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The Trans Golgi Network (TGN)/endosomal system is a sorting center for cargo brought via the anterograde secretory pathway and the endocytic pathway that internalizes material from the plasma membrane. As many of the cargo that transit this central trafficking hub are components of key homeostatic signaling pathways, TGN/endosomes define a critical signaling hub for cellular growth control. A particularly interesting yet incompletely understood aspect of regulation of TGN/endosome function is control of this system by two families of lipid exchange/lipid transfer proteins. The phosphatidylinositol transfer proteins promote pro-trafficking phosphoinositide (i.e. phosphatidylinositol-4-phosphate) signaling pathways whereas proteins of the oxysterol binding protein family play reciprocal roles in antagonizing those arms of phosphoinositide signaling. The precise mechanisms for how these lipid binding proteins execute their functions remain to be resolved. Moreover, information regarding the coupling of individual members of the oxysterol binding protein family to specific biological activities is particularly sparse. Herein, we review what is being learned regarding functions of the oxysterol binding protein family in the yeast model system. Focus is primarily directed at a discussion of the Kes1/Osh4 protein for which the most information is available., Competing Interests: Declaration of competing interest This work was supported by National Institutes of Health grant R35 GM131804 and BE0017 from the Robert A. Welch Foundation to VAB. The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

The ER is a large multifunctional organelle of eukaryotic cells. Malfunction of the ER in various disease states, such as atherosclerosis, diabetes, cancer, Alzheimer's and Parkinson's and amyotrophic lateral sclerosis, often correlates with alterations in its morphology. The ER exhibits regionally variable membrane morphology that includes, at the extremes, large relatively flat surfaces and interconnected tubular structures highly curved in cross-section. ER morphology is controlled by shaping proteins that associate with membrane lipids. To investigate the role of these lipids, we developed a sea urchin oocyte model, a relatively quiescent cell in which the ER consists mostly of tubules. We altered levels of endogenous diacylglycerol (DAG), phosphatidylethanolamine (PtdEth), and phosphatidylcholine by microinjection of enzymes or lipid delivery by liposomes and evaluated shape changes with 2D and 3D confocal imaging and 3D electron microscopy. Decreases and increases in the levels of lipids such as DAG or PtdEth characterized by negative spontaneous curvature correlated with conversion to sheet structures or tubules, respectively. The effects of endogenous alterations of DAG were reversible upon exogenous delivery of lipids of negative spontaneous curvature. These data suggest that proteins require threshold amounts of such lipids and that localized deficiencies of the lipids could contribute to alterations of ER morphology. The oocyte modeling system should be beneficial to studies directed at understanding requirements of lipid species in interactions leading to alterations of organelle shaping.

The discovery of the phosphatidylinositol-3-kinase (PI3K) pathway was a major advance in understanding growth factor signaling. The high frequency of PI3K pathway mutations in many cancers has encouraged a new field targeting cancer driver mutations. Although there have been many successes, targeting PI3K itself has proven challenging, in part because of its multiple isoforms with distinct roles. Despite promising preclinical results, development of PI3K inhibitors as pharmacologic anticancer agents has been limited by modest single-agent efficacy and significant adverse effects. If we could overcome these limitations, PI3K inhibitors would be a powerful cancer-fighting tool. Data from phase III clinical trials yields insight into some of the problems with PI3K inhibitors. Recent advances have shed light on the mechanisms of tumor resistance to PI3K inhibitors via feedback pathways that cause elevated insulin levels that then activate the same PI3K pathways that are the targets of inhibition. Improving our understanding of the complex regulatory feedback pathways that activate in response to PI3K inhibition will reveal ways to increase the efficacy of PI3K inhibitors and reduce adverse effects, increasing the usefulness of this class as a treatment option for multiple cancer types.

Virtually all aspects of T and B lymphocyte development, homeostasis, activation, and effector function are impacted by the interaction of their clonally distributed antigen receptors with antigens encountered in their respective environments. Antigen receptors mediate their effects by modulating intracellular signaling pathways that ultimately impinge on the cytoskeleton, bioenergetic pathways, transcription, and translation. Although these signaling pathways are rather well described at this point, especially those steps that are most receptor-proximal, how such pathways contribute to more quantitative aspects of lymphocyte function is still being elucidated. One of the signaling pathways that appears to be involved in this “tuning” process is controlled by the lipid kinase PI3K. Here we review recent key findings regarding both the triggering/enhancement of PI3K signals (via BCAP and ICOS) as well as their regulation (via PIK3IP1 and PHLPP) and how these signals integrate and determine cellular processes. Lymphocytes display tremendous functional plasticity, adjusting their metabolism and gene expression programs to specific conditions depending on their tissue of residence and the nature of the infectious threat to which they are responding. We give an overview of recent findings that have contributed to this model, with a focus on T cells, including what has been learned from patients with gain-of-function mutations in PI3K as well as lessons from cancer immunotherapy approaches.

Intercellular communication has been known for decades to involve either direct contact between cells or to operate via circulating molecules, such as cytokines, growth factors, or lipid mediators. During the last decade, we have begun to appreciate the increasing importance of intercellular communication mediated by extracellular vesicles released by viable cells either from plasma membrane shedding (microvesicles, also named microparticles) or from an intracellular compartment (exosomes). Exosomes and microvesicles circulate in all biological fluids and can trigger biological responses at a distance. Their effects include a large variety of biological processes, such as immune surveillance, modification of tumor microenvironment, or regulation of inflammation. Extracellular vesicles can carry a large array of active molecules, including lipid mediators, such as eicosanoids, proteins, and nucleic acids, able to modify the phenotype of receiving cells. This review will highlight the role of the various lipidic pathways involved in the biogenesis and functions of microvesicles and exosomes.

Phosphoinositides (PPIns) are lipid signaling molecules that act as master regulators of cellular signaling. Recent studies have revealed novel roles of PPIns in myriad cellular processes and multiple human diseases mediated by misregulation of PPIn signaling. This review will present a timely summary of recent discoveries in PPIn biology, specifically their role in regulating unexpected signaling pathways, modification of signaling outcomes downstream of integral membrane proteins, and novel roles in lipid transport. This has revealed new roles of PPIns in regulating membrane trafficking, immunity, cell polarity, and response to extracellular signals. A specific focus will be on novel opportunities to target PPIn metabolism for treatment of human diseases, including cancer, pathogen infection, developmental disorders, and immune disorders. [ABSTRACT FROM AUTHOR]

Diacylglycerol kinases (DGKs) regulate the balance between diacylglycerol (DAG) and phosphatidic acid. DGKζ is highly abundant in skeletal muscle and induces fiber hypertrophy. We hypothesized that DGKζ influences functional and metabolic adaptations in skeletal muscle and whole-body fuel utilization. DAG content was increased in skeletal muscle and adipose tissue, but unaltered in liver of DGKζ KO mice. Linear growth, body weight, fat mass, and lean mass were reduced in DGKζ KO versus wild-type mice. Conversely, male DGKζ KO and wild-type mice displayed a similar robust increase in plantaris weight after functional overload, suggesting that DGKζ is dispensable for muscle hypertrophy. Although glucose tolerance was similar, insulin levels were reduced in high-fat diet (HFD)-fed DGKζ KO versus wild-type mice. Submaximal insulin-stimulated glucose transport and p-Akt Ser473 were increased, suggesting enhanced skeletal muscle insulin sensitivity. Energy homeostasis was altered in DGKζ KO mice, as evidenced by an elevated respiratory exchange ratio, independent of altered physical activity or food intake. In conclusion, DGKζ deficiency increases tissue DAG content and leads to modest growth retardation, reduced adiposity, and protection against insulin resistance. DGKζ plays a role in the control of growth and metabolic processes, further highlighting specialized functions of DGK isoforms in type 2 diabetes pathophysiology.

Phospholipids and phospholipases play important roles in several cellular processes and responses to adverse growth conditions. However, the mechanism of action of phosphatidylinositol-specific phospholipase C (PI-PLC), and its resulting minor lipid function in response to pollutant-induced stress, remains to be elucidated. In this work, we studied the effects of phenol treatment on the PLC pathway, using two lines of hairy root cultures (HRs) from Nicotiana tabacum as plant model systems: wild-type (WT) and double-transgenic (DT) HRs. We quantified several product formations such as PIP, PIP2, DGPP, and PA, which are mainly synthesized by specific lipid kinases belonging to the PLC pathway. In both HRs, phenol treatment significantly increased the formation of these compounds in a differential manner. In WT HRs, PA formation was twofold higher than in control. PIP2 and PIP levels were about onefold higher than those of the controls while DGPP levels increased by 50%. In DT HRs, PIP levels were onefold higher than those of the controls while PIP2, DGPP, and PA levels increased by 120%. Phenol treatment also upregulated the PLC4 gene expression mainly in the first hours of exposure in both HRs, while the DGK1 gene expression was only upregulated in WT HRs after 24 h of treatment. These results show an active participation of the PLC pathway under phenol treatment suggesting that this signal pathway could be important in plant cell responses to phenol-induced stress. [ABSTRACT FROM AUTHOR]

Diacylglycerol kinase (DGK) consists of 10 isozymes. The α-isozyme enhances the proliferation of cancer cells. However, DGKα facilitates the nonresponsive state of immunity known as T-cell anergy; therefore, DGKα enhances malignant traits and suppresses immune surveillance. The aim of this study was to identify a novel small molecule that selectively and potently inhibits DGKα activity. We screened a library containing 9,600 chemical compounds using a newly established high-throughput DGK assay. As a result, we have obtained a promising compound, 5-[(2E)-3-(2-furyl)prop-2-enylidene]-3-[(phenylsulfonyl)amino]2-thioxo-1,3-thiazolidin-4-one) (CU-3), which selectively inhibited DGKα with an IC50 value of 0.6 μM. CU-3 targeted the catalytic region, but not the regulatory region, of DGKα. CU-3 competitively reduced the affinity of DGKα for ATP, but not diacylglycerol or phosphatidylserine. Moreover, this compound induced apoptosis in HepG2 hepatocellular carcinoma and HeLa cervical cancer cells while simultaneously enhancing the interleukin-2 production of Jurkat T cells. Taken together, these results indicate that CU-3 is a selective and potent inhibitor for DGKα and can be an ideal anticancer drug candidate that attenuates cancer cell proliferation and simultaneously enhances immune responses including anticancer immunity.

Signaling through the phosphoinositide 3-kinase pathways mediates the actions of a plethora of hormones, growth factors, cytokines, and neurotransmitters upon their target cells following receptor occupation. Overactivation of these pathways has been implicated in a number of pathologies, in particular a range of malignancies. The tight regulation of signaling pathways necessitates the involvement of both stimulatory and terminating enzymes; inappropriate activation of a pathway can thus result from activation or inhibition of the two signaling arms. The focus of this review is to discuss, in detail, the activities of the identified families of phosphoinositide phosphatase expressed in humans, and how they regulate the levels of phosphoinositides implicated in promoting malignancy.

Steatohepatitis occurs in up to 20% of patients with fatty liver disease and leads to its primary disease outcomes, including fibrosis, cirrhosis, and increased risk of hepatocellular carcinoma. Mechanisms that mediate this inflammation are of major interest. We previously showed that overload of saturated fatty acids, such as that which occurs with metabolic syndrome, induced sphingosine kinase 1 (SphK1), an enzyme that generates sphingosine-1-phosphate (S1P). While data suggest beneficial roles for S1P in some contexts, we hypothesized that it may promote hepatic inflammation in the context of obesity. Consistent with this, we observed 2-fold elevation of this enzyme in livers from humans with nonalcoholic fatty liver disease and also in mice with high saturated fat feeding, which recapitulated the human disease. Mice exhibited activation of NFκB, elevated cytokine production, and immune cell infiltration. Importantly, SphK1-null mice were protected from these outcomes. Studies in cultured cells demonstrated saturated fatty acid induction of SphK1 message, protein, and activity, and also a requirement of the enzyme for NFκB signaling and increased mRNA encoding TNFα and MCP1. Moreover, saturated fat-induced NFκB signaling and elevation of TNFα and MCP1 mRNA in HepG2 cells was blocked by targeted knockdown of S1P receptor 1, supporting a role for this lipid signaling pathway in inflammation in nonalcoholic fatty liver disease.

Target residence time ( τ ) has been suggested to be a better predictor of the biological activity of kinase inhibitors than inhibitory potency (IC 50 ) in enzyme assays. Surface plasmon resonance binding assays for 46 human protein and lipid kinases were developed. The association and dissociation constants of 80 kinase inhibitor interactions were determined. τ and equilibrium affinity constants ( K D ) were calculated to determine kinetic selectivity. Comparison of τ and K D or IC 50 values revealed a strikingly different view on the selectivity of several kinase inhibitors, including the multi-kinase inhibitor ponatinib, which was tested on 10 different kinases. In addition, known pan-Aurora inhibitors resided much longer on Aurora B than on Aurora A, despite having comparable affinity for Aurora A and B. Furthermore, the γ/δ-selective PI3K inhibitor duvelisib and the δ-selective drug idelalisib had similar 20-fold selectivity for δ- over γ-isoform but duvelisib resided much longer on both targets. [ABSTRACT FROM AUTHOR]

Choline kinase catalyzes the phosphorylation of choline, the first step of phospholipid synthesis. Increased phosphorylation of choline is a hallmark characteristic of the malignant phenotype in a variety of neoplasms. However, in hypoxic cancer cells, choline phosphorylation is decreased. To understand the mechanism behind this altered metabolic state, we examined the expression and regulation of the major choline kinase isoform, choline kinase α (ChKα), in hypoxic PC-3 human prostate cancer cells. Hypoxia decreased choline phosphorylation, choline kinase activity, and ChKα mRNA and protein levels. Promoter analysis studies revealed a region upstream of the ChKα gene bearing a conserved DNA consensus binding motif, hypoxia response element-7 (HRE7), at position −222 relative to +1 translation start site, for binding the hypoxia dependent master regulator transcription factor, hypoxia-inducible factor 1α (HIF-1α). Electrophoretic mobility shift competition/supershift assay and chromatin immunoprecipitation assay confirmed binding of HIF-1α to HRE7. A putative promoter of ChKα was isolated from PC-3 genomic DNA and cloned into a luciferase-based reporter vector system. In PC-3 cells, hypoxia decreased the expression of luciferase under the control of the ChKα promoter. Mutation of HRE7 abrogated this hypoxia effect, further demonstrating the involvement of HRE7 in hypoxia-sensitive regulation of ChKα. The results strongly suggest that transcriptional control of choline phosphorylation is largely mediated via HIF-1α binding to the newly identified HRE7.

Despite the ubiquitous roles of lipids in biology, the detection of lipids has relied on invasive techniques, population measurements, or nonspecific labeling. Such difficulties can be circumvented by a label-free imaging technique known as coherent anti-Stokes Raman (CARS) microscopy, which is capable of chemically selective, highly sensitive, and high-speed imaging of lipid-rich structures with submicron three-dimensional spatial resolution. We review the broad applications of CARS microscopy to studies of lipid biology in cell cultures, tissue biopsies, and model organisms. Recent technical advances, limitations of the technique, and perspectives are discussed.

The phosphatidylinositol phosphate kinase (PIPK) family of enzymes is primarily responsible for converting singly phosphorylated phosphatidylinositol derivatives to phosphatidylinositol bisphosphates. As such, these kinases are central to many signaling and membrane trafficking processes in the eukaryotic cell. The three types of phosphatidylinositol phosphate kinases are homologous in sequence but differ in catalytic activities and biological functions. Type I and type II kinases generate phosphatidylinositol 4,5-bisphosphate from phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate, respectively, whereas the type III kinase produces phosphatidylinositol 3,5-bisphosphate from phosphatidylinositol 3-phosphate. Based on crystallographic analysis of the zebrafish type I kinase PIP5Kα, we identified a structural motif unique to the kinase family that serves to recognize the monophosphate on the substrate. Our data indicate that the complex pattern of substrate recognition and phosphorylation results from the interplay between the monophosphate binding site and the specificity loop: the specificity loop functions to recognize different orientations of the inositol ring, whereas residues flanking the phosphate binding Arg244 determine whether phosphatidylinositol 3-phosphate is exclusively bound and phosphorylated at the 5-position. This work provides a thorough picture of how PIPKs achieve their exquisite substrate specificity. [ABSTRACT FROM AUTHOR]

Soil salinity is one of the major abiotic stresses that affect the crop productivity. Understanding the mechanisms by which plants transmit the signals to cellular machinery to trigger adaptive responses is essential to develop more stress tolerant crops. Barley ( Hordeum vulgare L.) is an important cereal and its production is affected by increasing dryland salinity. This severely limits growth and reduces yields. In barley seedling, NaCl and mannitol stresses modulated the level of phosphatidic acid (PA), the proline accumulation and the reduced root length. PA is a well-known lipid signal and an intermediary in the lipid synthesis. However, little is known about their role during the saline and osmotic stresses in barley roots. PA increased by phospholipase D (PLD, E.C. 3.1.4.4) and by diacylglycerol kinase activities (DAG-k, EC 2.7.1.107) and its conversion to DGPP suggested that they are part of stress responses to salinity in barley. In contrast, saline stress decreased the activity of the Mg 2+ -independent, NEM-insensitive form of phosphatidate phosphohydrolase (PAP2, E.C. 3.1.3.4), keeping the PA levels. The application of 1-butanol also stimulated proline accumulation while the DGK-inhibitor treatment decreased proline levels. Endogenous phytohormone levels measured by liquid chromatography-tandem mass spectrometry revealed that, under stress, the barley roots decreased the GA 3 and ABA levels and increased the SA and JA endogenous amounts. The results presented here suggest that PA may modulate the cellular signal of barley roots by differentially affecting components of the abiotic stress − response cascade. [ABSTRACT FROM AUTHOR]

The sphingosine and diacylglycerol kinases form a superfamily of structurally related lipid signaling kinases. One of the striking features of these kinases is that although they are clearly involved in agonist-mediated signaling, this signaling is accomplished with only a moderate (and sometimes no) increase in the enzymatic activity of the enzymes. Here, we summarize findings that indicate that signaling by these kinases is strongly dependent on their localization to specific intracellular sites rather than on increases in enzyme activity. Both the substrates and products of these enzymes are bioactive lipids. Moreover, many of the metabolic enzymes that act on these lipids are found in specific organelles. Therefore, changes in the membrane localization of these signaling kinases have profound effects not only on the production of signaling lipid phosphates but also on the metabolism of the upstream signaling lipids.

Alterations in PI3K/AKT signaling are known to be implicated with tumorigenesis. The PI3 kinases family of lipid kinases has been an attractive therapeutic target for cancer treatment. Imidazopyridine compound 1 , a potent, selective, and orally available pan-PI3K inhibitor, identified by scaffold morphing of a benzothiazole hit, was further optimized in order to achieve efficacy in a PTEN-deleted A2780 ovarian cancer mouse xenograft model. With a hypothesis that a planar conformation between the core and the 6-heteroaryl ring will allow for the accommodation of larger 5′-substituents in a hydrophobic area under P-loop, SAR efforts focused on 5′-alkoxy heteroaryl rings at the 6-position of imidazopyridine and imidazopyridazine cores that have the same dihedral angle of zero degrees. 6′-Alkoxy 5′-aminopyrazines in the imidazopyridine series were identified as the most potent compounds in the A2780 cell line. Compound 14 with 1,1,1-trifluoroisopropoxy group at 6′-position demonstrated excellent potency and selectivity, good oral exposure in rats and in vivo efficacy in A2780 tumor-bearing mouse. Also, we disclose the X-ray co-crystal structure of one enantiomer of compound 14 in PI3Kα, confirming that the trifluoromethyl group fits nicely in the hydrophobic hot spot under P-loop. [ABSTRACT FROM AUTHOR]

Type 2 diabetes is a complex disease. It results from a failure of the body to maintain energy homoeostasis. Multicellular organisms have evolved complex strategies to preserve a relatively stable internal nutrient environment, despite fluctuations in external nutrient availability. This complex strategy involves the coordinated responses of multiple organs to promote storage or mobilization of energy sources according to the availability of nutrients and cellular bioenergetics needs. The endocrine pancreas plays a central role in these processes by secreting insulin and glucagon. When this co-ordinated effort fails, hyperglycaemia and hyperlipidaemia develops, characterizing a state of metabolic imbalance and ultimately overt diabetes. Although diabetes is most likely a collection of diseases, scientists are starting to identify genetic components and environmental triggers. Genome-wide association studies revealed that by and large, gene variants associated with type 2 diabetes are implicated in pancreatic β-cell function, suggesting that the β-cell may be the weakest link in the chain of events that results in diabetes. Thus, it is critical to understand how environmental cues affect the β-cell. Phosphoinositides are important 'decoders' of environmental cues. As such, these lipids have been implicated in cellular responses to a wide range of growth factors, hormones, stress agents, nutrients and metabolites. Here we will review some of the well-established and potential new roles for phosphoinositides in β-cell function/dysfunction and discuss how our knowledge of phosphoinositide signalling could aid in the identification of potential strategies for treating or preventing type 2 diabetes. [ABSTRACT FROM AUTHOR]

The class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that transduce a host of cellular signals and regulate a broad range of essential functions including growth, proliferation, and migration. As such, PI3Ks have pivotal roles in diseases such as cancer, diabetes, primary immune disorders, and inflammation. These enzymes are activated downstream of numerous activating stimuli including receptor tyrosine kinases, G protein-coupled receptors (GPCRs), and the Ras superfamily of small G proteins. A major challenge is to decipher how each PI3K isoform is able to successfully synergize these inputs into their intended signaling function. This article highlights recent progress in characterizing the molecular mechanisms of PI3K isoform-specific activation pathways, as well as novel roles for PI3Ks in human diseases, specifically cancer and immune diseases. [ABSTRACT FROM AUTHOR]

Virulent fungi represent a particularly difficult problem in the infectious disease arena as these organisms are eukaryotes that share many orthologous activities with their human hosts. The fact that these activities are often catalyzed by conserved proteins places additional demands on development of pharmacological strategies for specifically inhibiting target fungal activities without imposing undesirable secondary effects on the host. While deployment of a limited set of anti-mycotics has to date satisfied the clinical needs for treatment of fungal infections, the recent emergence of multi-drug resistant fungal 'superbugs' now poses a serious global health threat with rapidly diminishing options for treatment. This escalating infectious disease problem emphasizes the urgent need for development of new classes of anti-mycotics. In that regard, Sec14 phosphatidylinositol transfer proteins offer interesting possibilities for interfering with fungal phosphoinositide signaling with exquisite specificity and without targeting the highly conserved lipid kinases responsible for phosphoinositide production. Herein, we review the establishment of proof-of-principle that demonstrates the feasibility of such an approach. We also describe the lead compounds of four chemotypes that directly target fungal Sec14 proteins. The rules that pertain to the mechanism(s) of Sec14 inhibition by validated small molecule inhibitors, and the open questions that remain, are discussed – as are the challenges that face development of next generation Sec14-directed inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: The early signaling events in T cell activation through CD3 receptor include a rapid change in intra cellular free calcium concentration and reorganization of actin cytoskeleton. Phosphatidylinositol 4-kinases (PtdIns 4-kinases) are implicated as key components in these early signaling events. The role of type II PtdIns 4-kinase β in CD3 receptor signaling was investigated with the help of short hairpin RNA sequences. Cross-linking of CD3 receptors on Jurkat T Cells with monoclonal antibodies showed an early increase in type II PtdIns 4-kinase activity and co-localization of type II PtdIns 4-kinase β with CD3 ζ. Transfection of Jurkat T Cells with shRNAs inhibited CD3 receptor mediated type II PtdIns 4-kinase activation with a concomitant reduction in intra cellular calcium release, suggesting a role for type II PtdIns 4-kinase β in CD3 receptor signal transduction. Knock-down of type II PtdIns 4-kinase β with shRNAs also correlated with a decrease in PtdIns 4-kinase activity in cytoskeleton fractions and reduced adhesion to matrigel surfaces. These results indicate that type II PtdIns 4-kinase β is a key component in early T cell activation signaling cascades. [Copyright &y& Elsevier]

Abstract: We analyzed lipid kinase and lipid phosphatase activities and determined endogenous phytohormone levels by liquid chromatography–tandem mass spectrometry in root and coleoptile tissues following germination of barley (Hordeum vulgare) seeds. The enzymes showing highest activity in aleurone cells were diacylglycerol kinase (DAG-k, EC 2.7.1.107) and phosphatidate kinase (PA-k). The ratio of gibberellins (GAs) to abscisic acid (ABA) was 2-fold higher in aleurone than in coleoptile or root tissues. In coleoptiles, phosphatidylinositol 4-kinase (PI4-k, EC 2.7.1.67) showed the highest enzyme activity, and jasmonic acid (JA) level was higher than in aleurone. In roots, activities of PI4-k, DAG-k, and PA-k were similar, and salicylic acid (SA) showed the highest concentration. In the assays to evaluate the hydrolysis of DGPP (diacylglycerol pyrophosphate) and PA (phosphatidic acid) we observed that PA hydrolysis by LPPs (lipid phosphate phosphatases) was not modified; however, the diacylglycerol pyrophosphate phosphatase (DGPPase) was strikingly higher in coleoptile and root tissues than to aleurone. Relevance of these findings in terms of signaling responses and seedling growth is discussed. [Copyright &y& Elsevier]

Abstract: The phospholipidic signal transduction system involves generation of second messengers by hydrolysis or changes in phosphorylation state. Several studies have shown that the signaling pathway forms part of plant response to phytoregulators such as salicylic acid (SA) and methyl jasmonate (MJ), which have been widely used to stimulate secondary metabolite production in cell cultures. An evaluation was made of the effect of SA and MJ on phospholipidic signaling and capsaicinoid production in Capsicum chinense Jacq. suspension cells. Treatment with SA inhibited phospholipase C (PLC) (EC: 3.1.4.3) and phospholipase D (PLD) (EC: 3.1.4.4) activities in vitro, but increased lipid kinase activities in vitro at different SA concentrations. Treatment with MJ produced increases in PLC and PLD activities, while lipid kinase activities were variable and dose-dependent. The production of vanillin, a precursor of capsaicinoids, increased at specific SA or MJ doses. Preincubation with neomycin, a phospholipase inhibitor, before SA or MJ treatment inhibits increase in vanillin production which suggests that phospholipidic second messengers may participate in the observed increase in vanillin production. [ABSTRACT FROM AUTHOR]

Abstract: Background: Eosinophils are characteristic participants in allergic inflammation. The intracellular signalling mechanisms involved in the migration of eosinophils to sites of allergic inflammation are poorly understood. Chemotactic responses of eosinophils to platelet-activating factor (PAF), but not eotaxin, have been demonstrated to be dependent upon the activation of phosphoinositide 3-kinase (PI3K) but the specific isoform of PI3K involved has not been identified. Objective: To determine the roles of the leukocyte-specific PI3Kγ and PI3Kδ isoforms of PI3K in PAF-induced chemotaxis of human eosinophils. Methods: Chemotactic responses of the EoL-1 eosinophilic cell line and human peripheral blood eosinophils were measured. The effects of a PI3Kγ-selective inhibitor (5-[2,2-difluorobenzo(1,3)dioxol-5-ylmethylene]-thiazolidine-2,4-dione; AS604850) and gene knock-down of PI3Kγ and PI3Kδ on chemotactic responses were determined. Results: AS604850 caused a concentration-dependent suppression of chemotactic responses of EoL-1 cells and blood eosinophils to PAF but not eotaxin. Specific siRNAs reduced the expression of PI3Kγ and PI3Kδ in EoL-1 cells. Knock-down of PI3Kγ by siRNA resulted in a 75% inhibition of the chemotactic response to PAF but had no effect on the response to eotaxin. Knock-down of endogenous PI3Kδ by siRNA resulted in a 38% inhibition of the chemotactic response to PAF but had no effect on the response to eotaxin. Conclusion: PI3Kγ plays a major role in the induction of chemotaxis in PAF-stimulated eosinophils, while PI3Kδ plays a lesser role. Interventions which reduce the activity of PI3Kγ may have therapeutic potential in allergic diseases. [ABSTRACT FROM AUTHOR]

Abstract: Phosphoinositides play important roles in Golgi traffic and structural integrity. Specific lipid kinases and phosphatases associate with the Golgi complex and regulate the multiplicity of trafficking routes from this organelle. Work in different model systems showed that the basic elements that regulate lipid signaling at the Golgi are conserved from yeast to humans. Many of the enzymes involved in Golgi phosphoinositide metabolism are essential for viability or cause severe human disease when malfunctioning. Phosphoinositide effectors at the Golgi control both non-vesicular transfer of lipids and sorting of secretory and membrane proteins. In addition, Golgi phosphoinositides were recently implicated in the metabolic and cell growth-dependent regulation of the secretory pathway. [Copyright &y& Elsevier]

Abstract: Recent results, fundamentally obtained from animal tissues, suggest that polyamines (Pas), essential compounds for the growth and development of all life organisms, may interact with a signal transduction cascade. Because Pas are highly positive charged compounds, their binding with phospholipids involved in signal transduction is likely to be the case. In this work, the in vivo effect of Pas on some important components of phospholipid signal transduction pathway was studied, by the first time, in plant tissue. Endogenous Pas content varied during the culture cycle of Coffea arabica cells: putrescine (Put) levels increased at the end of the stationary phase, both spermidine (Spd) and spermine (Spm) accumulated at the beginning of the linear growth phase. Cells that were incubated with Put presented a significant increase in phospholipase D (PLD) (EC: 3.1.4.4) activity, phospholipase C (PLC) (EC: 3.1.4.3) activity decreased, and the effect on lipid kinases was less marked. However, the incubation of the cells with Spd and Spm significantly stimulated the lipid kinases activities, fundamentally increased the formation of phosphatidyl inositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2), while the effect on PLC and PLD activities was minor when compared with the cells treated with Put. The results presented here suggest that Pas may modulate the cellular signal of C. arabica cells by differentially affecting components of the phospholipid cascade. [Copyright &y& Elsevier]

Annexin VI is a 68-kDa protein of the Annexin family, a group of Ca2+-dependent phospholipid-binding proteins widely distributed in mammalian tissues including skeletal muscle. We investigated a) which membrane system contributes Annexin VI to skeletal muscle triads, and b) whether Annexin VI removal affects triad integrity or function. Annexin VI was present in isolated triads and transverse tubules but not in heavy sarcoplasmic reticulum vesicles, indicating that Annexin VI binds to either free or triad-attached transverse tubules. Extraction with EGTA of Annexin VI from triads did not alter their migration as a single band in sucrose density gradients or their ouabain binding-site density, indicating that triad integrity does not require Annexin VI. Caffeine-induced Ca2+ release kinetics and Ca2+ uptake rates were likewise not affected by Annexin VI removal from triads, suggesting that Annexin VI is not involved in these functions. Annexin VI purified from rabbit skeletal muscle displayed Ca2+-dependent binding to liposomes containing phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine. Binding saturated at 1/20 molar ratio phosphatidylinositol 4,5-bisphosphate/phosphatidylcholine and was optimal at free [Ca2+] ? 20 mM. Extraction of Annexin VI from triads did not affect the generation of phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, or phosphatidic acid by endogenous lipid kinases, suggesting that despite its capacity to bind to negatively charged phospholipids, Annexin VI does not affect the kinase activities responsible for their generation. [ABSTRACT FROM AUTHOR]

The synthetic peptide carrying residues 1–40 of chicken αD-globin, which promotes differentiation in Trypanosoma cruzi epimastigote, stimulated PPtdIns-k, DAG-k, and PA-k activities in a dose-dependent manner. A biphasic behavior only for PPtdIns-k and DAG-k was demonstrated by changes in [32P]PPtdIns and PtdOH levels, the earlier phase peaking at 3 min with a return to basal levels by 6 min and then a second phase with a sustained increase in time. This behavior was not observed for PA-k; the DGPP levels peaked at 6 min and were sustained in time. PMA pretreatment only abolished the first peak of PPtdIns-k, DAG-k activities, and InsPs/InsP3 levels. There was also a transient elevation in intracellular calcium concentration, but this variation was modified only 50% by PMA. The results suggest that peptide 1–40 induces activation of the inositol cycle through lipid kinase activation in a biphasic manner. In this response, the early increase of enzymatic activities would be regulated by PKC and the InsP3 may only be responsible, in part, for the calcium signaling. [Copyright &y& Elsevier]

The article analyzes Inositol lipids as spatial regulators of membrane traffic. The intact lipid molecule functions as a reversible recruiting device for proteins to transiently bind to membranes and as an allosteric regulator of many proteins. The Inositol head-group of phosphatidylinositol can be phosphorylated at a single or a combination of positions to give rise to different phosphoinositides. The first hint that phosphoinositides were important in membrane traffic came from studies showing that a bacterial phospholipase C inhibited regulated exocytosis in PC12 cells and from genetic studies in Saccharomyces cerevisiae.

The possible participation of several major components of the signal transduction pathway in photoperiodic flower induction was examined in Pharbitis cotyledons. Exogenous applications of GTP‐γ‐S (1–10 μM) or of the phorbol ester, phorbol 12‐myristate‐13‐acetate (PMA, 0.1–5.0 μM) to Pharbitis plants held under a marginal inductive period (11.5 h dark) significantly increased their flowering response. Membrane lipid fluidity, GTP‐binding and protein kinase activity were increased following a single flowering‐inducing dark period of 16 h; however, a light‐break of 10 min that abolished flower induction failed to reverse the dark‐induced increase in these processes. Photo‐inductive dark conditions significantly increased the content of diacylglycerol (DAG) and phosphoinositides in the cotyledon membranes, together with the activities of their kinases, and a light break decreased them to control levels and below. In addition, a single spraying with GTP‐γ‐S or PMA at 1 μM significantly increased both the lipid content and the kinase activities. These compounds also enhanced the kinase activities in vitro. It is concluded that DAG and phosphoinositide metabolism play a role in the linking of the photoperiodic induction of the phytochrome with the flowering response in Pharbitis nil. [ABSTRACT FROM AUTHOR]

The specific activities of 6 enzymes, which are involved in the synthesis and catabolism of membrane lipids, were monitored in plasma membranes isolated from petunia petals during senescence. These included phosphatidylinositol (PI) kinase (EC 2.7.1.67), phosphatidylinositol monophosphate (PIP) kinase (EC 2.7.1.68), diacylglycerol (DAG) kinase (EC 2.7.1.107), phospholipase A (EC 3.1.1.4) and PIP- and PIP2-phospholipase C (EC 3.1.4.3). Using endogenous substrate, the [32P]PA and [32P]PIP2 formation increased to 140 and 200%, respectively, of the day 1 value by 4 days after harvest. There was no significant change in [32P]PIP formation during the same time period. On the fifth day the petals wilted and the [32P]PA and [32P]PIP formation declined significantly. In contrast, the [32P]PIP2 formation remained high in the day 5 petals. When the lipid kinase activities were assayed in the membranes in the presence of exogenous substrate the specific activity of all of the enzymes increased, and the changes in [32P]PA production over the 5-day period were similar to those observed with endogenous substrate. When exogenous PI and PIP were added, however, there was no longer an increase in [32P]PIP2 formation by plasma membranes of day 4 petals and [32P]PIP formation significantly decreased. The relative decrease in PIP and PIP2 formation by day 4 membranes when exogenous substrate was added may have resulted from differences in the lipase activities in the day 1 and day 4 membranes. The plasma membrane A-type phospholipase activity increased throughout the 5 day period, and phospholipase C activity increased two-fold between day 1 and day 4. Such changes in the metabolism of the plasma membrane lipids during flower senescence would affect the ability of the petals to use inositol phospholipid-based signal transduction pathways. [ABSTRACT FROM AUTHOR]