Substrate Phosphatidylinositol Research Articles

INPP5K controls the dynamic structure and signaling of wild-type and mutated, leukemia-associated IL-7 receptors

AbstractThe downstream signaling of the interleukin-7 (IL-7) receptor (IL-7R) plays important physiological and pathological roles, including the differentiation of lymphoid cells and proliferation of acute lymphoblastic leukemia cells. Gain-of-function mutations in the IL-7Rα chain, the specific component of the receptor for IL-7, result in constitutive, IL-7–independent signaling and trigger acute lymphoblastic leukemia. Here, we show that the loss of the phosphoinositide 5-phosphatase INPP5K is associated with increased levels of the INPP5K substrate phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) and causes an altered dynamic structure of the IL-7 receptor. We discovered that the IL-7Rα chain contains a very conserved positively charged polybasic amino acid sequence in its cytoplasmic juxtamembrane region; this region establish stronger ionic interactions with negatively charged PtdIns(4,5)P2 in the absence of INPP5K, freezing the IL-7Rα chain structure. This dynamic structural alteration causes defects in IL-7R signaling, culminating in decreased expressions of EBF1 and PAX5 transcription factors, in microdomain formation, cytoskeletal reorganization, and bone marrow B-cell differentiation. Similar alterations after the reduced INPP5K expression also affected mutated, constitutively activated IL-7Rα chains that trigger leukemia development, leading to reduced cell proliferation. Altogether, our results indicate that the lipid 5-phosphatase INPP5K hydrolyzes PtdIns(4,5)P2, allowing the requisite conformational changes of the IL-7Rα chain for optimal signaling.

Phosphatidylcholine Cation-Tyrosine π Complexes: Motifs for Membrane Binding by a Bacterial Phospholipase C.

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes are a virulence factor in many Gram-positive organisms. The specific activity of the Bacillus thuringiensis PI-PLC is significantly increased by adding phosphatidylcholine (PC) to vesicles composed of the substrate phosphatidylinositol, in part because the inclusion of PC reduces the apparent Kd for the vesicle binding by as much as 1000-fold when comparing PC-rich vesicles to PI vesicles. This review summarizes (i) the experimental work that localized a site on BtPI-PLC where PC is bound as a PC choline cation—Tyr-π complex and (ii) the computational work (including all-atom molecular dynamics simulations) that refined the original complex and found a second persistent PC cation—Tyr-π complex. Both complexes are critical for vesicle binding. These results have led to a model for PC functioning as an allosteric effector of the enzyme by altering the protein dynamics and stabilizing an ‘open’ active site conformation.

RABENOSYN separation-of-function mutations uncouple endosomal recycling from lysosomal degradation, causing a distinct Mendelian disorder

Rabenosyn (RBSN) is a conserved endosomal protein necessary for regulating internalized cargo. Here, we present clinical, genetic, cellular and biochemical evidence that two distinct RBSN missense variants are responsible for a novel Mendelian disorder consisting of progressive muscle weakness, facial dysmorphisms, ophthalmoplegia and intellectual disability. Using exome sequencing, we identified recessively acting germline alleles p.Arg180Gly and p.Gly183Arg, which are both situated in the FYVE domain of RBSN. We find that these variants abrogate binding to its cognate substrate phosphatidylinositol 3-phosphate (PI3P) and thus prevent its translocation to early endosomes. Although the endosomal recycling pathway was unaltered, mutant p.Gly183Arg patient fibroblasts show accumulation of cargo tagged for lysosomal degradation. Our results suggest that these variants are separation-of-function alleles, which cause a delay in endosomal maturation without affecting cargo recycling. We conclude that distinct germline mutations in RBSN cause non-overlapping phenotypes with specific and discrete endolysosomal cellular defects.

Combining data integration and molecular dynamics for target identification in α-Synuclein-aggregating neurodegenerative diseases: Structural insights on Synaptojanin-1 (Synj1)

Parkinson’s disease (PD), Alzheimer’s disease (AD) and Amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases hallmarked by the formation of toxic protein aggregates. However, targeting these aggregates therapeutically have thus far shown no success. The treatment of AD has remained particularly problematic since no new drugs have been approved in the last 15 years. Therefore, novel therapeutic targets need to be identified and explored. Here, through the integration of genomic and proteomic data, a set of proteins with strong links to α-synuclein-aggregating neurodegenerative diseases was identified. We propose 17 protein targets that are likely implicated in neurodegeneration and could serve as potential targets. The human phosphatidylinositol 5-phosphatase synaptojanin-1, which has already been independently confirmed to be implicated in Parkinson’s and Alzheimer’s disease, was among those identified. Despite its involvement in PD and AD, structural aspects are currently missing at the molecular level. We present the first atomistic model of the 5-phosphatase domain of synaptojanin-1 and its binding to its substrate phosphatidylinositol 4,5-bisphosphate (PIP2). We determine structural information on the active site including membrane-embedded molecular dynamics simulations. Deficiency of charge within the active site of the protein is observed, which suggests that a second divalent cation is required to complete dephosphorylation of the substrate. The findings in this work shed light on the protein’s binding to phosphatidylinositol 4,5-bisphosphate (PIP2) and give additional insight for future targeting of the protein active site, which might be of interest in neurodegenerative diseases where synaptojanin-1 is overexpressed.

Nuclear Phosphatidylinositol 5-Phosphatase Is Essential for Allelic Exclusion of Variant Surface Glycoprotein Genes in Trypanosomes.

Allelic exclusion of variant surface glycoprotein (VSG) genes is essential for African trypanosomes to evade the host antibody response by antigenic variation. The mechanisms by which this parasite expresses only one of its ∼2,000 VSG genes at a time are unknown. We show that nuclear phosphatidylinositol 5-phosphatase (PIP5Pase) interacts with repressor activator protein 1 (RAP1) in a multiprotein complex and functions in the control of VSG allelic exclusion. RAP1 binds PIP5Pase substrate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], and catalytic mutation of PIP5Pase that inhibits PI(3,4,5)P3 dephosphorylation results in simultaneous transcription of VSGs from all telomeric expression sites (ESs) and from silent subtelomeric VSG arrays. PIP5Pase and RAP1 bind to telomeric ESs, especially at 70-bp repeats and telomeres, and their binding is altered by PIP5Pase inactivation or knockdown, implying changes in ES chromatin organization. Our data suggest a model whereby PIP5Pase controls PI(3,4,5)P3 binding by RAP1 and, thus, RAP1 silencing of telomeric and subtelomeric VSG genes. Hence, allelic exclusion of VSG genes may entail control of nuclear phosphoinositides.

A Phospholipase C-Like Protein From Ricinus communis Increases Hydroxy Fatty Acids Accumulation in Transgenic Seeds of Camelina sativa.

There have been strong interests in producing unusual fatty acids in oilseed crops to provide renewable industrial feedstock. Results are so far largely disappointing since much lower amounts of such fatty acids accumulate in genetically engineered seeds than in their original natural sources. It has been suggested that the flux of unusual fatty acids through phosphatidylcholine (PC) represents a major bottleneck for high accumulation of such fatty acids in triacylglycerol (TAG). We show here that a phospholipase C-like protein (RcPLCL1) from castor bean, which accumulates nearly 90% of the hydroxylated ricinoleic acid in its seed TAG, increases the amount of hydroxy fatty acids (HFAs) when co-expresses with the fatty acid hydroxylase (RcFAH12) in transgenic seed of Camelina sativa. RcPLCL1 shows hydrolyzing activities on both PC and phosphatidylinositol substrates in our in vitro assay conditions. The PC-PLC activity of the RcPLCL1 may have increased the efficiency of HFA-PC to diacylglycerol conversion, which explains our observation of increased HFA contents in TAG concomitant with decreased HFA in the membrane lipid PC during seed development. Consequently, this may also alleviate the potential detrimental effect of HFA on germination of the engineered camelina seeds. Our results provide new knowledge that will help design effective strategies to engineer high levels of HFAs in transgenic oilseeds.

PI-273, a Substrate-Competitive, Specific Small-Molecule Inhibitor of PI4KIIα, Inhibits the Growth of Breast Cancer Cells.

While phosphatidylinositol 4-kinase (PI4KIIα) has been identified as a potential target for antitumor therapy, the clinical applications of PI4KIIα are limited by a lack of specific inhibitors. Here we report the first small-molecule inhibitor (SMI) of human PI4KIIα. Docking-based and ligand-based virtual screening strategies were first employed to identify promising hits, followed by two rounds of kinase activity inhibition validation. 2-(3-(4-Chlorobenzoyl)thioureido)-4-ethyl-5-methylthiophene-3-carboxamide (PI-273) exhibited the greatest inhibitory effect on PI4KIIα kinase activity (IC50 = 0.47 μmol/L) and suppressed cell proliferation. Surface plasmon resonance and thermal shift assays indicated that PI-273 interacted directly with PI4KIIα. Kinetic analysis identified PI-273 as a reversible competitive inhibitor with respect to the substrate phosphatidylinositol (PI), which contrasted with most other PI kinase inhibitors that bind the ATP binding site. PI-273 reduced PI4P content, cell viability, and AKT signaling in wild-type MCF-7 cells, but not in PI4KIIα knockout MCF-7 cells, indicating that PI-273 is highly selective for PI4KIIα. Mutant analysis revealed a role of palmitoylation insertion in the selectivity of PI-273 for PI4KIIα. In addition, PI-273 treatment retarded cell proliferation by blocking cells in G2-M, inducing cell apoptosis and suppressing colony-forming ability. Importantly, PI-273 significantly inhibited MCF-7 cell-induced breast tumor growth without toxicity. PI-273 is the first substrate-competitive, subtype-specific inhibitor of PI4KIIα, the use of which will facilitate evaluations of PI4KIIα as a cancer therapeutic target. Cancer Res; 77(22); 6253-66. ©2017 AACR.

Calmodulin Interacts and Regulates Enzyme Activity of the Mammalian Sperm Phospholipase C

Sperm-specific Phospholipase C zeta (PLCζ) is widely considered to be the sole, physiological stimulus responsible for the generation of Ca2+ oscillations that induce egg activation and early embryo development during mammalian fertilization. PLCζ, which is delivered from the fertilizing sperm into the egg cytoplasm, catalyzes the hydrolysis of its membrane-bound phospholipid substrate phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], triggering the cytoplasmic Ca2+ oscillations through the inositol 1,4,5-trisphosphate (InsP3) signaling pathway. Despite the recent advances the detailed regulatory mechanism of PLCζ is still unclear, as binding partners of this protein within the sperm or the fertilizing egg have not yet been identified. Calmodulin (CaM) is a ubiquitous Ca2+ sensor in eukaryotic cells. A previous study has reported that CaM directly interacts and regulates the activity of PLC delta 1 protein, a somatic PLC isoform with structural similarities to sperm PLCζ. Bioinformatics analysis revealed putative CaM-binding sites on PLCζ sequence. In the present study, we have used co-immunoprecipitation analysis and we show that in the presence of Ca2+, human PLCζ directly interacts with CaM. Isothermal titration calorimetry (ITC) experiments were performed to map the interaction. Three different peptides corresponding to disparate sequences within human PLCζ were used and it was shown that PLCζ interacts with CaM via one region of the molecule. In addition, recombinant proteins corresponding to the N- and C-lobe of human CaM were used for ITC experiments, which revealed that CaM interacts with PLCζ in the presence of Ca2+, only through one of its lobe domains. In vitro PIP2 hydrolysis assays revealed that CaM alters PLCζ PIP2 hydrolytic activity at high Ca2+ concentrations and, as suggested by liposome binding assays, this appears to be due to CaM binding to PLCζ affecting proper access of the enzyme active site to its substrate PI(4,5)P2.

Arabidopsis AtPLC2 Is a Primary Phosphoinositide-Specific Phospholipase C in Phosphoinositide Metabolism and the Endoplasmic Reticulum Stress Response.

Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response.

Characterization of tocopherol kinase activity in primary human coronary artery smooth muscle cells

The vitamin E derivative, alpha‐tocopheryl phosphate (αTP), is detectable in tissues in small quantity (30–300 nM) suggesting the existence of enzyme(s) with α‐tocopherol (αT) kinase activity. Here, we have characterized the production of αTP from αT and [γ‐32P]‐ATP in primary human coronary artery smooth muscle cells (HCA‐SMC) using thin layer chromatography (TLC) followed by ultra performance liquid chromatography (UPLC). In addition to αT, γT is also phosphorylated. γTP inhibits proliferation of THP‐1 cells and reduces CD36 expression more potently than αTP (10 μM). The recombinant human tocopherol associated protein (TAP1/SEC14L2) binds both αT and αTP and stimulates phosphorylation of αT possibly by facilitating its transport and presentation to the putative αT kinase in the correct spatial orientation. Recombinant hTAP1 reduces the in vitro activity of the phosphatidylinositol‐3‐kinase gamma (PI3Kγ) potentially by sequestering the substrate phosphatidylinositol (PI). Indeed, the addition αT or αTP to the reaction stimulates PI3Kγ, most likely by facilitating egress of PI from hTAP1 and its binding to the enzyme. It is suggested that the continuous mutual exchange of different ligands in the metabolic nanoreactor formed by hTAPs may be a way to make these lipophiles more accessible as substrates to enzymes and as constituents to specific membrane domains. Supported by USDA Contract #58–1950‐0–014.

Phosphatidylinositol-4-phosphate 5-Kinase Isoforms Exhibit Acyl Chain Selectivity for Both Substrate and Lipid Activator

Phosphatidylinositol 4,5-bisphosphate is mostly produced in the cell by phosphatidylinositol-4-phosphate 5-kinases (PIP5K) and has a crucial role in numerous signaling events. Here we demonstrate that in vitro all three isoforms of PIP5K, α, β, and γ, discriminate among substrates with different acyl chains for both the substrates phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) although to different extents, with isoform γ being the most selective. Fully saturated dipalmitoyl-PtdIns4P was a poor substrate for all three isoforms, but both the 1-stearoyl-2-arachidonoyl and the 1-stearoyl-2-oleoyl forms of PtdIns4P were good substrates. V(max) was greater for the 1-stearoyl-2-arachidonoyl form compared with the 1-stearoyl-2-oleoyl form, although for PIP5Kβ the difference was small. For the α and γ isoforms, K(m) was much lower for 1-stearoyl-2-oleoyl PtdIns4P, making this lipid the better substrate of the two under most conditions. Activation of PIP5K by phosphatidic acid is also acyl chain-dependent. Species of phosphatidic acid with two unsaturated acyl chains are much better activators of PIP5K than those containing one saturated and one unsaturated acyl chain. PtdIns is a poor substrate for PIP5K, but it also shows acyl chain selectivity. Curiously, there is no acyl chain discrimination among species of phosphatidic acid in the activation of the phosphorylation of PtdIns. Together, our findings indicate that PIP5K isoforms α, β, and γ act selectively on substrates and activators with different acyl chains. This could be a tightly regulated mechanism of producing physiologically active unsaturated phosphatidylinositol 4,5-bisphosphate species in the cell.

Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq

Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq

Characterisation of the PTEN inhibitor VO-OHpic

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a phosphatidylinositol triphosphate 3-phosphatase that counteracts phosphoinositide 3-kinases and has subsequently been implied as a valuable drug target for diabetes and cancer. Recently, we demonstrated that VO-OHpic is an extremely potent inhibitor of PTEN with nanomolar affinity in vitro and in vivo. Given the importance of this inhibitor for future drug design and development, its mode of action needed to be elucidated. It was discovered that inhibition of recombinant PTEN by VO-OHpic is fully reversible. Both K(m) and V(max) are affected by VO-OHpic, demonstrating a noncompetitive inhibition of PTEN. The inhibition constants K(ic) and K(iu) were determined to be 27 ± 6 and 45 ± 11 nM, respectively. Using the artificial phosphatase substrate 3-O-methylfluorescein phosphate (OMFP) or the physiological substrate phosphatidylinositol 3,4,5-triphosphate (PIP(3)) comparable parameters were obtained suggesting that OMFP is a suitable substrate for PTEN inhibition studies and PTEN drug screening.

Reduced NO-cGMP Signaling Contributes to Vascular Inflammation and Insulin Resistance Induced by High-Fat Feeding

Diet-induced obesity (DIO) in mice causes vascular inflammation and insulin resistance that are accompanied by decreased endothelial-derived NO production. We sought to determine whether reduced NO-cGMP signaling contributes to the deleterious effects of DIO on the vasculature and, if so, whether these effects can be blocked by increased vascular NO-cGMP signaling. By using an established endothelial cell culture model of insulin resistance, exposure to palmitate, 100 micromol/L, for 3 hours induced both cellular inflammation (activation of IKK beta-nuclear factor-kappaB) and impaired insulin signaling via the insulin receptor substrate-phosphatidylinositol 3-kinase pathway. Sensitivity to palmitate-induced endothelial inflammation and insulin resistance was increased when NO signaling was reduced using an endothelial NO synthase inhibitor, whereas endothelial responses to palmitate were blocked by pretreatment with either an NO donor or a cGMP analogue. To investigate whether endogenous NO-cGMP signaling protects against vascular responses to nutrient excess in vivo, adult male mice lacking endothelial NO synthase were studied. As predicted, both vascular inflammation (phosphorylated I kappaB alpha and intercellular adhesion molecule levels) and insulin resistance (phosphorylated Akt [pAkt] and phosphorylated eNOS [peNOS] levels) were increased in endothelial NO synthase(-/-) (eNOS(-/-)) mice, reminiscent of the effect of DIO in wild-type controls. Next, we asked whether the vascular response to DIO in wild-type mice can be reversed by a pharmacological increase of cGMP signaling. C57BL6 mice were either fed a high-fat diet or remained on a low-fat diet for 8 weeks. During the final 2 weeks of the study, mice on each diet received either placebo or the phosphodiesterase-5 inhibitor sildenafil, 10 mg/kg per day orally. In high-fat diet-fed mice, vascular inflammation and insulin resistance were completely prevented by sildenafil administration at a dose that had no effect in mice fed the low-fat diet. Reduced signaling via the NO-cGMP pathway is a mediator of vascular inflammation and insulin resistance during overnutrition induced by high-fat feeding. Therefore, phosphodiesterase-5, soluble guanylyl cyclase, and other molecules in the NO-cGMP pathway (eg, protein kinase G) constitute potential targets for the treatment of vascular dysfunction in the setting of obesity.

Inhibition of Inositol Phosphorylceramide Synthase by the Cyclic Peptide Aureobasidin A

By using a detergent-washed membrane preparation, the interaction of the fungal natural product inhibitor aureobasidin A (AbA) with inositol phosphorylceramide synthase (IPC synthase) was studied by kinetic analysis of wild-type and mutant enzyme-catalyzed reactions. AbA inhibited the wild-type enzyme from both Candida albicans and Saccharomyces cerevisiae in an irreversible, time-dependent manner, with apparent K(i) values of 183 and 234 pM, respectively. Three synthetic chemistry-derived AbA derivatives, PHA-533179, PHA-556655, and PHA-556656, had affinities 4 to 5 orders of magnitude lower and were reversible inhibitors that competed with the donor substrate phosphatidylinositol (PI). AbA was a reversible, apparently noncompetitive inhibitor, with a K(i) of 1.4 microM, of the IPC synthase from an AbA-resistant S. cerevisiae mutant. The K(m) values for both substrates (ceramide and PI) were similar when they interacted with the mutant and the wild-type enzymes. By contrast, the V(max) for the mutant enzyme was less than 10% of that for the wild-type enzyme. A comparison of the results obtained with AbA with those obtained with two other natural products inhibitors, rustmicin and khafrefungin, revealed that while rustmicin appeared to be a reversible, noncompetitive inhibitor of the wild-type enzyme, with a K(i) of 16.0 nM, khafrefungin had the kinetic properties of a time-dependent inhibitor and an apparent K(i) of 0.43 nM. An evaluation of the efficiencies of these compounds as inhibitors of the mutant enzyme revealed for both a drop in the apparent affinity for the enzyme of more than 2 orders of magnitude.

Regulation of PLCβ1a membrane anchoring by its substrate phosphatidylinositol (4,5)-bisphosphate

Basic knowledge as to the subcellular location and dynamics of PLCbeta isozymes is scant. Here, we report on the subcellular location of GFP-PLCbeta1a and the use of total internal reflection fluorescence (TIRF) microscopy to examine the dynamics of GFP-PLCbeta1a at the plasma membrane upon stimulation of Gq-coupled receptors. Using this technique, we observed PLCbeta1a dissociation from the plasma membrane upon addition of agonist. An increase in intracellular calcium and a decrease in PtdIns(4,5)P2 both coincided with a translocation of PLCbeta1a from the plasma membrane into the cytosol. In order to differentiate between calcium and PtdIns(4,5)P2, rapamycin-induced heterodimerization of FRB and FKBP12 fused to 5-phosphatase IV was used to instantaneously convert PtdIns(4,5)P2 into PtdIns(4)P. Addition of rapamycin caused PLCbeta1a to dissociate from the plasma membrane, indicating that removal of PtdIns(4,5)P2 is sufficient to cause translocation of PLCbeta1a from the plasma membrane. In conclusion, PLCbeta1a localization is regulated by its own substrate.

Expression and evolution of the phosphoinositide-specific phospholipase C gene family in Arabidopsis thaliana

Phosphoinositide-specific phospholipase C cleaves the substrate phosphatidylinositol 4,5-bisphosphate and generates inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, both of which are second messengers in the phosphoinositide signal transduction pathways operative in animal cells. Five PI-PLC isoforms, beta, gamma, delta, epsilon and zeta, have been identified in mammals. Plant PI-PLCs are structurally close to the mammalian PI-PLC-zeta isoform. The Arabidopsis genome contains nine AtPLC genes. Expression patterns of all nine genes in different organs and in response to various environmental stimuli were studied by applying a quantitative RT-PCR approach. Multiple members of the gene family were differentially expressed in Arabidopsis organs, suggesting putative roles for this enzyme in plant development, including tissue and organ differentiation. This study also shows that a majority of the AtPLC genes are induced in response to various environmental stimuli, including cold, salt, nutrients Murashige-Skoog salts, dehydration, and the plant hormone abscisic acid. Results of this and previous studies strongly suggest that transcriptional activation of the PI-PLC gene family is important for adapting plants to stress environments. Expression patterns and phylogenetic relationships indicates that AtPLC gene members probably evolved through multiple rounds of gene duplication events, with AtPLC4 and AtPLC5 and AtPLC8 and AtPLC9 being duplicated in tandem in recent times.

Polyunsaturated Fatty Acids Influence Synaptojanin Localization to Regulate Synaptic Vesicle Recycling

The lipid polyunsaturated fatty acids are highly enriched in synaptic membranes, including synaptic vesicles, but their precise function there is unknown. Caenorhabditis elegans fat-3 mutants lack long-chain polyunsaturated fatty acids (LC-PUFAs); they release abnormally low levels of serotonin and acetylcholine and are depleted of synaptic vesicles, but the mechanistic basis of these defects is unclear. Here we demonstrate that synaptic vesicle endocytosis is impaired in the mutants: the synaptic vesicle protein synaptobrevin is not efficiently retrieved after synaptic vesicles fuse with the presynaptic membrane, and the presynaptic terminals contain abnormally large endosomal-like compartments and synaptic vesicles. Moreover, the mutants have abnormally low levels of the phosphoinositide phosphatase synaptojanin at release sites and accumulate the main synaptojanin substrate phosphatidylinositol 4,5-bisphosphate at these sites. Both synaptobrevin and synaptojanin mislocalization can be rescued by providing exogenous arachidonic acid, an LC-PUFA, suggesting that the endocytosis defect is caused by LC-PUFA depletion. By showing that the genes fat-3 and synaptojanin act in the same endocytic pathway at synapses, our findings suggest that LC-PUFAs are required for efficient synaptic vesicle recycling, probably by modulating synaptojanin localization at synapses.

SLP-65 Signal Transduction Requires Src Homology 2 domain-mediated Membrane Anchoring and a Kinase-independent Adaptor Function of Syk

The family of SLPs (Src homology 2 domain-containing leukocyte adaptor proteins) are cytoplasmic signal effectors of lymphocyte antigen receptors. A main function of SLP is to orchestrate the assembly of Ca(2+)-mobilizing enzymes at the inner leaflet of the plasma membrane. For this purpose, SLP-76 in T cells utilizes the transmembrane adaptor LAT, but the mechanism of SLP-65 membrane anchoring in B cells remains an enigma. We now employed two genetic reconstitution systems to unravel structural requirements of SLP-65 for the initiation of Ca(2+) mobilization and subsequent activation of gene transcription. First, mutational analysis of SLP-65 in DT40 B cells revealed that its C-terminal Src homology 2 domain controls efficient tyrosine phosphorylation by the kinase Syk, plasma membrane recruitment, as well as downstream signaling to NFAT activation. Second, we dissected these processes by expressing SLP-65 in SLP-76-deficient T cells and found that a kinase-independent adaptor function of Syk is required to link phosphorylated SLP-65 to Ca(2+) mobilization. These approaches unmask a mechanistic complexity of SLP-65 activation and coupling to signaling cascades in that Syk is upstream as well as downstream of SLP-65. Moreover, membrane anchoring of the SLP-65-assembled Ca(2+) initiation complex, which appears to be fundamentally different from that of closely related SLP-76, does not necessarily involve a B cell-specific component.

Epigallocatechin Gallate, a Green Tea Polyphenol, Mediates NO-dependent Vasodilation Using Signaling Pathways in Vascular Endothelium Requiring Reactive Oxygen Species and Fyn

Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, mimics metabolic actions of insulin to inhibit gluconeogenesis in hepatocytes. Because signaling pathways regulating metabolic and vasodilator actions of insulin are shared in common, we hypothesized that EGCG may also have vasodilator actions to stimulate production of nitric oxide (NO) from endothelial cells. Acute intra-arterial administration of EGCG to mesenteric vascular beds isolated ex vivo from WKY rats caused dose-dependent vasorelaxation. This was inhibitable by L-NAME (NO synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), or PP2 (Src family kinase inhibitor). Treatment of bovine aortic endothelial cells (BAEC) with EGCG (50 microm) acutely stimulated production of NO (assessed with NO-specific fluorescent dye DAF-2) that was inhibitable by l-NAME, wortmannin, or PP2. Stimulation of BAEC with EGCG also resulted in dose- and time-dependent phosphorylation of eNOS that was inhibitable by wortmannin or PP2 (but not by MEK inhibitor PD98059). Specific knockdown of Fyn (but not Src) with small interfering RNA inhibited both EGCG-stimulated phosphorylation of Akt and eNOS as well as production of NO in BAEC. Treatment of BAEC with EGCG generated intracellular H(2)O(2) (assessed with H(2)O(2)-specific fluorescent dye CM-H(2)DCF-DA), whereas treatment with N-acetylcysteine inhibited EGCG-stimulated phosphorylation of Fyn, Akt, and eNOS. We conclude that EGCG has endothelial-dependent vasodilator actions mediated by intracellular signaling pathways requiring reactive oxygen species and Fyn that lead to activation of phosphatidylinositol 3-kinase, Akt, and eNOS. This mechanism may explain, in part, beneficial vascular and metabolic health effects of green tea consumption.