Regulation of phospholipase D activity, membrane targeting and intracellular trafficking by phosphoinositides

Generation of PA (phosphatidic acid) by PLD (phospholipase D)-catalysed hydrolysis of phosphatidylcholine plays a pivotal role in cellular signalling pathways that regulate organization of the actin cytoskeleton, vesicular transport and exocytosis and stimulation of cell growth and survival. PLD regulation and function are intimately linked with phosphoinositide metabolism. Phosphatidyl 4-phosphate 5-kinase is stimulated by PA in vitro and this enzyme is the downstream effector of a significant subset of PLD signalling pathways. Yeast and mammalian PLDs are potently and specifically activated by the product of this kinase, PtdIns(4,5)P2, through interactions mediated by a polybasic motif within the catalytic core of the enzyme. Integrity of this motif is critical for agonist activation of mammalian PLD and for PLD function in secretion, sporulation and exocytosis in vivo. Although dispensable for catalysis in vitro, these PLD enzymes also contain N-terminal PH (pleckstrin) and PX (phox) homology domains. Binding studies using recombinantly expressed PLD fragments indicate that the PH and PX domains also interact specifically with distinct phosphoinositide ligands. Both the PX and PH domains are important for PLD function by controlling the dynamic association of the enzyme with the plasma membrane and its intracellular trafficking by the endocytic pathway. These results identify two distinct modes of regulation of PLD by phosphoinositides: stimulation of catalysis mediated by the polybasic domain and dynamic regulation of membrane targeting mediated primarily by the PH and PX domains.     

Interaction of the type Ialpha PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4, 5-bisphosphate in the regulation of PLD2 activity

Phosphoinositides are localized in various intracellular compartments and can regulate a number of intracellular functions, such as cytoskeletal dynamics and membrane trafficking. Phospholipase Ds (PLDs) are regulated enzymes that hydrolyse phosphatidylcholine (PtdCho) to generate the putative second messenger phosphatidic acid (PtdOH). In vitro, PLDs have an absolute requirement for higher phosphorylated inositides, such as phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)]. Whether this lipid is able to regulate the activity of PLD in vivo is contentious. To examine this hypothesis we studied the relationship between PLD and an enzyme critical for the intracellular synthesis of PtdIns(4,5)P(2): phosphatidylinositol 4-phosphate 5-kinase alpha (Type Ialpha PIPkinase). We find that both PLD1 and PLD2 interact with the Type Ialpha PIPkinase and that PLD2 activity in vivo can be regulated solely by the expression of this lipid kinase. Moreover, PLD2 is able to recruit the Type Ialpha PIPkinase to its intracellular location. We show that the physiological requirement of PLD enzymes for PtdIns(4,5)P(2) is critical and that PLD2 activity can be regulated solely by the levels of this key intracellular lipid.     

Regulation of phospholipase D activity by actin. Actin exerts bidirectional modulation of Mammalian phospholipase D activity in a polymerization-dependent,isoform-specific manner

Many critical cellular processes, including proliferation, vesicle trafficking, and secretion, are regulated by both phospholipase D (PLD) and the actin microfilament system. Stimulation of human PLD1 results in its association with the detergent-insoluble actin cytoskeleton, but the molecular mechanisms and functional consequences of PLD-actin interactions remain incompletely defined. Biochemical and pharmacologic modulation of actin polymerization resulted in complex bidirectional effects on PLD activity, both in vitro and in vivo. Highly purified G-actin inhibited basal and stimulated PLD activity, whereas F-actin produced the opposite effects. Actin-induced modulation of PLD activity was independent of the activating stimulus. The efficacy and potency of the effects of actin were isoform-specific but broadly conserved among actin family members. Human betagamma-actin was only 45% as potent and 40% as efficacious as rabbit skeletal muscle alpha-actin, whereas its inhibitory profile was similar to the single actin species from the yeast, Saccharomyces cerevisiae. Use of actin polymerization-specific reagents indicated that PLD1 binds both monomeric G-actin, as well as actin filaments. These data are consistent with a model in which the physical state of the actin cytoskeleton is a critical determinant of its regulation of PLD activity.     

Continual production of phosphatidic acid by phospholipase D is essential for antigen-stimulated membrane ruffling in cultured mast cells

Phospholipase Ds (PLDs) are regulated enzymes that generate phosphatidic acid (PA), a putative second messenger implicated in the regulation of vesicular trafficking and cytoskeletal reorganization. Mast cells, when stimulated with antigen, show a dramatic alteration in their cytoskeleton and also release their secretory granules by exocytosis. Butan-1-ol, which diverts the production of PA generated by PLD to the corresponding phosphatidylalcohol, was found to inhibit membrane ruffling when added together with antigen or when added after antigen. Inhibition by butan-1-ol was completely reversible because removal of butan-1-ol restored membrane ruffling. Measurements of PLD activation by antigen indicate a requirement for continual PA production during membrane ruffling, which was maintained for at least 30 min. PLD1 and PLD2 are both expressed in mast cells and green fluorescent protein-tagged proteins were used to identify PLD2 localizing to membrane ruffles of antigen-stimulated mast cells together with endogenous ADP ribosylation factor 6 (ARF6). In contrast, green fluorescent protein-PLD1 localized to intracellular vesicles and remained in this location after stimulation with antigen. Membrane ruffling was independent of exocytosis of secretory granules because phorbol 12-myristate 13-acetate increased membrane ruffling in the absence of exocytosis. Antigen or phorbol 12-myristate 13-acetate stimulation increased both PLD1 and PLD2 activity when expressed individually in RBL-2H3 cells. Although basal activity of PLD2-overexpressing cells is very high, membrane ruffling was still dependent on antigen stimulation. In permeabilized cells, antigen-stimulated phosphatidylinositol(4,5)bisphosphate synthesis was dependent on both ARF6 and PA generated from PLD. We conclude that both activation of ARF6 by antigen and a continual PLD2 activity are essential for local phosphatidylinositol(4,5)bisphosphate generation that regulates dynamic actin cytoskeletal rearrangements.     

Coordination between RAB GTPase and phosphoinositide regulation and functions

Membrane trafficking relies on dynamic changes in membrane identities that are determined by the regulation of distinct RAB GTPases and phosphoinositides. RABs and phosphoinositides both act to spatiotemporally recruit effectors of membrane remodelling, including sequential RAB and phosphoinositide activities. New ideas on coordinated regulation of specific RABs and phosphoinositides, achieved by direct physical and functional interactions between their regulatory enzymes, are emerging as a central mechanism to ensure precision and fidelity of membrane trafficking.     

Localization and possible functions of phospholipase D isozymes

The activation of PLD is believed to play an important role in the regulation of cell function and cell fate by extracellular signal molecules. Multiple PLD activities have been characterized in mammalian cells and, more recently, several PLD genes have been cloned. Current evidence indicates that diverse PLD activities are localized in most, if not all, cellular organelles, where they are likely to subserve different functions in signal transduction, membrane vesicle trafficking and cytoskeletal dynamics.     

Localization and possible functions of phospholipase D isozymes.

The activation of PLD is believed to play an important role in the regulation of cell function and cell fate by extracellular signal molecules. Multiple PLD activities have been characterized in mammalian cells and, more recently, several PLD genes have been cloned. Current evidence indicates that diverse PLD activities are localized in most, if not all, cellular organelles, where they are likely to subserve different functions in signal transduction, membrane vesicle trafficking and cytoskeletal dynamics.     

Regulation of phospholipase D1 subcellular cycling through coordination of multiple membrane association motifs

The signaling enzyme phospholipase D1 (PLD1) facilitates membrane vesicle trafficking. Here, we explore how PLD1 subcellular localization is regulated via Phox homology (PX) and pleckstrin homology (PH) domains and a PI4,5P2-binding site critical for its activation. PLD1 localized to perinuclear endosomes and Golgi in COS-7 cells, but on cellular stimulation, translocated to the plasma membrane in an activity-facilitated manner and then returned to the endosomes. The PI4,5P2-interacting site sufficed to mediate outward translocation and association with the plasma membrane. However, in the absence of PX and PH domains, PLD1 was unable to return efficiently to the endosomes. The PX and PH domains appear to facilitate internalization at different steps. The PH domain drives PLD1 entry into lipid rafts, which we show to be a step critical for internalization. In contrast, the PX domain appears to mediate binding to PI5P, a lipid newly recognized to accumulate in endocytosing vesicles. Finally, we show that the PH domain-dependent translocation step, but not the PX domain, is required for PLD1 to function in regulated exocytosis in PC12 cells. We propose that PLD1 localization and function involves regulated and continual cycling through a succession of subcellular sites, mediated by successive combinations of membrane association interactions.     

磷酸肌醇家族与细胞功能

磷酸肌醇具有信号前体的作用,在激动剂作用下可产生第二信使,近年研究表明磷酸肌醇家族成员要 也可作为脂质第二信使将效应器蛋白聚集于特定膜区以调控它们的组装或活性。磷酸肌醇与蛋白的结合主要依赖于ＰＨ、ＦＹＶＥ及ＰＴＢ结构域。磷酸肌醇家族成员参与许多细胞活动诸如离子转运,膜泡运输,信号转导,细胞骨架再组装和核基因调控。     

The role of phosphoinositides in membrane transport.

Phosphoinositides serve as intrinsic membrane signals that regulate intracellular membrane trafficking. Recently, phosphoinositides have been found to direct the localization and activity of effector proteins containing consensus sequence motifs such as FYVE, PH and ENTH domains. In addition, recent results show that regulated synthesis and turnover of phosphoinositides by membrane-associated phosphoinoside kinases and phosphatases spatially restrict the location of effectors critical for cellular transport processes, such as clathrin-mediated endocytosis, autophagy, phagocytosis, macropinocytosis and biosynthetic trafficking.     

EGF regulation of PITP dynamics is blocked by inhibitors of phospholipase C and of the Ras-MAP kinase pathway

Phosphatidylinositol transfer proteins (PITP) function in signal transduction and in membrane traffic. Studies aimed at elucidating the mechanism of action of PITP have yielded a singular theme; the activity of PITP stems from its ability to transfer phosphatidylinositol (PI) from its site of synthesis to sites of cellular activity and to stimulate the local synthesis of phosphorylated forms of PI. The participation of various phosphoinositides in EGF signal transduction and in the trafficking of the EGF receptors is well documented. Using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET) between EGFP-PITP proteins and fluorescently labeled phospholipids, we report that PITPalpha and PITPbeta can dynamically interact with PI or PC at the plasma membrane when stimulated with EGF. Additionally, PITPbeta is localized at the Golgi, and EGF stimulation resulted in enhanced FRET. Inhibitors of the PLC and the Ras/MAP kinase pathway were both able to inhibit the EGF-stimulated interaction of PITPalpha with PI at the plasma membrane. The mobility of PITP proteins was determined by using fluorescence recovery after photobleaching (FRAP), and EGF stimulation reduced the mobility at the plasma membrane. We conclude that the dynamic behavior of PITPalpha and PITPbeta in vivo is a regulated process involving multiple mechanisms.     

Analysis of phosphoinositides in protein trafficking

Phosphoinositides are key regulators of vesicle-mediated protein trafficking. Their roles include recruiting vesicle coat and effector proteins to the site of budding and promoting vesicle fusion. The intracellular levels of phosphoinositides and their localization to intracellular membranes are critical to their functions. An analytical procedure was developed that optimizes the recovery of radiolabeled cellular phosphoinositides. Quantitative analyses of yeast cellular phosphoinositides indicated that this approach is useful for examining the intracellular membrane phosphoinositide compositions related to trafficking phenomena. The approach will also enable investigators to determine whole-plant phosphoinositide compositions that have been difficult to achieve in the past. These analytical advances should be generally applicable to studies of phosphoinositide dynamics related to membrane trafficking in yeast, plant, and animal cells.     

Tied up: Does altering phosphoinositide-mediated membrane trafficking influence neurodegenerative disease phenotypes?

Phosphoinositides are a class of membrane lipids that are found on several intracellular compartments and play diverse roles inside cells, such as vesicle formation, protein trafficking, endocytosis etc. Intracellular distribution and levels of phosphoinositides are regulated by enzymes that generate and breakdown these lipids as well as other proteins that associate with phosphoinositides. These events lead to differing levels of specific phosphoinositides on different intracellular compartments. At these intracellular locations, phosphoinositides and their associated proteins, such as Rab GTPases, dynamin and BAR domain-containing proteins, regulate a variety of membrane trafficking pathways. Neurodegenerative phenotypes in disorders such as Parkinson’s disease (PD) can arise as a consequence of altered or hampered intracellular trafficking. Altered trafficking can cause proteins such as \(\upalpha \)-synuclein to aggregate intracellularly. Several trafficking pathways are regulated by master regulators such as LRRK2, which is known to regulate the activity of phosphoinositide effector proteins. Perturbing either the levels of phosphoinositides or their interactions with different proteins disrupts intracellular trafficking pathways, contributing to phenotypes often observed in disorders such as Alzheimer’s or PDs. Thus, studying phosphoinositide regulation and its role in trafficking can give us a deeper understanding of the contribution of disrupted trafficking to neurodegenerative phenotypes.     

Mechanism of membrane binding of the phospholipase D1 PX domain

Mammalian phospholipases D (PLD), which catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid (PA), have been implicated in various cell signaling and vesicle trafficking processes. Mammalian PLD1 contains two different membrane-targeting domains, pleckstrin homology and Phox homology (PX) domains, but the precise roles of these domains in the membrane binding and activation of PLD1 are still unclear. To elucidate the role of the PX domain in PLD1 activation, we constructed a structural model of the PX domain by homology modeling and measured the membrane binding of this domain and selected mutants by surface plasmon resonance analysis. The PLD1 PX domain was found to have high phosphoinositide specificity, i.e. phosphatidylinositol 3,4,5-trisphosphate (PtdIns-(3,4,5)P(3)) > phosphatidylinositol 3-phosphate > phosphatidylinositol 5-phosphate > other phosphoinositides. The PtdIns(3,4,5)P(3) binding was facilitated by the cationic residues (Lys(119), Lys(121), and Arg(179)) in the putative binding pocket. Consistent with the model structure that suggests the presence of a second lipid-binding pocket, vesicle binding studies indicated that the PLD1 PX domain could also bind with moderate affinity to PA, phosphatidylserine, and other anionic lipids, which were mediated by a cluster of cationic residues in the secondary binding site. Simultaneous occupancy of both binding pockets synergistically increases membrane affinity of the PX domain. Electrostatic potential calculations suggest that a highly positive potential near the secondary binding site may facilitate the initial adsorption of the domain to the anionic membrane, which is followed by the binding of PtdIns(3,4,5)P(3) to its binding pocket. Collectively, our results suggest that the interaction of the PLD1 PX domain with PtdIns(3,4,5)P(3) and/or PA (or phosphatidylserine) may be an important factor in the spatiotemporal regulation and activation of PLD1.     

Pathways for phosphoinositide synthesis.

In eukaryotic cells, phosphatidylinositol can be phosphorylated on the inositol ring by a series of kinases to produce at least seven distinct phosphoinositides. These lipids have been implicated in a variety of cellular processes, including calcium regulation, actin rearrangement, vesicle trafficking, cell survival and mitogenesis. The phosphorylated lipids can act as precursors of second messengers or act directly to recruit specific signaling proteins to the membrane. A number of the kinases responsible for producing these lipids have been purified and their cDNA clones have been isolated. The most well characterized of these enzymes are the phosphoinositide 3-kinases. However, progress has also been made in the characterization of phosphatidylinositol 4-kinases and phosphatidylinositol-4-phosphate 5-kinases. In addition, new pathways involving phosphatidylinositol-5-phosphate 4-kinases, phosphatidylinositol-3-phosphate 5-kinases and phosphatidylinositol-3-phosphate 4-kinases have recently been described. The various enzymes and pathways involved in the synthesis of cellular phosphoinositides will be discussed.     

Interaction of the small G protein RhoA with the C terminus of human phospholipase D1

Mammalian phosphatidylcholine-specific phospholipase D1 (PLD1) is a signal transduction-activated enzyme thought to function in multiple cell biological settings including the regulation of membrane vesicular trafficking. PLD1 is activated by the small G proteins, ADP-ribosylation factor (ARF) and RhoA, and by protein kinase C-alpha (PKC-alpha). This stimulation has been proposed to involve direct interaction and to take place at a distinct site in PLD1 for each activator. In the present study, we employed the yeast two-hybrid system to attempt to identify these sites. Successful interaction of ARF and PKC-alpha with PLD1 was not achieved, but a C-terminal fragment of human PLD1 (denoted "D4") interacted with the active mutant of RhoA, RhoAVal-14. Deletion of the CAAX box from RhoAVal-14 decreased the strength of the interaction, suggesting that lipid modification of RhoA is important for efficient binding to PLD1. The specificity of the interaction was validated by showing that the PLD1 D4 fragment interacts with glutathione S-transferase-RhoA in vitro in a GTP-dependent manner and that it associates with RhoAVal-14 in COS-7 cells, whereas the N-terminal two-thirds of PLD1 does not. Finally, we show that recombinant D4 peptide inhibits RhoA-stimulated PLD1 activation but not ARF- or PKC-alpha-stimulated PLD1 activation. These results conclusively demonstrate that the C-terminal region of PLD1 contains the RhoA-binding site and suggest that the ARF and PKC interactions occur elsewhere in the protein.     

Mammalian phospholipase D: Function,and therapeutics

Despite being discovered over 60 years ago, the precise role of phospholipase D (PLD) is still being elucidated. PLD enzymes catalyze the hydrolysis of the phosphodiester bond of glycerophospholipids producing phosphatidic acid and the free headgroup. PLD family members are found in organisms ranging from viruses, and bacteria to plants, and mammals. They display a range of substrate specificities, are regulated by a diverse range of molecules, and have been implicated in a broad range of cellular processes including receptor signaling, cytoskeletal regulation and membrane trafficking. Recent technological advances including: the development of PLD knockout mice, isoform-specific antibodies, and specific inhibitors are finally permitting a thorough analysis of the in vivo role of mammalian PLDs. These studies are facilitating increased recognition of PLD's role in disease states including cancers and Alzheimer's disease, offering potential as a target for therapeutic intervention.     

A role for rho-kinase in rho-controlled phospholipase D stimulation by the m3 muscarinic acetylcholine receptor.

Stimulation of phospholipase D (PLD) by membrane receptors is now recognized as a major signal transduction pathway involved in diverse cellular functions. Rho proteins control receptor signaling to PLD, and these GTPases have been shown to directly stimulate purified recombinant PLD1 enzymes in vitro. Here we report that stimulation of PLD activity, measured in the presence of phosphatidylinositol 4,5-bisphosphate, by RhoA in membranes of HEK-293 cells expressing the m3 muscarinic acetylcholine receptor (mAChR) is phosphorylation-dependent. Therefore, the possible involvement of the RhoA-stimulated serine/threonine kinase, Rho-kinase, was investigated. Overexpression of Rho-kinase and constitutively active Rho-kinase (Rho-kinase-CAT) but not of kinase-deficient Rho-kinase-CAT markedly increased m3 mAChR-mediated but not protein kinase C-mediated PLD stimulation, similar to overexpression of RhoA. Expression of the Rho-inactivating C3 transferase abrogated the stimulatory effect of wild-type Rho-kinase, but not of Rho-kinase-CAT. Recombinant Rho-kinase-CAT mimicked the phosphorylation-dependent PLD stimulation by RhoA in HEK-293 cell membranes. Finally, the Rho-kinase inhibitor HA-1077 largely inhibited RhoA-induced PLD stimulation in membranes as well as PLD stimulation by the m3 mAChR but not by protein kinase C in intact HEK-293 cells. We conclude that Rho-kinase is involved in Rho-dependent PLD stimulation by the G protein-coupled m3 mAChR in HEK-293 cells. Thus, our findings identify Rho-kinase as a novel player in the receptor-controlled PLD signaling pathway.     

HS1BP3 inhibits autophagy by regulation of PLD1

Macroautophagy/autophagy is a membrane trafficking and intracellular degradation process involving the formation of double-membrane autophagosomes and their ultimate fusion with lysosomes. Much is yet to be learned about the regulation of this process, especially at the level of the membranes and lipids involved. We have recently found that the PX domain protein HS1BP3 (HCLS1 binding protein 3) is a negative regulator of autophagosome formation. HS1BP3 depletion increases the formation of LC3-positive autophagosomes both in human cells and zebrafish. HS1BP3 localizes to ATG16L1- and ATG9-positive autophagosome precursors deriving from recycling endosomes, which appear to fuse with LC3-positive phagophores. The HS1BP3 PX domain interacts with phosphatidic acid (PA) and 3’-phosphorylated phosphoinositides. When HS1BP3 is depleted, the total cellular PA content is upregulated stemming from increased activity of the PA-producing enzyme PLD (phospholipase D) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 negatively regulates autophagy by decreasing the PA content of the ATG16L1-positive autophagosome precursor membranes through inhibition of PLD1 activity and localization.     

Phosphatidylinositol 4-kinases: old enzymes with emerging functions

Phosphoinositides account for only a tiny fraction of cellular phospholipids but are extremely important in the regulation of the recruitment and activity of many signaling proteins in cellular membranes. Phosphatidylinositol (PtdIns) 4-kinases generate PtdIns 4-phosphate, the precursor of important regulatory phosphoinositides but also an emerging regulatory molecule in its own right. The four mammalian PtdIns 4-kinases regulate a diverse array of signaling events, as well as vesicular trafficking and lipid transport, but the mechanisms by which their lipid product PtdIns 4-phosphate controls these processes is only beginning to unfold.