Inositol phospholipid metabolism in Arabidopsis. Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C

Phosphoinositides (PIs) constitute a minor fraction of total cellular lipids in all eukaryotic cells. They fulfill many important functions through interaction with a wide range of cellular proteins. Members of distinct inositol lipid kinase families catalyze the synthesis of these phospholipids from phosphatidylinositol. The hydrolysis of PIs involves phosphatases and isoforms of PI-specific phospholipase C. Although our knowledge of the roles played by plant PIs is clearly limited at present, there is no doubt that they are involved in many physiological processes during plant growth and development. In this review, we concentrate on inositol lipid-metabolizing enzymes from the model plant Arabidopsis for which biochemical characterization data are available, namely the inositol lipid kinases and PI-specific phospholipase Cs. The biochemical properties and structure of characterized and genome-predicted isoforms are presented and compared with those of the animal enzymes to show that the plant enzymes have some features clearly unique to this kingdom.     

Cloning of a human type II phosphatidylinositol 4-kinase reveals a novel lipid kinase family

Phosphoinositide lipids regulate numerous cellular processes in all eukaryotes. The versatility of this phospholipid is provided by combinations of phosphorylation on the 3', 4', and 5' positions of the inositol head group. Two distinct structural families of phosphoinositide (PI) kinases have so far been identified and named after their prototypic members, the PI 3-kinase and phosphatidylinositol (PtdIns) phosphate kinase families, both of which have been found to contain structural homologues possessing PI 4-kinase activity. Nevertheless, the prevalent PtdIns 4-kinase activity in many mammalian cell types is conferred by the widespread type II PtdIns 4-kinase, which has so far resisted molecular characterization. We have partially purified the human type II isoform from plasma membrane rafts of human A431 epidermoid carcinoma cells and obtained peptide mass and sequence data. The results allowed the cDNA containing the full open reading frame to be cloned. The predicted amino acid sequence revealed that the type II enzyme is the prototypic member of a novel, third family of PI kinases. We have named the purified protein type IIalpha and a second human isoform, type IIbeta. The type IIalpha mRNA appears to be expressed ubiquitously in human tissues, and homologues appear to be expressed in all eukaryotes.     

p110beta and p110delta phosphatidylinositol 3-kinases up-regulate Fc(epsilon)RI-activated Ca2+ influx by enhancing inositol 1,4,5-trisphosphate production

Fc(epsilon)RI-induced Ca2+ signaling in mast cells is initiated by activation of cytosolic tyrosine kinases. Here, in vitro phospholipase assays establish that the phosphatidylinositol 3-kinase (PI 3-kinase) lipid product, phosphatidylinositol 3,4,5-triphosphate, further stimulates phospholipase Cgamma2 that has been activated by conformational changes associated with tyrosine phosphorylation or low pH. A microinjection approach is used to directly assess the consequences of inhibiting class IA PI 3-kinases on Ca2+ responses after Fc(epsilon)RI cross-linking in RBL-2H3 cells. Injection of antibodies to the p110beta or p110delta catalytic isoforms of PI 3-kinase, but not antibodies to p110alpha, lengthens the lag time to release of Ca2+ stores and blunts the sustained phase of the calcium response. Ca2+ responses are also inhibited in cells microinjected with recombinant inositol polyphosphate 5-phosphatase I, which degrades inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), or heparin, a competitive inhibitor of the Ins(1,4,5)P3 receptor. This indicates a requirement for Ins(1,4,5)P3 to initiate and sustain Ca2+ responses even when PI 3-kinase is fully active. Antigen-induced cell ruffling, a calcium-independent event, is blocked by injection of p110beta and p110delta antibodies, but not by injection of 5-phosphatase I, heparin, or anti-p110alpha antibodies. These results suggest that the p110beta and p110delta isoforms of PI 3-kinase support Fc(epsilon)RI-induced calcium signaling by modulating Ins(1,4,5)P3 production, not by directly regulating the Ca2+ influx channel.     

Molecular and biochemical characterization of three WD-repeat-domain-containing inositol polyphosphate 5-phosphatases in Arabidopsis thaliana

Type II inositol polyphosphate 5-phosphatases (5PTases) in animals and yeast have been known to be important for regulating inositol and phospholipid signaling by hydrolyzing phosphate from both inositol polyphosphates and phosphoinositides. However, the molecular and biochemical properties of type II 5PTases in plants have not yet been studied. In this report, we show that three Arabidopsis genes, At5PTase12, At5PTase13 and At5PTase14, encode proteins with a 5PTase domain and a WD-repeat domain, a novel combination present only in plant 5PTases. We demonstrate that these genes are differentially expressed in Arabidopsis organs and At5PTase13 is induced in response to ABA and wounding treatments. Our biochemical studies reveal that although both At5PTase12 and At5PTase13 exhibit phosphatase activity toward only Ins(1,4,5)P3, At5PTase14 hydrolyzes phosphate from PI(4,5)P2, PI(3,4,5)P3 and Ins(1,4,5)P3 with the highest substrate affinity toward PI(4,5)P2. All three At5PTases require Mg2+ for their phosphatase activities. Our molecular and biochemical characterization of three WD-repeat-domain-containing At5PTases provides a foundation for further elucidation of their cellular functions in Arabidopsis.     

PI3-kinase inhibition: a target for drug development?

The phosphoinositide 3-kinases (PI3-kinases) are a ubiquitously expressed enzyme family that, through the generation of phospholipid second messengers, play a key role in the regulation of many cellular processes. These include motility, proliferation and survival, and carbohydrate metabolism. Members of the PI3-kinase family and related kinases, their mechanism of activation and the cellular events that they influence are described in this review. As knowledge of their involvement in disease processes increases, the PI3-kinases appear to be an increasingly attractive target for drug development, particularly in the fields of cancer and other proliferative diseases, and in the treatment of inflammatory and immunological conditions. Evidence of the functional specialization of PI3-kinase isoforms suggests that selective inhibition with acceptable toxicity might be possible.     

Phylogenomics of phosphoinositide lipid kinases: perspectives on the evolution of second messenger signaling and drug discovery

Background  

Phosphoinositide lipid kinases (PIKs) generate specific phosphorylated variants of phosatidylinositols (PtdIns) that are critical for second messenger signaling and cellular membrane remodeling. Mammals have 19 PIK isoforms spread across three major families: the PtIns 3-kinases (PI3Ks), PtdIns 4-kinases (PI4Ks), and PtdIns-P (PIP) kinases (PIPKs). Other eukaryotes have fewer yet varying PIK complements. PIKs are also an important, emerging class of drug targets for many therapeutic areas including cancer, inflammatory and metabolic diseases and host-pathogen interactions. Here, we report the genomic occurrences and evolutionary relationships or phylogenomics of all three PIK families across major eukaryotic groups and suggest potential ramifications for drug discovery.     

Serum and glucocorticoid-regulated protein kinases: variations on a theme

The phosphatidylinositol 3' kinase (PI3K)-signaling pathway plays a critical role in a variety of cellular responses such as modulation of cell survival, glucose homeostasis, cell division, and cell growth. PI3K generates important lipid second messengers-phosphatidylinositides that are phosphorylated at the 3' position of their inositol ring head-group. These membrane restricted lipids act by binding with high affinity to specific protein domains such as the pleckstrin homology (PH) domain. Effectors of PI3K include molecules that harbor such domains such as phosphoinositide-dependent kinase (PDK1) and protein kinase B (PKB), also termed Akt. The mammalian genome encodes three different PKB genes (alpha, beta, and gamma; Akt1, 2, and 3, respectively) and each is an attractive target for therapeutic intervention in diseases such as glioblastoma and breast cancer. A second family of three protein kinases, termed serum and glucocorticoid-regulated protein kinases (SGKs), is structurally related to the PKB family including regulation by PI3K but lack a PH domain. However, in addition to PH domains, a second class of 3' phosphorylated inositol phospholipid-binding domains exists that is termed Phox homology (PX) domain: this domain is found in one of the SGKs (SGK3). Here, we summarize knowledge of the three SGK isoforms and compare and contrast them to PKB with respect to their possible importance in cellular regulation and potential as therapeutic targets.     

拟南芥中myo-肌醇-多磷酸合成与代谢及其信号

在酵母、真菌、动物和植物等真核生物中, 以myo-肌醇为基石通过不同位点的磷酸化形成各种myo-肌醇-多磷酸及其衍生物。过去10年的研究发现这些肌醇多磷酸参与了膜脂定向转运、蛋白结构稳定、离子通道调控、RNA转运以及DNA修复和染色质重塑等细胞生物学的基本进程。近些年在模式植物拟南芥(Arabidopsis thaliana)的研究中, 许多调控植物生长发育和环境胁迫应答的重要基因被发现, 并证实这些基因参与myo-肌醇-多磷酸的合成与代谢。该文概述了拟南芥中myo-肌醇-多磷酸合成与代谢的基因调控机理, 综述了不同肌醇多磷酸作为信号分子的功能, 提出肌醇多磷酸如同一类信息代码传递着植物细胞有序进程的基本指令。     

Class I phosphoinositide 3-kinases

Phosphoinositide 3-kinases (PI3-kinases) are a family of enzymes that 3'-phosphorylate the inositol head group of membrane phosphoinositides. They are subdivided into three major classes (I, II and III) based on their structural homology, regulation and substrate specificity. It is now becoming clear that PI3-kinase isoforms are subject to differential regulation and may play distinct roles within the cell. PI3-kinases and their second messenger lipid products have been implicated in a plethora of cellular responses with increasing evidence for involvement in the pathogenesis of human diseases. The future development of specific PI3-kinase isoform inhibitors may offer therapeutic benefit in a broad range of clinical settings, related to cancer, inflammatory and immunological diseases.     

Diacylglycerol Kinase Inhibition and Vascular Function

Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structural features. These diverse isoforms of DGK are considered to activate distinct cellular functions according to extracellular stimuli. Each DGK isoform is thought to play various roles inside the cell, depending on its subcellular localization (nuclear, ER, Golgi complex or cytoplasm). In vascular smooth muscle, vasoconstrictors such as angiotensin II, endothelin-1 and norepinephrine stimulate contraction by increasing inositol trisphosphate (IP(3)), calcium, DG and PKC activity. Inhibition of DGK could increase DG availability and decrease PA levels, as well as alter intracellular responses, including calcium-mediated and PKC-mediated vascular contraction. The purpose of this review is to demonstrate a role of DGK in vascular function. Selective inhibition of DGK isoforms may represent a novel therapeutic approach in vascular dysfunction.     

Endoplasmic reticulum stress-induced apoptosis accompanies enhanced expression of multiple inositol polyphosphate phosphatase 1 (Minpp1): a possible role for Minpp1 in cellular stress response

Inositol polyphosphates represent a group of differentially phosphorylated inositol metabolites, many of which are implicated to regulate diverse cellular processes such as calcium mobilization, vesicular trafficking, differentiation, apoptosis, etc. The metabolic network of these compounds is complex and tightly regulated by various kinases and phosphatases present predominantly in the cytosol. Multiple inositol polyphosphate phosphatase 1 (Minpp1) is the only known endoplasmic reticulum (ER) luminal enzyme that hydrolyzes various inositol polyphosphates in vitro as well as in vivo conditions. However, access of the Minpp1 to cytosolic substrates has not yet been demonstrated clearly and hence its physiological function. In this study, we examined a potential role for Minpp1 in ER stress-induced apoptosis. We generated a custom antibody and characterized its specificity to study the expression of Minpp1 protein in multiple mammalian cells under experimentally induced cellular stress conditions. Our results demonstrate a significant increase in the expression of Minpp1 in response to a variety of cellular stress conditions. The protein expression was corroborated with the expression of its mRNA and enzymatic activity. Further, in an attempt to link the role of Minpp1 to apoptotic stress, we studied the effect of Minpp1 expression on apoptosis following silencing of the Minpp1 gene by its specific siRNA. Our results suggest an attenuation of apoptotic parameters following knockdown of Minpp1. Thus, in addition to its known role in inositol polyphosphate metabolism, we have identified a novel role for Minpp1 as a stress-responsive protein. In summary, our results provide, for the first time, a probable link between ER stress-induced apoptosis and Minpp1 expression.     

植物信号传导中的磷脂酶

20世纪 80年代早期人们意识到构成细胞膜的磷脂不只是一道将细胞物质与外界隔开的屏障,而且是细胞对外界环境刺激作出应答的物质基础。磷脂酰肌醇（ｐｈｏｓｐｈｏｔｉｄｙｌｉｎｏｓｉｔｏｌ,ＰＩ）不但是构成细胞膜的重要组分（约占细胞膜组分的 10 %）,在细胞内外环境信号的传递方面也起着重要的作用［1］。磷脂酶（ｐｈｏｓｐｈｏｌｉｐａｓｅ）水解磷脂后产生的三磷酸肌醇 （ｉｎｏｓ ｉｔｏｌｔｒｉｓｐｈｏｓｐｈａｔｅ,ＩＰ3 ）／二酰基甘油（ｄｉａｃｙｌｇｌｙｃｅｒｏｌ,ＤＡＧ）、磷脂酸（ｐｈｏｓｐｈａｔｉｄｉｃａｃｉ…     

Phosphoinositide 3-kinases in immunity: lessons from knockout mice

Phosphoinositide 3-kinases (PI3Ks) constitute a family of evolutionarily conserved lipid kinases that phosphorylate the D3 position of the inositol ring of phosphoinositides and produce PI(3)P, PI(3,4)P(2), and PI(3,4,5)P(3). Intense in vitro research over the last decade has unequivocally demonstrated that PI3Ks, in particular those belonging to class I, regulate a vast array of fundamental cellular responses. Given the pleiotropic roles of PI3Ks and the lipid product PI(3,4,5)P(3) in plethora of cellular responses, it is pertinent to explore the significance of PI3K signaling in vivo. In the past two or three years, the components of this signaling pathway have been genetically manipulated in mouse. This review briefly summarizes the immunological significance of PI3K signaling as revealed by the study of gene-targeted "knockout" mice.     

Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2β, an inositol polyphosphate 6-/3-kinase from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Inositol phosphates (IPs) and their turnover products have been implicated to play important roles in stress signaling in eukaryotic cells. In higher plants genes encoding inositol polyphosphate kinases have been identified previously, but their physiological functions have not been fully resolved. Here we expressed Arabidopsis inositol polyphosphate 6-/3-kinase (AtIpk2β) in two heterologous systems, i.e. the yeast Saccharomyces cerevisiae and in tobacco (Nicotiana tabacum), and tested the effect on abiotic stress tolerance. Expression of AtIpk2β rescued the salt-, osmotic- and temperature-sensitive growth defects of a yeast mutant strain (arg82Δ) that lacks inositol polyphosphate multikinase activity encoded by the ARG82/IPK2 gene. Transgenic tobacco plants constitutively expressing AtIpk2β under the control of the Cauliflower Mosaic Virus 35S promoter were generated and found to exhibit improved tolerance to diverse abiotic stresses when compared to wild type plants. Expression patterns of various stress responsive genes were enhanced, and the activities of anti-oxidative enzymes were elevated in transgenic plants, suggesting a possible involvement of AtIpk2β in plant stress responses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

磷脂酰肌醇3-激酶γ/δ与炎症相关疾病

磷脂酰肌醇3-激酶(PI3K)是一类脂质与蛋白激酶家族,其主要通过在磷脂酰肌醇的肌醇环三位进行磷酸化产生胞内重要的第二信使——磷脂酰肌醇-3,4,5-三磷酸(phosphatidyl inositol 3,4,5-trisphosphate,PIP3)而发挥作用.磷脂酰肌醇3-激酶γ/δ(PI3Kγ/δ)是I类PI3K家族中的成员,其主要表达于免疫相关细胞中,这2种PI3K亚型参与先天性与获得性免疫应答.因此,PI3Kγ/PI3Kδ被视为因免疫反应调控异常导致的炎症疾病的治疗药物靶点.目前,利用特异性抑制剂靶向干预PI3Kγ和/或PI3Kδ,成为炎症相关疾病治疗的新策略.本文简介了PI3Kγ与PI3Kδ在不同类型免疫细胞中的功能;并就采用小分子特异性抑制剂,靶向抑制PI3Kγ和/或PI3Kδ在各类炎症相关疾病中的治疗作用和效果进行综述.     

Déjà vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins

After reading many 2-DE-based articles featuring lists of the differentially expressed proteins, one starts experiencing a disturbing déjà vu. The same proteins seem to predominate regardless of the experiment, tissue or species. To quantify the occurrence of individual differentially expressed proteins in 2-DE experiment reports, we compiled the identities of differentially expressed proteins identified in human, mouse, and rat tissues published in three recent volumes of Proteomics and calculated the appearance of the most predominant proteins in the dataset. The most frequently identified protein is a highly abundant glycolytic enzyme enolase 1, differentially expressed in nearly every third experiment on both human and rodent tissues. Heat-shock protein 27 (HSP27) and heat-shock protein 60 (HSP60) were differentially expressed in about 30 percent of human and rodent samples, respectively. Considering protein families as units, keratins and peroxiredoxins are the most frequently identified molecules, with at least one member of the group being differentially expressed in about 40 percent of all experiments. We suggest that the frequent identification of these proteins must be considered in the interpretation of any 2-DE studies. We consider if these commonly observed changes represent common cellular stress responses or are a reflection of the technical limitations of 2-DE.     

Genomewide analysis of phospholipid signaling genes in Phytophthora spp.: novelties and a missing link

Phospholipids are cellular membrane components in eukaryotic cells that execute many important roles in signaling. Genes encoding enzymes required for phospholipid signaling and metabolism have been characterized in several organisms, but only a few have been described for oomycetes. In this study, the genome sequences of Phytophthora sojae and P. ramorum were explored to construct a comprehensive genomewide inventory of genes involved in the most universal phospholipid signaling pathways. Several genes and gene families were annotated, including those encoding phosphatidylinositol synthase (PIS), phosphatidylinositol (phosphate) kinase (PI[P]K), diacylglycerol kinase (DAG), and phospholipase D (PLD). The most obvious missing link is a gene encoding phospholipase C (PLC). In all eukaryotic genomes sequenced to date, PLC genes are annotated based on certain conserved features; however, these genes seem to be absent in Phytophthora spp. Analysis of the structural and regulatory domains and domain organization of the predicted isoforms of PIS, PIK, PIPK, DAG, and PLD revealed many novel features compared with characterized representatives in other eukaryotes. Examples are transmembrane proteins with a C-terminal catalytic PLD domain, secreted PLD-like proteins, and PIPKs that have an N-terminal G-protein-coupled receptor-transmembrane signature. Compared with other sequenced eukaryotes, the genus Phytophthora clearly has several exceptional features in its phospholipid-modifying enzymes.