proteins is often correlated with changes in the level of inositol phospholipids. A number of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) are activated by PI(3,4,5)P3, the product of PI 3-kinases, and some by PI(4,5)P2. In collaboration with investigators in NHLBI (led by Dr. Julie Donaldson) we investigated the role of PI(4,5)P2 in plasma membrane dynamics regulated by ADP-ribosylation factor (Arf) 6. Since Arf 6 activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates PI(4,5)P2, we used the pleckstrin homology domain of PLCdelta fused to the green fluorescent protein (PLCdelta1PH-GFP) to visualize this lipid during Arf 6 activation. Activation of Arf6 by expression of its exchange factor EFA6 stimulated formation of membrane protrusions and the uptake of PM into macropinosomes enriched in PI(4,5)P2, with recycling of this membrane back to the PM. In contrast, expression of Arf6 Q67L, a GTP hydrolysis-resistant mutant, induced the formation of PIP2-positive actin-coated vacuoles that were unable to recycle membrane back to the PM. Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6 Q67L. These results demonstrated that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway. We also explored the possibility that we could develop additional research tools to analyze the role of inositol lipids in the regulation of another important small GTP binding protein, Ras. It has been shown that H-Ras is present in special membrane subdomains, termed RAFTs, that are also enriched in inositol phospholipids. The active, GTP-bound form of Ras has been shown to activate PI 3-kinases and PI(3,4,5)P3 to recruit both GEFs and GAPs that regulate the active state of Ras. To study the dynamics of Ras activation in live cells and explore its connection with RAFTs and the various inositol lipids, we investigated whether the minimum molecular determinants of Ras recognition by the Raf-1 serine/threonine kinase, the best-known downstream target of Ras, could be used to visualize Ras activation in live cells by following the distribution of such domain fused to the green fluorescent protein (GFP). When the Ras binding domain (RBD) of Raf-1 was fused to GFP [Raf-1(51-131)GFP] very little localization of the fluorescence was observed in the plasma membrane of Ras-transformed NIH 3T3 cells. However, addition of the cystein-rich region (CRD) to the construct [Raf-1(51-220)GFP] showed clear localization to membrane ruffles of Ras-transformed NIH 3T3 cells. In normal NIH 3T3 cells, [Raf-1(51-220)GFP] showed minimal membrane localization that was enhanced after stimulation with PDGF or PMA. Mutations within either the RBD (R89L) or CRD (C168S) disrupted the membrane localization of [Raf-1(51-220)GFP], suggesting that both domains contribute to the recruitment of the fusion protein to Ras at the plasma membrane. The abilities of the various constructs to localize to the plasma membrane closely correlated with their inhibitory effects on MEK1- or MAP-kinase activation. Membrane localization of full-length Raf-1-GFP was less prominent than that of [Raf-1(51-220)GFP], in spite of its strong binding to RasV12 and potent activation of MAP-kinase. These finding indicate that both RBD and CRD are needed to recruit Raf-1 to active Ras at the plasma membrane, and that these domains are not fully exposed in the Raf-1 molecule. Visualization of activated Ras in live cells will help us to better understand the dynamics of Ras activation under various physiological and pathological conditions, and, combined with our other GFP-fused domains that recognize phosphoinositides, will allow analysis of the role of inositides in Ras activation.

Tamas Balla指导的分子信号转导单位研究了介导哺乳动物细胞中激素和生长因子的作用的信号转导途径，并特别强调了磷酸辅助剂衍生的使者的作用。当前的研究针对（1）了解几种磷脂酰肌醇（PI）4-激酶在控制激素敏感磷酸固醇池的功能和调节中； （2）表征确定PI 3-和PI 4-激酶的催化特异性和抑制剂敏感性的结构特征； （3）通过Pleckstrin同源性和选定的调节蛋白的其他结构域定义蛋白磷酸肌醇相互作用的分子基础； （4）开发工具来分析活细胞中的肌醇脂质动力学； （5）确定G蛋白偶联受体和受体酪氨酸激酶在细胞反应激活中脂质蛋白相互作用的重要性。 神经元钙传感器1调节哺乳动物细胞中的磷脂酰肌醇4-激酶β-肌醇脂质激酶越来越多地被认为是膜重塑事件的调节剂，包括高尔基膜到脱铂膜的转运，外胞周增生或内核病。 PI 4-激酶（PI4K）是催化PI（4）P的形成的酶，PI（4）P是具有重要调节功能的其他几种多磷酸肌醇的主要前体。该单元的研究人员最近纯化并克隆了两个哺乳动物PI4K，一个较大（约200 kDa）的α和牛肾上腺和大脑的较小（〜100 kDa）β形式。这些酶分别是酵母stt4和Pik1基因产物的哺乳动物同源物，并且在包括植物在内的所有真核生物中都非常保守。最近，据报道，Ca2+依赖性调节蛋白NCS-1的酵母同源物能够通过与PIK1蛋白质相互作用来刺激酵母均质物的Pi 4-激酶活性。 NCS-1首先在Drosophila（命名为频率素）中鉴定为突触可塑性的重要决定因素和突触发育的调节剂。在一系列研究中，研究了哺乳动物NCS-1是否能够相互作用和调节哺乳动物细胞中的PI4KBETA。当与重组NCS-1孵育时，重组PI4KBETA，但没有其GST融合形式显示出增强的PI激酶活性，但前提是后者被肉豆蔻酰菌霉。同样，发现在PI4KBETA-免疫沉淀物中，发现与重组或体外翻译的PI4KBETA相关，而不是其肉豆蔻酰化缺陷的突变体。当在COS-7细胞中表达时，PI4KBETA和NCS-1形成了一种复合物，可以用抗蛋白质的抗体进行免疫沉淀，而PI 4-激酶活性则存在于抗NCS-1免疫剂中。对表达的NCS-1-YFP分布的共聚焦分析表明，NCS-1与内源性PI4KBETA共定位于高尔基体中，但也存在于质膜中，并且在未转染的cos-7细胞中未观察到的许多大型核周囊泡的壁。催化无效的PI4KBETA的共表达抑制了囊泡表型的发展，这表明这些囊泡的形成是NCS-1激活PI4KBETA的结果。 PI4KBETA和NCS-1的转染对透化的COS-7细胞中的基底PIP合成没有影响，但在Ca2+诱导的磷脂酶C活化期间，在Ca2+诱导的CA2+诱导的CA2+期间，增加了麦芽素敏感的32p-磷酸4-磷酸盐。这些结果共同表明，NCS-1能够与哺乳动物细胞中的PI4KBETA相互作用，并且可能在影响囊泡运输的特定细胞室的调节中起作用。 分析与小GTP结合蛋白激活有关的肌醇磷脂变化 - 小GTP结合蛋白的激活通常与肌醇磷脂水平的变化相关。许多鸟嘌呤核苷酸交换因子（GEFS）和GTPase激活蛋白（GAP）被PI（3,4,5）P3，PI 3-激酶的乘积和PI（4,5）P2激活。在与NHLBI的研究人员（由朱莉·唐纳森（Julie Donaldson）博士领导）中，我们调查了Pi（4,5）P2在由ADP-核糖基化因子（ARF）调节的质膜动力学（ARF）6中的作用6。由于ARF 6激活了ARF 6激活了4-磷酸5-激酶（PIP 5-激酶）的磷酸磷酸5-激酶（pip 5-kinase），启用了we En Zyme（4）PI2，pi2 pi-eNEN-PI2 pi-py pi2 pi ins pi in sp pi-2 pi ins pi in s p2 Plcdelta的Pleckstrin同源域与绿色荧光蛋白（PLCDELTA1PH-GFP）融合在一起，可在ARF 6激活过程中可视化这种脂质。通过表达其交换因子EFA6激活ARF6刺激了膜突起的形成，并将PM摄入富含PI（4,5）P2的大辣酱中，并将该膜回收回到PM。相反，GTP水解突变体ARF6 Q67L的表达诱导了无法将膜回收回到PM的PIP2阳性肌动蛋白涂层液泡的形成。人PIP 5激酶α的过表达模仿了ARF6 Q67L所见的效果。这些结果表明，通过激活和灭活ARF6控制的PIP 5-激酶活性和PIP2周转对于通过ARF6 PM-粘体回收途径进行运输至关重要。 我们还探讨了我们可以开发其他研究工具来分析肌醇脂质在调节另一种重要的小型GTP结合蛋白RAS中的作用的可能性。已经表明，H-RAS存在于特殊的膜亚域中，称为筏，它们也富含肌醇磷脂。活跃的GTP结合形式的RA已显示可激活PI 3-激酶和PI（3,4,5）P3，以募集调节RAS活性状态的GEF和间隙。 To study the dynamics of Ras activation in live cells and explore its connection with RAFTs and the various inositol lipids, we investigated whether the minimum molecular determinants of Ras recognition by the Raf-1 serine/threonine kinase, the best-known downstream target of Ras, could be used to visualize Ras activation in live cells by following the distribution of such domain fused to the green fluorescent protein (GFP).当将RAF-1的RAS结合结构域（RBD）融合到GFP [RAF-1（51-131）GFP]中时，在Ras转化的NIH 3T3细胞的质膜中观察到荧光的定位很少。然而，将富含Cystein的区域（CRD）添加到构建体[RAF-1（51-220）GFP]中显示在Ras转化的NIH 3T3细胞的膜褶皱中明显定位。在正常的NIH 3T3细胞中，[RAF-1（51-220）GFP]显示出最小的膜定位，这些膜定位在用PDGF或PMA刺激后得到了增强。 RBD（R89L）或CRD（C168S）内的突变破坏了[RAF-1（51-220）GFP]的膜定位，这表明两个结构域都有助于将融合蛋白募集到质膜膜上的RAS。各种构建体与质膜定位的能力与它们对MEK1或MAP-激酶激活的抑制作用密切相关。全长RAF-1-GFP的膜定位不如[RAF-1（51-220）GFP]突出，尽管它与RASV12具有很强的结合和MAP-激酶的有效激活。这些发现表明，RBD和CRD都需要将RAF-1募集到质膜的活性Ras，并且这些结构域在RAF-1分子中均未完全暴露。活性细胞中活化RA的可视化将有助于我们更好地了解在各种生理和病理条件下RAS激活的动力学，并结合我们识别磷酸肌醇的其他GFP融合结构域的结合，将允许分析Insitides在RAS活化中的作用。