Phosphatidic Acid (PA)-preferring Phospholipase A1 Regulates Mitochondrial Dynamics

Recent studies have suggested that phosphatidic acid (PA), a cone-shaped phospholipid that can generate negative curvature of lipid membranes, participates in mitochondrial fusion. However, precise mechanisms underling the production and consumption of PA on the mitochondrial surface are not fully understood. Phosphatidic acid-preferring phospholipase A₁ (PA-PLA₁)/DDHD1 is the first identified intracellular phospholipase A₁ and preferentially hydrolyzes PA in vitro. Its cellular and physiological functions have not been elucidated. In this study, we show that PA-PLA₁ regulates mitochondrial dynamics. PA-PLA₁, when ectopically expressed in HeLa cells, induced mitochondrial fragmentation, whereas its depletion caused mitochondrial elongation. The effects of PA-PLA₁ on mitochondrial morphology appear to counteract those of MitoPLD, a mitochondrion-localized phospholipase D that produces PA from cardiolipin. Consistent with high levels of expression of PA-PLA₁ in testis, PA-PLA₁ knock-out mice have a defect in sperm formation. In PA-PLA₁-deficient sperm, the mitochondrial structure is disorganized, and an abnormal gap structure exists between the middle and principal pieces. A flagellum is bent at that position, leading to a loss of motility. Our results suggest a possible mechanism of PA regulation of the mitochondrial membrane and demonstrate an in vivo function of PA-PLA₁ in the organization of mitochondria during spermiogenesis.     

Regulation of Phospholipase D Activity and Phosphatidic Acid Production after Purinergic (P2Y6) Receptor Stimulation

Phosphatidic acid (PA) is a lipid second messenger located at the intersection of several lipid metabolism and cell signaling events including membrane trafficking, survival, and proliferation. Generation of signaling PA has long been primarily attributed to the activation of phospholipase D (PLD). PLD catalyzes the hydrolysis of phosphatidylcholine into PA. A variety of both receptor-tyrosine kinase and G-protein-coupled receptor stimulations have been shown to lead to PLD activation and PA generation. This study focuses on profiling the PA pool upon P2Y₆ receptor signaling manipulation to determine the major PA producing enzymes. Here we show that PLD, although highly active, is not responsible for the majority of stable PA being produced upon UDP stimulation of the P2Y₆ receptor and that PA levels are tightly regulated. By following PA flux in the cell we show that PLD is involved in an initial increase in PA upon receptor stimulation; however, when PLD is blocked, the cell compensates by increasing PA production from other sources. We further delineate the P2Y₆ signaling pathway showing that phospholipase Cβ3 (PLCβ3), PLCδ1, DGKζ and PLD are all downstream of receptor activation. We also show that DGKζ is a novel negative regulator of PLD activity in this system that occurs through an inhibitory mechanism with PKCα. These results further define the downstream events resulting in PA production in the P2Y₆ receptor signaling pathway.     

Partially Purified RhoA-Stimulated Phospholipase D Activity Specifically Binds to Phosphatidylinositol 4,5-Bisphosphate

Abstract: Phosphatidylinositol 4,5-bisphosphate (PIP₂) is absolutely required for the ADP-ribosylation factor-stimulated phospholipase D (PLD) activity. In the present study, partially purified rat brain PLD was found to be activated by another PLD activator, RhoA, when PIP₂, but not other acidic phospholipids, was included in vesicles comprising phosphatidylethanolamine (PE) and the PLD substrate phosphatidylcholine (PC) (PE/PC vesicles), demonstrating the absolute requirement of PIP₂ for the RhoA-stimulated PLD activation, too. It is interesting that the RhoA-dependent PLD activity in the partially purified preparation was drastically decreased after the preparation was incubated with and separated from PE/PC vesicles containing PIP₂. The PLD activity was extracted by higher concentrations of NaCl from the vesicles containing PIP₂ that were incubated with and then separated from the partially purified PLD preparation. These results demonstrate that RhoA-dependent PLD binds to PE/PC vesicles with PIP₂. The degree of binding of the RhoA-dependent PLD activity to the vesicles was totally dependent on the amount of PIP₂ in the vesicles and correlated well with the extent of the enzyme activation. Furthermore, it was found that a recombinant peptide of the pleckstrin homology domain of β-adrenergic receptor kinase fused to glutathione S-transferase, which specifically binds to PIP₂, inhibited the PIP₂-stimulated, RhoA-dependent PLD activity in a concentration-dependent manner. From these results, it is concluded that in vitro rat brain PLD translocates to the vesicles containing PIP₂, owing to its specific interaction with PIP₂, to access its substrate PC, thereby catalyzing the hydrolysis of PC. PLD appears to localize exclusively on plasma membranes of cells and tissues. An aminoglycoside, neomycin, that has high affinity for PIP₂ effectively extracted the RhoA-dependent PLD activity from rat brain membranes. This indicates that PIP₂ serves as an anchor to localize PLD on plasma membranes in vivo.     

Phospholipase D and the Maintenance of Phosphatidic Acid Levels for Regulation of Mammalian Target of Rapamycin (mTOR)

Phosphatidic acid (PA) is a critical metabolite at the heart of membrane phospholipid biosynthesis. However, PA also serves as a critical lipid second messenger that regulates several proteins implicated in the control of cell cycle progression and cell growth. Three major metabolic pathways generate PA: phospholipase D (PLD), diacylglycerol kinase (DGK), and lysophosphatidic acid acyltransferase (LPAAT). The LPAAT pathway is integral to de novo membrane phospholipid biosynthesis, whereas the PLD and DGK pathways are activated in response to growth factors and stress. The PLD pathway is also responsive to nutrients. A key target for the lipid second messenger function of PA is mTOR, the mammalian/mechanistic target of rapamycin, which integrates both nutrient and growth factor signals to control cell growth and proliferation. Although PLD has been widely implicated in the generation of PA needed for mTOR activation, it is becoming clear that PA generated via the LPAAT and DGK pathways is also involved in the regulation of mTOR. In this minireview, we highlight the coordinated maintenance of intracellular PA levels that regulate mTOR signals stimulated by growth factors and nutrients, including amino acids, lipids, glucose, and Gln. Emerging evidence indicates compensatory increases in one source of PA when another source is compromised, highlighting the importance of being able to adapt to stressful conditions that interfere with PA production. The regulation of PA levels has important implications for cancer cells that depend on PA and mTOR activity for survival.     

Phosphatidic Acid-Dependent Phosphorylation of a 29-kDa Protein by Protein Kinase Cα in Bovine Brain Cytosol

Abstract: Activation of phospholipase D (PLD) is involved in receptor-mediated signal transduction responses. Signaling from PLD to a downstream molecule(s) appears to be mediated by the PLD product phosphatidic acid (PA). A target molecule(s) of PA, however, has not yet been identified. The present study sought to define such a target molecule(s) of PA. In bovine brain cytosol, proteins with apparent molecular weights of 29,000 (p29) and 32,000 (p32) were prominently phosphorylated in the presence of PA, but not in its absence, indicating that there is a PA-regulated protein kinase (PARK) in bovine brain that phosphorylates p29 and p32. One of these substrates, p29, was purified to near homogeneity. Its partial amino acid sequence was determined and found to be identical to that of a known brain-specific 25-kDa protein (p25). The purified p29 was also readily recognized by and immunoprecipitated with an anti-p25 antibody. These results suggest that p29 is very similar to or identical with p25. Using the purified p29 as a substrate, PARK was purified to near homogeneity. The purified PARK had an apparent molecular weight of 80,000, was strongly recognized by an anti-protein kinase C (PKC)α antibody, and was activated by phosphatidylserine (PS) as well as PA. The PA- and PS-stimulated PARK activity was extremely augmented by the presence of 1 µM free Ca²⁺. In the presence of 1 mM EGTA, phorbol 12-myristate 13-acetate activated PARK synergistically with PA or PS. Similar results were obtained with the purified recombinant PKCα. From these results, it is suggested that the PARK activity purified might be attributed to PKCα. In p25-depleted bovine brain cytosol, which was prepared by treatment of bovine brain cytosol with the anti-p25 antibody, PA-dependent phosphorylation of p29, but not p32, was almost completely eliminated. When PKCα in bovine brain cytosol was depleted by its precipitation with the anti-PKCα antibody, neither p29 nor p32 in this PKCα-depleted cytosol was phosphorylated in the presence of PA. These results indicate that in bovine brain cytosol PA activates PKCα, which, in turn, phosphorylates p29, which may be identical with p25.     

Lipin 2 Binds Phosphatidic Acid by the Electrostatic Hydrogen Bond Switch Mechanism Independent of Phosphorylation

Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for the penultimate step of triglyceride synthesis and dephosphorylation of phosphatidic acid (PA) to generate diacylglycerol. The lipin family of PA phosphatases is composed of lipins 1–3, which are members of the conserved haloacid dehalogenase superfamily. Although genetic alteration of LPIN2 in humans is known to cause Majeed syndrome, little is known about the biochemical regulation of its PAP activity. Here, in an attempt to gain a better general understanding of the biochemical nature of lipin 2, we have performed kinetic and phosphorylation analyses. We provide evidence that lipin 2, like lipin 1, binds PA via the electrostatic hydrogen bond switch mechanism but has a lower rate of catalysis. Like lipin 1, lipin 2 is highly phosphorylated, and we identified 15 phosphosites. However, unlike lipin 1, the phosphorylation of lipin 2 is not induced by insulin signaling nor is it sensitive to inhibition of the mammalian target of rapamycin. Importantly, phosphorylation of lipin 2 does not negatively regulate either membrane binding or PAP activity. This suggests that lipin 2 functions as a constitutively active PA phosphatase in stark contrast to the high degree of phosphorylation-mediated regulation of lipin 1. This knowledge of lipin 2 regulation is important for a deeper understanding of how the lipin family functions with respect to lipid synthesis and, more generally, as an example of how the membrane environment around PA can influence its effector proteins.     

The Molecular Basis of Leukocyte Adhesion Involving Phosphatidic Acid and Phospholipase D

Defining how leukocytes adhere to solid surfaces, such as capillary beds, and the subsequent migration through the extracellular matrix, is a central biological issue. We show here that phospholipase D (PLD) and its enzymatic reaction product, phosphatidic acid (PA), regulate cell adhesion of immune cells (macrophages and neutrophils) to collagen and have defined the underlying molecular mechanism in a spatio-temporal manner that coincides with PLD activity timing. A rapid (t_½ = 4 min) and transient activation of the PLD1 isoform occurs upon adhesion, and a slower (t_½ = 7.5 min) but prolonged (>30 min) activation occurs for PLD2. Importantly, PA directly binds to actin-related protein 3 (Arp3) at EC₅₀ = 22 nm, whereas control phosphatidylcholine did not bind. PA-activated Arp3 hastens actin nucleation with a kinetics of t_½ = 3 min at 300 nm (compared with controls of no PA, t_½ = 5 min). Thus, PLD and PA are intrinsic components of cell adhesion, which reinforce each other in a positive feedback loop and react from cues from their respective solid substrates. In nascent adhesion, PLD1 is key, whereas a sustained adhesion in mature or established focal points is dependent upon PLD2, PA, and Arp3. A prolonged adhesion could effectively counteract the reversible intrinsic nature of this cellular process and constitute a key player in chronic inflammation.     

Pharmacological and Genetic Targeting of the PI4KA Enzyme Reveals Its Important Role in Maintaining Plasma Membrane Phosphatidylinositol 4-Phosphate and Phosphatidylinositol 4,5-Bisphosphate Levels

Phosphatidylinositol 4-kinase type IIIα (PI4KA) is a host factor essential for hepatitis C virus replication and hence is a target for drug development. PI4KA has also been linked to endoplasmic reticulum exit sites and generation of plasma membrane phosphoinositides. Here, we developed highly specific and potent inhibitors of PI4KA and conditional knock-out mice to study the importance of this enzyme in vitro and in vivo. Our studies showed that PI4KA is essential for the maintenance of plasma membrane phosphatidylinositol 4,5-bisphosphate pools but only during strong stimulation of receptors coupled to phospholipase C activation. Pharmacological blockade of PI4KA in adult animals leads to sudden death closely correlating with the drug''s ability to induce phosphatidylinositol 4,5-bisphosphate depletion after agonist stimulation. Genetic inactivation of PI4KA also leads to death; however, the cause in this case is due to severe intestinal necrosis. These studies highlight the risks of targeting PI4KA as an anti-hepatitis C virus strategy and also point to important distinctions between genetic and pharmacological studies when selecting host factors as putative therapeutic targets.     

Phosphatidylinositol Phosphodiesterase (Phospholipase C) Activity in the Pineal Gland: Characterization and Photoneural Regulation

Phosphatidylinositol phosphodiesterase (PL-C) appears to be a key element in the adrenergic regulation of pineal cyclic AMP levels. In the present study, the rat pineal enzyme was characterized using exogenous [3H]phosphatidylinositol (0.5 mM) as substrate. Half the enzyme activity was found in the cytosolic fraction, but the highest specific concentration was associated with the membrane fraction. Two pH optima (5.5 and 7.5) of enzyme activity were observed for the membrane fraction but only one in the cytosol fraction (pH 5.5). Enzyme activity in both fractions was Ca2+ dependent. In the case of the membrane protein in pH 7.5, the enzyme activity was sensitive to changes in Ca2+ in the 10-100 nM range. Addition of an equimolar concentration of phosphatidylinositol 4-phosphate nearly completely inhibited the hydrolysis of [3H]phosphatidylinositol; other phospholipids (1.0 mM) were less potent. This may reflect our present finding that [3H]phosphatidylinositol 4-phosphate is a better substrate than [3H]phosphatidylinositol for the enzyme. Stimulus deprivation (2 weeks of constant light or superior cervical ganglionectomy) reduced the cytosolic activity by 30% and had no effect on the membrane-associated enzyme.     

Phosphatidic Acid-dependent Recruitment and Function of the Rac Activator DOCK1 during Dorsal Ruffle Formation

Activation of receptor tyrosine kinases leads to the formation of two different types of plasma membrane structures: peripheral ruffles and dorsal ruffles. Although the formation of both ruffle types requires activation of the small GTPase Rac, the difference in kinetics suggests that a distinct regulatory mechanism operates for their ruffle formation. DOCK1 and DOCK5 are atypical Rac activators and are both expressed in mouse embryonic fibroblasts (MEFs). We found that although PDGF-induced Rac activation and peripheral ruffle formation were coordinately regulated by DOCK1 and DOCK5 in MEFs, DOCK1 deficiency alone impaired dorsal ruffle formation in MEFs. Unlike DOCK5, DOCK1 bound to phosphatidic acid (PA) through the C-terminal polybasic amino acid cluster and was localized to dorsal ruffles. When this interaction was blocked, PDGF-induced dorsal ruffle formation was severely impaired. In addition, we show that phospholipase D, an enzyme that catalyzes PA synthesis, is required for PDGF-induced dorsal, but not peripheral, ruffle formation. These results indicate that the phospholipase D-PA axis selectively controls dorsal ruffle formation by regulating DOCK1 localization.     

Phosphorylation of Lipin 1 and Charge on the Phosphatidic Acid Head Group Control Its Phosphatidic Acid Phosphatase Activity and Membrane Association

The lipin gene family encodes a class of Mg²⁺-dependent phosphatidic acid phosphatases involved in the de novo synthesis of phospholipids and triglycerides. Unlike other enzymes in the Kennedy pathway, lipins are not integral membrane proteins, and they need to translocate from the cytosol to intracellular membranes to participate in glycerolipid synthesis. The movement of lipin 1 within the cell is closely associated with its phosphorylation status. Although cellular analyses have demonstrated that highly phosphorylated lipin 1 is enriched in the cytosol and dephosphorylated lipin 1 is found on membranes, the effects of phosphorylation on lipin 1 activity and binding to membranes has not been recapitulated in vitro. Herein we describe a new biochemical assay for lipin 1 using mixtures of phosphatidic acid (PA) and phosphatidylethanolamine that reflects its physiological activity and membrane interaction. This depends on our observation that lipin 1 binding to PA in membranes is highly responsive to the electrostatic charge of PA. The studies presented here demonstrate that phosphorylation regulates the ability of the polybasic domain of lipin 1 to recognize di-anionic PA and identify mTOR as a crucial upstream signaling component regulating lipin 1 phosphorylation. These results demonstrate how phosphorylation of lipin 1 together with pH and membrane phospholipid composition play important roles in the membrane association of lipin 1 and thus the regulation of its enzymatic activity.     

Phosphatidic acid as the biosynthetic precursor of the endocannabinoid 2-arachidonoylglycerol in intact mouse neuroblastoma cells stimulated with ionomycin

In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2-arachidonoylglycerol (2-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2-AG. [3H]2-AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg2+/Ca2(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]2-AG formation. These data indicate that DAG precursors for 2-AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]2-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.     

Amino-terminal Cysteine Residues Differentially Influence RGS4 Protein Plasma Membrane Targeting, Intracellular Trafficking, and Function

Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.     

Lack of Phospholipase D Activity in Chromaffin Cells: Bradykinin-Stimulated Phosphatidic Acid Formation Involves Phospholipase C in Chromaffin Cells but Phospholipase D in PC12 Cells

The role of lipid-bound second messengers in the regulation of neurotransmitter secretion is an important but poorly understood subject. Both bovine adrenal chromaffin cells and rat phoeochromocytoma (PC12) cells, two widely studied models of neuronal function, respond to bradykinin by generating phosphatidic acid (PA). This putative second messenger may be produced by two receptor-linked pathways: sequential action of phospholipase C (PLC) and diacylglycerol kinase (DAG kinase), or directly by phospholipase D (PLD). Here we show that bradykinin stimulation of chromaffin cells prelabelled (24 h) with 32Pi leads to production of [32P]PA which is not affected by 50 mM butanol. However, bradykinin stimulation of PC12 cells leads to [32P]PA formation, all of which is converted to phosphatidylbutanol in the presence of butanol. When chromaffin cells prelabelled with [3H]choline were stimulated with bradykinin there was no enhancement of formation of water soluble products of phosphatidylcholine hydrolysis. When chromaffin cells were permeabilised with pneumolysin and incubated in the presence of [gamma-32P]ATP, the formation of [32P]PA was still stimulated by bradykinin. These results show that, although both neuronal models synthesize PA in response to bradykinin, they do so by quite different routes: PLC/DAG kinase for chromaffin cells and PLD for PC12 cells. The observation that neither bradykinin nor tetradecanoyl phorbol acetate stimulate PLD in chromaffin cells suggests that these cells lack PLD activity. The conservation of PA formation, albeit by different routes, may indicate an essential role of PA in the regulation of cellular events by bradykinin.     

The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts

Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus.     

Phospholipase D mediates nutrient input to mammalian target of rapamycin complex 1 (mTORC1)

The mammalian target of rapamycin (mTOR) is a critical sensor of nutritional sufficiency. Although much is known about the regulation of mTOR in response to growth factors, much less is known about the regulation of mTOR in response to nutrients. Amino acids have no impact on the signals that regulate Rheb, a GTPase required for the activation of mTOR complex 1 (mTORC1). Phospholipase D (PLD) generates a metabolite, phosphatidic acid, that facilitates association between mTOR and the mTORC1 co-factor Raptor. We report here that elevated PLD activity in human cancer cells is dependent on both amino acids and glucose and that amino acid- and glucose-induced increases in mTORC1 activity are dependent on PLD. Amino acid- and glucose-induced PLD and mTORC1 activity were also dependent on the GTPases RalA and ARF6 and the type III phosphatidylinositol-3-kinase hVps34. Thus, a key stimulatory event for mTORC1 activation in response to nutrients is the generation of phosphatidic acid by PLD.     

Similar Rates of Phosphatidylinositol Hydrolysis Following Activation of Wild-Type and Truncated Rat Neurokinin-1 Receptors

Abstract: The substance P (neurokinin-1) receptor belongs to the family of seven putative transmembrane domain receptors that are coupled via G proteins to phospholipase C activation. Homologous desensitization of substance P-stimulated responses has been described in various systems. The rat neurokinin-1 receptor and a truncated mutant lacking the carboxyl-terminal region were expressed in Chinese hamster ovary cells to examine the mechanisms of substance P-induced desensitization. Wild-type and truncated receptor-bearing cells were indistinguishable in agonist binding affinity and EC₅₀ of substance P-induced accumulation of ³H-inositol phosphates. Substance P-induced responses continued for 30–45 min in cells expressing wild-type and truncated receptors as well as in rat LRM-55 and human U373 cells, which express endogenous neurokinin-1 receptors. In transfected cells expressing the wild-type receptor, CP-96,345 added 15 min after substance P blocked further responses, demonstrating the continuing presence of responsive receptors. The rates of accumulation of ³H-inositol phosphates were four times greater in the initial 15 s of stimulation than for the next 20 min for both wild-type and truncated receptor types. This decrease in rate of substance P-stimulated phosphatidylinositol hydrolysis is therefore not dependent on the carboxyl-terminal region of the rat neurokinin-1 receptor, which contains 26 serine and threonine residues. These results are discussed in relation to current ideas regarding neurokinin-1 receptor desensitization.     

Phospholipase C epsilon scaffolds to muscle-specific A kinase anchoring protein (mAKAPbeta) and integrates multiple hypertrophic stimuli in cardiac myocytes

To define a role for phospholipase Cε (PLCε) signaling in cardiac myocyte hypertrophic growth, PLCε protein was depleted from neonatal rat ventricular myocytes (NRVMs) using siRNA. NRVMs with PLCε depletion were stimulated with endothelin (ET-1), norepinephrine, insulin-like growth factor-1 (IGF-1), or isoproterenol and assessed for development of hypertrophy. PLCε depletion dramatically reduced hypertrophic growth and gene expression induced by all agonists tested. PLCε catalytic activity was required for hypertrophy development, yet PLCε depletion did not reduce global agonist-stimulated inositol phosphate production, suggesting a requirement for localized PLC activity. PLCε was found to be scaffolded to a muscle-specific A kinase anchoring protein (mAKAPβ) in heart and NRVMs, and mAKAPβ localizes to the nuclear envelope in NRVMs. PLCε-mAKAP interaction domains were defined and overexpressed to disrupt endogenous mAKAPβ-PLCε complexes in NRVMs, resulting in significantly reduced ET-1-dependent NRVM hypertrophy. We propose that PLCε integrates multiple upstream signaling pathways to generate local signals at the nucleus that regulate hypertrophy.     

The Mechanism of Muscarinic Receptor-Stimulated Phosphatidylinositol Resynthesis in 1321N1 Astrocytoma Cells and Its Inhibition by Li+

Abstract: The coupling of muscarinic receptor-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C to resynthesis of phosphatidylinositol (PtdIns) and the ability of Li⁺ to inhibit this after cellular inositol depletion were studied in 1321N1 astrocytoma cells cultured in medium ± inositol (40 µM). In inositol-replete cells, 1 mM carbachol/10 mM LiCl evoked an initial (0–30 min) ～≥20-fold activation of phospholipase C, whereas prolonged (>60 min) stimulation turned over Ptdlns equal to the cellular total mass, involving ～80% of the cellular Ptdlns pool without reducing PtdIns concentrations significantly. PtdIns resynthesis was achieved by a similar, initial agonist activation of PtdIns synthase. The dose dependency for carbachol stimulation of PtdIns synthase and phospholipase C was similar (EC₅₀～ 20 µM) as was the relative intrinsic activity of muscarinic receptor partial agonists. This demonstrates the tight coupling of phosphoinositide hydrolysis to resynthesis and suggests this is achieved by a direct mechanism. In inositol-replete or depleted cells basal concentrations of inositol and CMP-phosphatidate were respectively ～20 mM or ≤100–500 µM and ～0.1 or ～≥1–10 pmol/mg of protein. Comparison of the effects of agonist ± Li⁺ on the concentrations of these cosubstrates for PtdIns synthase suggest that accelerated activity of this enzyme is differentially driven by stimulated increases in the amounts of CMP-phosphatidate or inositol in inositol-replete or depleted cells, respectively. Thus, the preferential capacity of Li⁺ to impair stimulated phosphoinositide turnover in systems expressing low cellular inositol can be attributed to its ability to attenuate the stimulated rise in inositol concentrations on which such systems selectively depend to trigger accelerated PtdIns resynthesis.