Possible regulation of phospholipase C activity in human platelets by phosphatidylinositol 4',5'-bisphosphate

Phospholipase C from human platelets was found to catalyze the Ca2+-dependent degradation of phosphatidylinositol (PI), phosphatidylinositol 4'-phosphate (DPI), and phosphatidylinositol 4',5'-bisphosphate (TPI) at Ca2+ concentrations from 150 microM to 5 mM. Both DPI and TPI inhibited the hydrolysis of [2-3H]inositol-labeled PI (250 microM) in a concentration-dependent manner. The use of DPI and TPI from beef brain, both of which have fatty acid compositions different from that of soybean PI, permitted an assessment of the inhibitory effect of polyphosphoinositides on the hydrolysis of PI by phospholipase C. Fatty acid analysis of the diacylglycerols formed demonstrated that DPI and TPI, when incubated in mixture with PI, were competitive substrates for PI hydrolysis. Increasing the DPI/PI ratio from 0 to 0.3 caused a shift in the degradation of PI to DPI without greatly affecting the formation of 1,2-diacylglycerol. TPI alone, or in mixture with PI, was a poor substrate for phospholipase C. Increasing the TPI/PI ratio from 0 to 0.21, on the other hand, inhibited both PI degradation (greater than or equal to 95%) and overall formation of 1,2-diacylglycerol (greater than or equal to 82%). Kinetic analysis revealed that TPI acts as a mixed-type inhibitor with a Ki of about 10 microM. The Ka for Ca2+ in PI hydrolysis was profoundly increased from 5 to 180 microM when TPI (36 microM) was included with PI (250 microM). Optimum PI degradation under these conditions was only attained when the calcium concentration approached 4 mM. Analysis of phospholipids from unstimulated human platelets from five different donors revealed DPI/PI and TPI/PI ratios of 0.42 and 0.16, respectively. These findings, combined with the observed inhibition of PI hydrolysis by TPI at a TPI/PI ratio of 0.16, would suggest that in unstimulated platelets phospholipase C activity may be inhibited by greater than or equal to 75%. Changes in 33P-prelabeled phospholipids of intact platelets upon stimulation with thrombin indicated a transient decline in 33P label of both TPI and DPI (15 s) followed by an increase in [33P]phosphatidic acid but no change in [33P]PI. The finding that DPI is selectively degraded by phospholipase C in mixture with PI at DPI/PI ratios determined to be present in unstimulated platelets indicates that DPI may be more important than PI in the formation of 1,2-diacylglycerol which is believed to serve as precursor of arachidonic acid for thromboxane biosynthesis. Furthermore, the results suggest that in human platelets TPI may serve as modulator for the formation of 1,2-diacylglycerol from inositol phospholipids.     

Interaction of cerebral-cortical membranes with exogenously added phosphatidylinositol 4,5-bisphosphate. Effects on measured phospholipase C activity.

Exogenously added phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is rapidly associated with cerebral-cortical membranes. Substrate association with membranes was promoted by Mg2+, but inhibited by bivalent chelators. Once associated with the membrane, the PtdInsP2 was resistant to displacement by EDTA. The apparent phospholipase C activity was dependent on the degree of association of substrate with membranes. After preincubation of membranes with substrate, PtdInsP2 hydrolysis was independent of the incubation volume, indicating that substrate and membrane-associated phospholipase C were not independently diluted. Hydrolysis of the membrane-associated substrate was stimulated by Ca2+, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), guanosine 5'[gamma-thio]triphosphate and carbachol in the presence of p[NH]ppG. Carbachol in the absence of guanine nucleotides, GDP, GTP, ATP and pyrophosphate was ineffective. These results demonstrate that exogenously added PtdInsP2 substrate is rapidly associated with membranes and hydrolysed by a phospholipase C whose activity is regulated by guanine nucleotides and agonist in the presence of guanine nucleotides. Use of exogenously added substrate for studies on the regulation of membrane phospholipase C requires consideration as to possible effects of incubation conditions on the partitioning of substrate into membranes.     

Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-alpha

Cytosolic phospholipase A(2)-alpha (cPLA(2)) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA(2) is dependent on a rise in cytosolic Ca(2+) concentration, membrane association via the Ca(2+)-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA(2) via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), including the role of a "pleckstrin homology (PH)-like" region of cPLA(2) (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate+the Ca(2+) ionophore, A23187, or epidermal growth factor+A23187, expression of the PH domain of phospholipase C-delta1 (which sequesters membrane PIP(2)) attenuated cPLA(2) activity. Stimulated cPLA(2) activity was also attenuated by the expression of cPLA(2) 135-366, or cPLA(2) 2-366, and expression of a PIP(2)-specific 5'-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP(2), with high affinity, only cPLA(2) 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA(2)s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA(2) activity can be modulated by sequestration or depletion of cellular PIP(2), although the interaction of cPLA(2) with membrane PIP(2) appears to be indirect, or of weak affinity.     

The differential regulation of phosphatidylinositol 4-phosphate 5-kinases and phospholipase D1 by ADP-ribosylation factors 1 and 6

Phosphatidylinositol 4-phosphate 5-kinases [PtdIns4P5Ks] synthesise the majority of cellular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and phospholipase D1 (PLD1) synthesises large amounts of phosphatidic acid (PtdOH). The activities of PtdIns4P5Ks and PLDs are thought to be coupled during cell signalling in order to support large simultaneous increases in both PtdIns(4,5)P(2) and PtdOH, since PtdOH activates PtdIns4P5Ks and PLD1 requires PtdIns(4,5)P(2) as a cofactor. However, little is known about the control of such a system. Membrane recruitment of ADP-ribosylation factors (Arfs) activates both PtdIns4P5Ks and PLDs, but it is not known if each enzyme is controlled in series by different Arfs or in parallel by a single form. We show through pull-down and vesicle sedimentation interaction assays that PtdIns4P5K activation may be facilitated by Arf-enhanced membrane association. However PtdIns4P5Ks discriminate poorly between near homogeneously myristoylated Arf1 and Arf6 although examples of all three known active isoforms (mouse alpha>beta, gamma) respond to these G-proteins. Conversely PLD1 genuinely prefers Arf1 and so the two lipid metabolising enzymes are differentially controlled. We propose that isoform selective Arf/PLD interaction and not Arf/PtdIns4P5K will be the critical trigger in the formation of distinct, optimal triples of Arf/PLDs/PtdIns4P5Ks and be the principle regulator of any coupled increases in the signalling lipids PtdIns(4,5)P(2) and PtdOH.     

Beta-arrestin scaffolding of phosphatidylinositol 4-phosphate 5-kinase Ialpha promotes agonist-stimulated sequestration of the beta2-adrenergic receptor

Members of the seven-transmembrane receptor (7TMR) superfamily are sequestered from the plasma membrane following stimulation both to limit cellular responses as well as to initiate novel G protein-independent signaling pathways. The best studied mechanism for 7TMR internalization is via clathrin-coated pits, where clathrin and adaptor protein complex 2 nucleate and polymerize upon encountering the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) to form the outer layer of the clathrin-coated vesicle. Activated receptors are recruited to clathrin-coated pits by beta-arrestins, scaffolding proteins that interact with agonist-occupied 7TMRs as well as adaptor protein complex 2 and clathrin. We report here that following stimulation of the beta2-adrenergic receptor (beta2-AR), a prototypical 7TMR, beta-arrestins bind phosphatidylinositol 4-phosphate 5-kinase (PIP5K) Ialpha, a PIP(2)-producing enzyme. Furthermore, beta-arrestin2 is required to form a complex with PIP5K Ialpha and agonist-occupied beta2-AR, and beta-arrestins synergize with the kinase to produce PIP(2) in response to isoproterenol stimulation. Interestingly, beta-arrestins themselves bind PIP(2), and a beta-arrestin mutant deficient in PIP(2) binding no longer internalizes 7TMRs, fails to interact with PIP5K Ialpha, and is not associated with PIP kinase activity assayed in vitro. However, a chimeric protein in which the core kinase domain of PIP5K Ialpha has been fused to the same beta-arrestin mutant rescues internalization of beta2-ARs. Collectively, these data support a model in which beta-arrestins direct the localization of PIP5K Ialpha and PIP(2) production to agonist-activated 7TMRs, thereby regulating receptor internalization.     

Phosphatidylinositol 4,5-bisphosphate mediates Ca2+-induced platelet alpha-granule secretion: evidence for type II phosphatidylinositol 5-phosphate 4-kinase function

To understand the molecular basis of granule release from platelets, we examined the role of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) in alpha-granule secretion. Streptolysin O-permeabilized platelets synthesized PtdIns(4,5)P(2) when incubated in the presence of ATP. Incubation of streptolysin O-permeabilized platelets with phosphatidylinositol-specific phospholipase C reduced PtdIns(4,5)P(2) levels and resulted in a dose- and time-dependent inhibition of Ca(2+)-induced alpha-granule secretion. Exogenously added PtdIns(4,5)P(2) inhibited alpha-granule secretion, with 80% inhibition at 50 microm PtdIns(4,5)P(2). Nanomolar concentrations of wortmannin, 33.3 microm LY294002, and antibodies directed against PtdIns 3-kinase did not inhibit Ca(2+)-induced alpha-granule secretion, suggesting that PtdIns 3-kinase is not involved in alpha-granule secretion. However, micromolar concentrations of wortmannin inhibited both PtdIns(4,5)P(2) synthesis and alpha-granule secretion by approximately 50%. Antibodies directed against type II phosphatidylinositol-phosphate kinase (phosphatidylinositol 5-phosphate 4-kinase) also inhibited both PtdIns(4,5)P(2) synthesis and Ca(2+)-induced alpha-granule secretion by approximately 50%. These antibodies inhibited alpha-granule secretion only when added prior to ATP exposure and not when added following ATP exposure, prior to Ca(2+)-mediated triggering. The inhibitory effects of micromolar wortmannin and anti-type II phosphatidylinositol-phosphate kinase antibodies were additive. These results show that PtdIns(4,5)P(2) mediates platelet alpha-granule secretion and that PtdIns(4,5)P(2) synthesis required for Ca(2+)-induced alpha-granule secretion involves the type II phosphatidylinositol 5-phosphate 4-kinase-dependent pathway.     

Presence of a phospholipase D (PLD) distinct from PLD1 or PLD2 in human neutrophils: immunobiochemical characterization and initial purification

Utilizing the transphosphatidylation reaction catalyzed by phospholipase D (PLD) in the presence of a primary alcohol and the short-chain phospholipid PC8, we have characterized the enzyme from human neutrophils. A pH optimum of 7.8-8.0 was determined. PIP(2), EDTA/EGTA, and ATP were found to enhance basal PLD activity in vitro. Inhibitory elements were: oleate, Triton X-100, n-octyl-beta-glucopyranoside, divalent cations, GTPgammaS and H(2)O(2). The apparent K(m) for the butanol substrate was 0.1 mM and the V(max) was 6.0 nmol mg(-1) h(-1). Immunochemical analysis by anti-pan PLD antibodies revealed a neutrophil PLD of approximately 90 kDa and other bands recognized minimally by anti-PLD1 or anti-PLD2 antibodies. The 90-kDa protein is tyrosine-phosphorylated upon cell stimulation with GM-CSF and formyl-Met-Leu-Phe. Protein partial purification using column liquid chromatography was performed after cell subfractionation. Based on the enzyme's regulatory and inhibitory factors, and its molecular weight, these data indicate an enzyme isoform that might be different from the mammalian PLD1/2 forms described earlier. The present results lay the foundation for further purification of this granulocyte PLD isoform.     

Phosphatidylinositol-specific phospholipase D: a pathway for generation of a second messenger

We have found a phospholipase D activity in the postnuclear fraction of human neutrophils which is stimulated by incubation of cells with the calcium ionophore A23187. The phospholipase D activity was assessed by both phosphatidate formation and free inositol release from phosphatidylinositol substrate. The phospholipase D activity shows an optimum pH of 7.5 and hydrolyzes specifically phosphatidylinositol. These results suggest that this phosphatidylinositol-specific phospholipase D can play a role in cell activating process.     

Type I phosphatidylinositol 4-phosphate 5-kinase directly interacts with ADP-ribosylation factor 1 and is responsible for phosphatidylinositol 4,5-bisphosphate synthesis in the golgi compartment

Phosphatidylinositol (PtdIns) 4,5-bisphosphate is involved in many aspects of membrane traffic, but the regulation of its synthesis is only partially understood. Golgi membranes contain PI 4-kinase activity and a pool of phosphatidylinositol phosphate (PIP), which is further increased by ADP-ribosylation factor 1 (ARF1). COS7 cells were transfected with alpha and beta forms of PI 4-kinase, and only membranes from COS7 cells transfected with PI 4-kinase beta increased their content of PIP when incubated with ARF1. PtdIns(4, 5)P(2) content in Golgi membranes was nonexistent but could be increased to a small extent upon adding either cytosol or Type I or Type II PIP kinases. However, when ARF1 was present, PtdIns(4,5)P(2) levels increased dramatically when membranes were incubated in the presence of cytosol or Type I, but not Type II, PIP kinase. To examine whether ARF1 could directly activate Type I PIP 5-kinase, we used an in vitro assay consisting of phosphatidycholine-containing liposomes, ARF1, and PIP 5-kinase. ARF1 increased Type I PIP 5-kinase activity in a guanine nucleotide-dependent manner, identifying this enzyme as a direct effector for ARF1.     

A role for phosphatidylinositol 3-kinase in the regulation of beta 1 integrin activity by the CD2 antigen

The rapid and reversible upregulation of the functional activity of integrin receptors on T lymphocytes is a vital step in the adhesive interactions that occur during successful T cell recognition of foreign antigen and transendothelial migration. Although the ligation of several different cell surface receptors, including the antigen- specific CD3/T cell receptor complex, the CD2, CD7, and CD28 antigens, as well as several chemokine receptors, has been shown to rapidly upregulate integrin function, the intracellular signaling events that initiate this increase in adhesion remain poorly defined. In this study, we have used DNA-mediated gene transfer to explore the role of phosphatidylinositol 3-kinase (PI 3-K) in the upregulation of beta 1 integrin functional activity mediated by the CD2 antigen. CD2 was expressed in the myelomonocytic cell line HL60, which expresses beta 1 integrins that mediate adhesion to fibronectin and VCAM-1 in an activation-dependent manner. Antibody stimulation of CD2 expressed on HL60 transfectants resulted within minutes in increased beta 1-mediated adhesion to fibronectin and VCAM-1 at levels comparable to that obtained upon stimulation with the phorbol ester PMA. A role for PI 3-K in CD2-mediated increases in beta 1 integrin function is suggested by: (a) the ability of the PI 3-K inhibitor wortmannin to completely inhibit CD2-induced increases in beta 1 integrin activity; (b) the association of PI 3-K with CD2; and (c) induced PI 3-K activity upon CD2 stimulation. The mode of association of PI 3-K with CD2 is not mediated by tyrosine phosphorylation-dependent binding of PI 3-K via SH2 domains, since: (a) PI 3-K is associated with CD2 in unstimulated cells; (b) CD2 stimulation fails to increase the amount of associated PI 3-K; and (c) the CD2 cytoplasmic domain lacks tyrosine residues. A role for both protein kinase C and cytoskeletal rearrangements in CD2 regulation of integrin activity is also suggested, since a PKC inhibitor partially inhibits CD2-induced increases in beta 1 integrin function, and CD2 stimulation increases F-actin content in a wortmannin- sensitive manner. Analysis of human peripheral T cells indicated that CD2 stimulation also results in PI 3-K-dependent upregulation of beta 1 integrin activity. Thus, these results demonstrate that CD2 can function as an adhesion regulator in the absence of expression of the CD3/T cell receptor complex; and directly implicate PI 3-K as a critical intracellular mediator involved in the regulation of beta 1 integrin functional activity by the CD2 antigen.     

The role of structural intersubunit microheterogeneity in the regulation of the activity in hysteresis of ribulose 1, 5-bisphosphate carboxylase/oxygenase

Many enzymes are composed of subunits with the identical primary structure. It has been believed that the protein structure of these subunits is the same. Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) comprises eight large subunits with the identical amino acid sequence and eight small subunits. Rotation of the side chains of the lysine residues, Lys-21 and Lys-305, in each of the eight large subunits in spinach RuBisCO in two ways produces microheterogeneity among the subunits. These structures are stabilized through hydrogen bonds by water molecules incorporated into the large subunits. This may cause different effects upon catalysis and a hysteretic, time-dependent decrease in activity in spinach RuBisCO. Changing the amino acid residues corresponding to Lys-21 and Lys-305 in non-hysteretic Chromatium vinosum RuBisCO to lysine induces hysteresis and increases the catalytic activity from 8.8 to 15.8 per site per second. This rate is approximately five times higher than that of the higher-plant enzyme.     

Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes by binding to a family of G protein-coupled receptors or as an intracellular second messenger. Mammalian S1P phosphatase (SPP-1), which degrades S1P to terminate its actions, was recently cloned based on homology to a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast. Confocal microscopy surprisingly revealed that epitope-tagged SPP-1 is intracellular and colocalized with the ER marker calnexin. Moreover, SPP-1 activity and protein appeared to be mainly enriched in the intracellular membranes with lower expression in the plasma membrane. Treatment of SPP-1 transfectants with S1P markedly increased ceramide levels, predominantly in the intracellular membranes, diminished survival, and enhanced apoptosis. Remarkably, dihydro-S1P, although a good substrate for SPP-1 in situ, did not cause significant ceramide accumulation or increase apoptosis. Ceramide accumulation induced by S1P was completely blocked by fumonisin B1, an inhibitor of ceramide synthase, but only partially reduced by myriocin, an inhibitor of serine palmitoyltransferase, the first committed step in de novo synthesis of ceramide. Furthermore, S1P, but not dihydro-S1P, stimulated incorporation of [3H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide. Collectively, our results suggest that SPP-1 functions in an unprecedented manner to regulate sphingolipid biosynthesis and is poised to influence cell fate.     

Rac homologues and compartmentalized phosphatidylinositol 4, 5-bisphosphate act in a common pathway to regulate polar pollen tube growth

Pollen tube cells elongate based on actin- dependent targeted secretion at the tip. Rho family small GTPases have been implicated in the regulation of related processes in animal and yeast cells. We have functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes. Expression of dominant negative Rac inhibited pollen tube elongation, whereas expression of constitutive active Rac induced depolarized growth. Pollen tube Rac was found to accumulate at the tip plasma membrane and to physically associate with a phosphatidylinositol monophosphate kinase (PtdIns P-K) activity. Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), the product of PtdIns P-Ks, showed a similar intracellular localization as Rac. Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-delta1, which binds specifically to PtdIns 4, 5-P2, inhibited pollen tube elongation. These results indicate that Rac and PtdIns 4, 5-P2 act in a common pathway to control polar pollen tube growth and provide direct evidence for a function of PtdIns 4, 5-P2 compartmentalization in the regulation of this process.     

Essential role of type I(alpha) phosphatidylinositol 4-phosphate 5-kinase in neurite remodeling

Rapid neurite remodeling is fundamental to nervous system development and plasticity and is regulated by Rho family GTPases that signal f-actin reorganization in response to various receptor ligands. Neuronal N1E-115 cells show dramatic neurite retraction and cell rounding in response to serum factors such as lysophosphatidic acid (LPA), sphingosine-1 phosphate (S1P), and thrombin, due to activation of the RhoA-Rho kinase pathway. Type I phosphatidylinositol 4-phosphate 5-kinases (PIPkinase), which regulate cellular levels of PtdIns(4,5)P(2), have been suggested as targets of the RhoA-Rho kinase pathway able to modulate cytoskeletal dynamics. Here, we show that the introduction of Type Ialpha PIPkinase into N1E-115 cells leads to cell rounding and complete inhibition of neurite outgrowth, perhaps through the dissociation of vinculin and the destabilization of focal adhesions. This occurs independently of RhoA, Rho kinase, and the activation of actomyosin contraction. Strikingly, expression of kinase-dead PIPkinase promotes the outgrowth of neurites, which fail to retract in response to LPA, S1P, thrombin, or active RhoA. Moreover, neurite retraction in response to an endogenous neuronal guidance cue, Semaphorin3A, was also dependent on Type Ialpha PIPkinase. Our results suggest an essential role for a Type I PIPkinase during neurite retraction in response to a number of diverse stimuli.     

Differential regulation of two Arabidopsis type III phosphatidylinositol 4-kinase isoforms. A regulatory role for the pleckstrin homology domain

Here, we compare the regulation and localization of the Arabidopsis type III phosphatidylinositol (PtdIns) 4-kinases, AtPI4Kalpha1 and AtPI4Kbeta1, in Spodoptera frugiperda (Sf9) insect cells. We also explore the role of the pleckstrin homology (PH) domain in regulating AtPI4Kalpha1. Recombinant kinase activity was found to be differentially sensitive to PtdIns-4-phosphate (PtdIns4P), the product of the reaction. The specific activity of AtPI4Kalpha1 was inhibited 70% by 0.5 mm PtdIns4P. The effect of PtdIns4P was not simply due to charge because AtPI4Kalpha1 activity was stimulated approximately 50% by equal concentrations of the other negatively charged lipids, PtdIns3P, phosphatidic acid, and phosphatidyl-serine. Furthermore, inhibition of AtPI4Kalpha1 by PtdIns4P could be alleviated by adding recombinant AtPI4Kalpha1 PH domain, which selectively binds to PtdIns4P (Stevenson et al., 1998). In contrast, the specific activity of AtPI4Kbeta1, which does not have a PH domain, was stimulated 2-fold by PtdIns4P but not other negatively charged lipids. Visualization of green fluorescent protein fusion proteins in insect cells revealed that AtPI4Kalpha1 was associated primarily with membranes in the perinuclear region, whereas AtPI4Kbeta1 was in the cytosol and associated with small vesicles throughout the cytoplasm. Expression of AtPI4Kalpha1 without the PH domain in the insect cells compromised PtdIns 4-kinase activity and caused mislocalization of the kinase. The green fluorescent protein-PH domain alone was associated with intracellular membranes and the plasma membrane. In vitro, the PH domain appeared to be necessary for association of AtPI4Kalpha1 with fine actin filaments. These studies support the idea that the Arabidopsis type III PtdIns 4-kinases are responsible for distinct phosphoinositide pools.     

Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia

Background

Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)₄-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4.

Methodology/Principal Findings

PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity.

Conclusions/Significance

Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.     

A role for calcium in sphingosine 1-phosphate-induced phospholipase D activity in C2C12 myoblasts

Receptor-regulated phospholipase D (PLD) is a key signaling pathway implicated in the control of fundamental biological processes. Here evidence is presented that in addition to protein kinase C (PKC) and Rho GTPases, Ca(2+) response evoked by sphingosine 1-phosphate (S1P) also participates to the enzyme regulation. Ca(2+) was found critical for PKC(alpha)-mediated PLD activation. Moreover, S1P-induced PLD activity resulted diminished by calmodulin inhibitors such as W-7 and CGS9343B implicating its involvement in the process. A plausible candidate for Ca(2+)-dependent PLD regulation by S1P was represented by calcineurin, in view of the observed reduction of the stimulatory effect by cyclosporin A. In contrast, monomeric GTP-binding protein Ral was translocated to membranes by S1P in a Ca(2+)-independent manner, ruling out its possible role in agonist-mediated regulation of PLD.