Lipid Phosphorylation Research Articles

Granulocyte/macrophage colony-stimulating factor affects myo-inositol metabolism in a novel manner. Implications for its priming action on human neutrophils.

Little is known about the signal transduction processes involved in the priming action of granulocyte/macrophage colony-stimulating factor (GM-CSF) on neutrophils. This study has used myo-[3H]inositol-labelled human neutrophils to determine whether preincubation with GM-CSF influences myo-inositol (Ins) metabolism in control cells, or in cells stimulated with the bacterial chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMetLeuPhe). GM-CSF pretreatment did not influence the total cellular 3H radioactivity content, demonstrating that the cytokine had no effect on Ins uptake. However, neutrophils pretreated with GM-CSF showed a dramatic 25-40% fall in the free [3H]Ins content of the cell, which was almost quantitatively recovered in a 2-4-fold increase in radioactivity within PtdIns. The remainder of the 3H radioactivity was found proportionately distributed throughout all other [3H]Ins-containing metabolites. Interestingly, in comparison with controls, the GM-CSF-stimulated increases in [3H]polyphosphoinositide (including 3-phosphorylated lipids) and [3H]Ins polyphosphate contents were consistently higher than that observed with PtdIns. This observation suggests that GM-CSF influences the hormone-sensitive pool of PtdIns, possibly through the activation of a PtdIns synthase which is rate-limiting to subsequent metabolic pathways. This is the first report of an action of GM-CSF on Ins metabolism, and highlights the conversion of Ins to PtdIns as a key regulatory metabolic step.

Association of Phosphatidylinositol 4-Kinase with the Plant Cytoskeleton.

In eukaryotic cells, phosphatidylinositol 4-hydroxy kinase and phosphatidylinositol-4-phosphate 5-hydroxy kinase are responsible for the formation of the two second messenger precursors phosphatidylinositol-4-phosphate (Ptdlns(4)P) and phosphatidylinositol-4,5-bisphosphate (Ptdlns(4,5)P2). In plant cells, these kinases have been considered to be exclusively membrane associated, with the majority of activity residing in the inner leaflet of the plasmalemma. By sequentially extracting carrot protoplasts with the detergent Nonidet P-40 then more rigorously with Triton X-100, we were able to remove the activity of three separate plasma membrane marker enzymes and to demonstrate that a significant proportion of cellular Ptdlns 4-kinase is associated with the cytoskeleton. When only endogenous substrates were present, Nonidet P-40-permeabilized protoplasts and Nonidet P-40-extracted cytoskeletons displayed a pattern of lipid phosphorylation similar to that obtained with isolated plant membranes or permeabilized cells, whereas the Triton X-100-extracted cytoskeletons showed little or no activity. In contrast, when exogenous substrates were added, a major proportion of PtdlnsP formed was due to kinase activity associated with the cytoskeleton as well as nuclei. However, by subtracting the activity of isolated nuclei, it could be demonstrated that a significant proportion of the detergent-resistant Ptdlns kinase activity resides with the cytoskeletal fraction. These findings suggest that the pathways of polyphosphoinositide biosynthesis in plant cells should be reevaluated to take account of the cytoskeleton and that Ptdlns(4)P itself may play a unique role in modulation of plant cytoskeletal integrity and cellular signal transduction.

Polyphoshoinositide synthesis and protein phosphorylation in the plasma membrane from full-grown Bufo arenarum oocytes

1. 1. Polyphosphoinositide content and phosphorylation of lipids and protein were analyzed in oocytes of the Bufo arenarum Hensel. 2. 2. Plasma membrane-enriched fractions obtained from full-grown, prophase-arrested oocytes incorporated [ 32P] into both phospholipids and protein after incubation with [° 32P] in an Mg 2+-containing medium. Phosphatidylinositol 4-phosphate (PIP), phosphatidate (PA) and phosphatidylinositol-4,5-bisphosphate (PIP 2) were the only labelled lipids. The [ 32P] incorporation depended on incubation time, the amount of protein, and the ATP concentration. 3. 3. Autoradiography of polyacrylamide gel electropherograms and scintillation counting showed that the radioactivity was mainly associated with a group of membrane proteins having an M r, of 87,000. 4. 4. This paper provides evidence for the capacity of prophase-arrested oocytes from Bufo arenarum to synthesize polyphosphoinositides and to phosphorylate distinct membrane proteins.

Age-related events in active T subpopulations. Changes in polyphosphoinositide metabolism during mitogenic activation

The active E rosette test allows the characterisation of a lymphocyte subset with high affinity receptors for sheep red blood cells. In this study, we compared active T lymphocytes of healthy subjects over 65 and under 30 years of age after mitogenic stimulation. Active E rosettes increased in both age groups after 48 h of phytohemoagglutinin (PHA) stimulation, mainly in young subjects. In addition, analysing the in vitro phosphorylation of inositol lipids, changes occurred inthe incorporation of radioactivity in both phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bis phosphate (PIP 2) in the early steps of the mitotic response in the elderly subjects as compared to young ones, hinting that modifications of this signal transduction system are related to mitogenic stimulation in this peculiar T subpopulation.

Interaction of pp60c-src, phospholipase C, inositol-lipid, and diacyglycerol kinases with the cytoskeletons of thrombin-stimulated platelets

Phosphatidylinositol (PtdIns)-4- and -3-kinases, PtdIns(4)P-5-kinase, diacylglycerol (DAG) kinase, and PtdIns-phospholipase C were all detected in cytoskeletons of resting human platelets. The total cytoskeletal enzyme activities were greatly increased upon thrombin stimulation of the intact cells. Those reached a maximum after a 60-s stimulation for PtdIns(4)P-5-kinase and phospholipase C, while the other kinases appeared to be slightly delayed. Specific activities were stimulated from about 4-fold (PtdIns-3-kinase) to about 6-fold (PtdIns-4-kinase). Thrombin treatment also promoted a co-extraction of pp60c-src with the cytoskeletons and its disappearance from the Triton X-100 soluble fraction. These results suggest that stimulation of platelets by thrombin causes the association of enzymes responsible for lipid phosphorylation and hydrolysis with the cytoskeletons. This could occur at cytoskeleton anchoring points to the membranes.

Inositol lipid phosphorylation in the cell nucleus

Inositol lipid metabolism has been analyzed in isolated rat liver nuclei and nuclear fractions, in order to determine the subcellular distribution of the sites of lipid phosphorylation and breakdown. Lipid kinases and phosphoesterases appear to be tightly bound nuclear components, and can utilize exogenous substrates administered to membrane-depleted structures. The possible involvement of specific carrier protein in the nuclear metabolism of inositol lipids has also been analysed by studying the uptake and processing of phosphatidylinositol transferred to the isolated nuclei by phosphatidylinositol transfer protein (PI-TP). PI-TP greatly stimulates the incorporation of phosphatidylinositol from microsomal membranes and synthetic vesicles, and the lipid taken up is available for phosphorylation and breakdown by enzymes associated to the nucleus. The results obtained support previous data on the metabolic and structural role of nuclear lipids, and suggest that the cell nucleus is a site of lipid phosphorylation, not necessarily involving enzymes and substrates located on the nuclear membrane. They also indicate that an integrated signalling pathway can exist at the nuclear level utilizing inositol lipid-derived second messengers and PKC to control replication and transcription.

Methyl Mercury Stimulates Protein32P Phospholabeling in Cerebellar Granule Cell Culture

Cultures of cerebellar granule neurons have been utilized to examine morphological and biochemical consequences of methyl mercury (MeHg). Exposure to MeHg for 24 h was found to exert toxic effects at concentrations below 1 microM characterized by neuron degeneration and neuritic varicosities. Dose-response and time course profiles for cell death were established using the 51Cr release assay, which revealed that 1 microM MeHg produced 15% cell death at 24 h, progressing to 50% at 48 h. Labeling of cultures with [32P]orthophosphate following 24-h exposure to 1 microM MeHg disclosed abnormalities in both protein and lipid phosphorylation. After 24-h exposure to 5 microM MeHg, phospholabeling of protein and lipid increased 174 and 128%, respectively, compared with controls. This stimulation of phosphorylation appeared to be neuron specific since cultures enriched in cerebellar glial cells and devoid of granule neurons displayed dose-dependent inhibition of total phosphorylation. Measurement of 32P labeling of ATP using a cyclic AMP-dependent protein kinase assay in conjunction with the firefly luciferase assay for ATP indicated no significant change in either total ATP levels or [32P]ATP specific activity at 1 or 4 h as a function of [MeHg]. Studies measuring 32P-phosphoprotein turnover indicated that MeHg had no effect on intracellular protein phosphatase activity. We conclude that one of the manifestations associated with in vitro cerebellar granule cell neurotoxicity is an abnormality in protein phosphorylation that is independent of [32P]ATP specific activity and protein phosphatase activity.

Inositol lipid phosphorylation in Golgi from liver

Inositol lipid phosphorylation in Golgi from liver

Lipid phosphorylation in isolated rat liver nuclei Synthesis of polyphosphoinositides at subnuclear level

Isolated rat liver nuclei and subnuclear fractions synthesize polyphosphoinositides in vitro in a mode dependent on the presence of nuclear membrane, detergent and exogenous substrates. The nuclear membrane is not essential as a source of lipid kinases, since the addition of erogenous phosphatidylinositol or phosphatidylinositol monophosphate to reaction mixtures lacking membranes restores the synthesis of phosphatidylinositol mono- and bisphosphate, respectively. Inositide phosphorylation is best accomplished by high-salt extracted nuclei and pre-detergent lamina. These data suggest that the nucleus, and especially the nuclear periphery, is a cell compartment in which polyphosphoinositide synthesis occurs; this might be related to the progression of phosphatidylinositol metabolism-dependent signals to the genetic apparatus.

Evidence of an auxin‐mediated phosphoinositide turnover and an inositol (1,4,5)trisphosphate effect on isolated membranes of Daucus carota L.

Microsomal membranes from carrot suspension cells were phosphorylated in vitro with [gamma-32P]ATP. In the presence of submicromolar concentrations of the natural auxin indoleacetic acid (IAA), a rapid, but transient decrease of the [32P] label could be detected in the phospholipid extracts of the membranes. The phytohormone effect was not the result of an inhibition of the lipid phosphorylation reactions, but was caused by a simultaneous release of water-soluble compounds, which, according to their chromatographic properties, were assumed to contain inositol polyphosphates. Although the [32P]-labeled lipids, as well as the inositol polyphosphates, were not identified unequivocally by chemical analysis, these findings point to an auxin-mediated control of a phosphoinositidase C-like reaction similar to the hormone-stimulated phosphoinositide response in animals. Exogenously applied inositol (1,4,5)trisphosphate [(1,4,5)IP3] was found to release 45Ca2+ from preloaded membrane vesicles of carrot cells. Both the detection of the auxin-stimulated phosphoinositide response and the (1,4,5)IP3-mediated Ca2+ release on isolated cell membranes offer new experimental approaches for the identification of the putative auxin receptor and its signal transduction pathway.

CTP-dependent lipid kinases of yeast

Membrane fractions from yeast Saccharomyces cerevisiae catalyzed a transfer of γ-phosphate from [γ- 32P]CTP into membranous lipids. Phosphorylated compounds were identified as phosphatidic acid and dolichyl phosphate (DolP). The membrane fraction also catalyzed phosphorylation of the exogenous dolichol. The activity of the phosphorylating enzymes could be modified by the yeast growing conditions; i.e., the enzyme from yeast grown aerobically favored the synthesis of phosphatidate over dolichyl phosphate in the ratio of 3:1, whereas the membrane fraction from anaerobically grown yeast synthesized PA and DolP in the ratio of 0.5:1. The activity of the phosphorylating enzymes could also be modified by divalent cations and the concentration of detergents. Phosphorylation of lipids does not occur in the presence of [γ- 32P]ATP and is not influenced by the presence of UTP or GTP. This result points to the specific role of CTP as a γ-phosphate donor for the synthesis of phosphatidate and dolichyl phosphates in the yeast System.

Phosphatidylinositol kinase activity of a plasma membrane-associated calcium-activated protein kinase from pea

A purified protein kinase preparation from pea is shown to contain phosphatidylinositol kinase activity. The preparation, produced by membrane solubilisation and ATP-agarose affinity chromatography, contains proteins of 18 kDa (ppl8, a calcium activated autophosphorylating protein-serine kinase), 67 kDa and 48 kDa and exhibits the phosphorylation of an endogenous lipid when supplied with [γ- 32P]ATP. This product is tentatively identified as lysophosphatidylinositol phosphate. When supplied with [γ- 32P]ATP and exogenous phosphatidylinositol, the production of phosphatidylinositol phosphate is observed.

Membrane bilayer balance and platelet shape: morphological and biochemical responses to amphipathic compounds

Activated platelets adopt a characteristic spiculate morphology. A wide variety of anionic an zwitterionic amphipathic compounds were found to effect a similar shape change and to cause the open canalicular system to become less prominent. Several cationic amphipaths reversed thrombin-, PAF-, and amphipath-induced spiculation and restored the discoid shape. Higher concentrations of cationic amphipaths caused the cells to assume spheroid and indented forms, and caused the canalicular system to appear more prominent. Three amphipaths were studied further to address possible mechanisms underlying their morphological effects. Dilauroylphosphatidylcholine was found to induce spiculation without causing the changes in protein phosphorylation and inositide metabolism generally associated with platelet activation. Two other amphipaths, chlorpromazine (which induced sphering) and dilauroylphosphatidylserine (which caused spiculation followed by sphering) caused specific changes in protein and/or lipid phosphorylation, which may be responsible for some, but not all, of the morphological effects of these compounds. To account for these findings, we propose that platelet shape can be influenced by changes in the plasma membrane bilayer balance. Agents that bind to the membrane outer monolayer are accomodated by spiculation; those that bind to the inner monolayer are accomodated by sphering.

Stimulation of polyphosphoinositide turnover upon activation of protein kinases in human erythrocytes

Activation of protein kinase C in erythrocytes by 4-β-phorbol 12-myristate 13-acetate (PMA) resulted in a parallel stimulation (time course and dose response) of the phosphorylation of both membrane proteins (heterodimers of 107 kDa and 97 kDa, protein 4.1 and 4.9, respectively) and of phosphatidylinositol 4-phosphate (PIP) and, to a lesser extent, of phosphatidylinositol 4,5-bisphosphate (PIP 2). Evidence that the effect on lipid was mediated by protein kinase C activation and not by a direct action of PMA was provided by (1) the lack of effect of a phorbol ester that did not activate protein kinase C or of PMA addition on isolated membranes from control erythrocytes, (2) the reversal of the effect in the presence of protein kinase C inhibitors (α-cobrotoxin, H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine) or trifluoperazine). PMA treatment did not change the specific activity of ATP or the content of PIP 2, but increased the content of PIP and decreased that of PI, indicating that the phosphorylation or dephosphorylation reactions linking PI and PIP were the target for the action of PMA. PMA treatment had no effect on the Ca 2+-dependent PIP/PIP 2 phospholipase C activity measured in isolated membranes. Mezerein, another protein kinase activator, had similar effects on both protein and lipid phosphorylation, when added with α-cobrotoxin. Activation of protein kinase A by cAMP also produced increases in phosphorylation, although quantitatively different from those induced by protein kinase C, in proteins and PIP. Simultaneous addition of PMA and cAMP at maximal doses resulted in only a partially additive effect on PIP labelling. These results show that inositol lipid turnover can be modulated by a protein kinase C and protein kinase A-dependent process involving the phosphorylation of a common protein. This could be PI kinase or PIP phosphatase or another protein regulating the activity of these enzymes.

Phosphatidylinositol and phosphatidylinositolphosphate kinases in plant plasma membranes

Both phosphatidylinositol (PI) and phosphatidylinositolphosphate (PIP) kinase activities were present in plasma membrane fractions isolated from shoots and roots of dark-grown wheat ( Triticum aestivum L.) by aqueous polymer two-phase partition. The enzymes phosphorylated their respective endogenous substrates as well as exogenously added substrates (PI and phosphatidylinositol 4-monophosphate, PI-4P), to form PIP and phosphatidylinositol diphosphate (PIP 2). The reactions were dependent on ATP. Phosphorylation of added PI reached maximum activity around 0.75 mM ATP, while the ATP requirement for maximal activity was higher both for phosphorylation of added PI-4P (1.25 mM ATP) and of endogenous lipids (1.5 mM ATP). Optimal Mg 2+ concentration varied between 5 mM (endogenous PI phosphorylation) and 15 mM (phosphorylation of exogenous PI). The Mg 2+ requirement could be substituted only partially by Mn 2+ and not at all by Ca 2+ Phosphorylation of endogenous lipid substrates was inhibited by Triton X-100 concentrations above 0.015%, while phosphorylation of exogenous substrates was stimulated several-fold by up to 0.5% Triton X-100. Triton X-100 also influenced the optimal pH range of the reactions. While phosphorylation of endogenous PI and PIP was optimal at pH 6.5–7 without Triton X-100 in the assay medium, addition of 0.010% Triton X-100 extended the optimal pH range up to pH 8.6. Phosphorylation of exogenous lipids were optimal at pH 7.8–8.2. At optimal conditions and with endogenous substrates, PIP formation was 125–225 and 40–90 pmol/mg protein per min in shoot and root plasma membranes, respectively, and PIP 2 formation 10–25 and 4–8 pmol/mg protein per min, respectively. With exogenous substrates, the corresponding rates increased 8–20-times. These results demonstrate the close resemblance between the characteristics of PI and PIP kinase activities in plant membranes with corresponding activities in animal plasma membranes. It is, however, not yet known if polyphosphoinositide metabolism in plant cells resembles the corresponding metabolism in animal cells also in function, that is, in acting as a signal-transducing system for internal Ca 2+ mobilization.

Endogenous hyperphosphorylation in plasma membrane from an ascites hepatocarcinoma cell line.

Plasma-membrane-bound kinases of AS-30D ascites from transplantable rat hepatocarcinoma were shown to extensively catalyze the phosphorylation of plasma membrane proteins and membrane lipids, using [gamma-32P]ATP or [gamma-32P]GTP as a phosphate donor. In contrast, plasma membranes from normal adult rat liver or fast-growing regenerating liver (24 h after partial hepatectomy) produce significantly less activity for protein phosphorylation and little phosphorylation of the lipids. However, neonatal (24 h old) rat liver plasma membrane preparations show levels of phosphorylation of proteins and lipids intermediate between those in the tumor cell line and normal adult plasma membrane preparations. Phosphatidic acid was identified as one of the 32P-labelled lipids in the tumor plasma membrane chloroform-methanol (2:1, v/v) extract. Phosphorylation of protein was not affected by cAMP or cGMP. However, calcium ion (in the presence or absence of calmodulin) significantly modifies the 32P labelling of a series of proteins in normal tissue but has little effect with the neoplastic preparations. Some plasma membrane proteins were capable of nucleotide binding, instead or in addition to being phosphorylated. Finally, the presence of membrane-bound phosphoprotein phosphatase(s) was also demonstrated in all the preparations examined by means of chase experiments with nonlabelled ATP or GTP, and (or) by the use of the phosphoprotein phosphatase inhibitor, orthovanadate.

Intracellular regulators of neuronal sprouting: II. Phosphorylation reactions in isolated growth cones

Mechanisms potentially involved in the regulation of neurite growth were investigated. Since both the phosphatidylinositol (PI) pathway and protein kinase C have been implicated in transmembrane signal transduction, protein and lipid phosphorylation reactions were examined in intact growth cone particles (GCPs) isolated from fetal rat brain. Three major substrates of Ca2+-dependent phosphorylation were observed: proteins of 40 and 46 kDa and an acidic 80 kDa species separated in 2D PAGE (pp40, pp46, and pp80ac). The pp40 and pp80ac substrates had similar rates of 32P incorporation, whereas that of pp46 was more rapid. The importance of protein kinase C in growth cone function is indicated by the enhancement of phosphorylation of the 3 major substrates by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). An examination of the Ca2+-dependent 32P incorporation into pp40 and pp46 revealed serine to be the only amino acid phosphorylated under these conditions. A rapidly metabolized pool of phosphoinositides was observed in GCPs. This suggests the presence of the Pl pathway's enzymes in this fraction. Inositol trisphosphate (IP3) was found to stimulate the phosphorylation of pp40 and pp80ac, indicating a possible link between the activation of the PI pathway and protein phosphorylation. Our findings demonstrate the prominence of the PI pathway and of Ca2+-dependent protein phosphorylation in the growth cone and may suggest the involvement of these mechanisms in growth-factor signal transduction.

Ca2+ regulation of 1-(3-sn-phosphatidyl)-1D-myo-inositol 4-phosphate formation and hydrolysis on sarcoplasmic-reticular Ca2+-transport ATPase. A new principle of phospholipid turnover regulation.

Lipid phosphorylation was shown to occur on the isolated sarcoplasmic-reticulum (SR) Ca2+-transport ATPase. More than 95% of the radioactivity incorporated on incubation of the SR ATPase with [gamma-32P]ATPMg can be extracted with acidic organic solvents and was identified as 1-(3-sn-phosphatidyl)-1D-myo-inositol 4-phosphate (PtdIns4P) [Varsányi, Toelle, Heilmeyer, Dawson & Irvine (1983) EMBO J. 2, 1543-1548]. This lipid phosphorylation is only observed at nanomolar concentrations of free Ca2+; in the presence of micromolar free Ca2+ PtdIns4P disintegrates rapidly. Also, upon blockade of the kinase reaction PtdIns4P decomposes, indicating a PtdIns/PtdIns4P turnover. The PtdIns4P concentration is dependent on the free Ca2+ concentration, being half-maximal at 35 nM-Ca2+. PtdIns4P hydrolysis is catalysed by a PtdIns4P phosphomonoesterase; accordingly no diacylglycerol is formed, which would be a product of a phosphodiesteratic cleavage. Fluoride inhibits this phosphomonoesterase. Ca2+ does not influence directly either the PtdIns kinase or the PtdIns4P phosphomonoesterase. PtdIns4P forms a tight complex with the transport ATPase, from which it can be removed only by chromatography on heparin-agarose in the presence of Triton X-100. It is concluded that Ca2+ regulates the PtdIns/PtdIns4P turnover by availability of substrate, depending on the Ca2+-transport-ATPase conformation, which traps or exposes the respective lipid head groups.

Phosphorylation of phosphatidylinositol associated with the nicotinic acetylcholine receptor of Torpedo Californica

When isolated, detergent solubilized and affinity chromatographically purified nicotinic acetylcholine receptor of Torpedo californica electric organ is incubated with [gamma-32P]ATP/Mg2+, phosphatidylinositol 4-phosphate (PIP) is formed from receptor associated phosphatidylinositol (PI). This receptor associated endogenous kinase activity is enhanced by orthovanadate and, remarkably, also by acetylcholine. Exogenously added PI-kinase only increases the phosphorylation rate if vanadate is present. PIP as the main phosphorylation product (up to 95%) remains bound to the beta-, gamma- and delta-subunits of the receptor and to the receptor associated v-protein. The alpha-subunits do not carry 32p phosphate; no phosphatidylinositol 4,5-bisphosphate formation has been observed. Concomitant to lipid phosphorylation tyrosine and serine residues are phosphorylated (5% of total incorporated 32P phosphate).

Kinase A does not activate phosphatidylinositol phosphorylation in rat lymphocyte membranes.

The interaction of the dissociated catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (kinase A) and phosphatidylinositol metabolism has been studied in rat spleen lymphocyte membranes. As reported previously (Sarkadi et al., FEBS Lett. 152: 195-198, 1983) addition of kinase A increased by about twofold the phosphorylation of phosphatidylinositol in lymphocyte membranes at low ATP concentrations. However, we have found that this increase is an artifact of the assay conditions, and that the increase is a consequence of an inhibition of membrane adenosine triphosphatase activity by the protein kinase A. When lipid phosphorylation was measured under initial rate conditions, at high ATP concentrations, the increase was abolished. No effect of kinase A was observed on initial rates of the synthesis or hydrolysis of phosphatidylinositol phosphate. No phosphatidylinositol bisphosphate was produced in the membranes under any of the assay conditions used.