Plasma membrane lipid metabolism of petunia petals during senescence

The specific activities of 6 enzymes, which are involved in the synthesis and catabolism of membrane lipids, were monitored in plasma membranes isolated from petunia petals during senescence. These included phosphatidylinositol (PI) kinase (EC 2.7.1.67), phosphatidylinositol monophosphate (PIP) kinase (EC 2.7.1.68). diacylglycerol (DAG) kinase (EC 2.7.1.107), phospholipase A (EC 3.1.1.4) and PIP- and PIP₂-phospholipase C˙(EC 3.1.4.3). Using endogenous substrate, the [³²P]PA and [³²P]PIP₂ formation increased to 140 and 200%, respectively, of the day 1 value by 4 days after harvest. There was no significant change in [³²P]PIP formation during the same time period. On the fifth day the petals wilted and the [³²P]PA and [³²P]PIP formation declined significantly. In contrast, the [³²P]PIP₂ formation remained high in the day 5 petals. When the lipid kinase activities were assayed in the membranes in the presence of exogenous substrate the specific activity of all of the enzymes increased. and the changes in [³²P]PA production over the 5-day period were similar to those observed with endogenous substrate. When exogenous PI and PIP were added, however, there was no longer an increase in [³²P]PIP₂ formation by plasma membranes of day 4 petals and [³²P]PIP formation significantly decreased. The relative decrease in PIP and PIP₂ formation by day 4 membranes when exogenous substrate was added may have resulted from differences in the lipase activities in the day 1 and day 4 membranes. The plasma membrane A-type phospholipase activity increased throughout the 5 day period, and phospholipase C activity increased two-fold between day 1 and day 4. Such changes in the metabolism of the plasma membrane lipids during flower senescence would affect the ability of the petals to use inositol phospholipid-based signal transduction pathways.     

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP₂) with specific activities of 5 and 10 μmol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6–7 (PIP) and pH 6–6.5 (PIP₂). The enzyme was dependent on micromolar concentrations of Ca²⁺ for activity, and millimolar Mg²⁺ further increased the activity. Other divalent cations (4 mM Ca²⁺, Mn²⁺ and Co²⁺) inhibited (PIP₂ as substrate) or enhanced (PIP as substrate) phospholipase C activity.     

Regulation of PI3K by PKC and MARCKS: Single-Molecule Analysis of a Reconstituted Signaling Pathway

In chemotaxing ameboid cells, a complex leading-edge signaling circuit forms on the cytoplasmic leaflet of the plasma membrane and directs both actin and membrane remodeling to propel the leading edge up an attractant gradient. This leading-edge circuit includes a putative amplification module in which Ca²⁺-protein kinase C (Ca²⁺-PKC) is hypothesized to phosphorylate myristoylated alanine-rich C kinase substrate (MARCKS) and release phosphatidylinositol-4,5-bisphosphate (PIP₂), thereby stimulating production of the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP₃) by the lipid kinase phosphoinositide-3-kinase (PI3K). We investigated this hypothesized Ca²⁺-PKC-MARCKS-PIP₂-PI3K-PIP₃ amplification module and tested its key predictions using single-molecule fluorescence to measure the surface densities and activities of its protein components. Our findings demonstrate that together Ca²⁺-PKC and the PIP₂-binding peptide of MARCKS modulate the level of free PIP₂, which serves as both a docking target and substrate lipid for PI3K. In the off state of the amplification module, the MARCKS peptide sequesters PIP₂ and thereby inhibits PI3K binding to the membrane. In the on state, Ca²⁺-PKC phosphorylation of the MARCKS peptide reverses the PIP₂ sequestration, thereby releasing multiple PIP₂ molecules that recruit multiple active PI3K molecules to the membrane surface. These findings 1) show that the Ca²⁺-PKC-MARCKS-PIP₂-PI3K-PIP₃ system functions as an activation module in vitro, 2) reveal the molecular mechanism of activation, 3) are consistent with available in vivo data, and 4) yield additional predictions that are testable in live cells. More broadly, the Ca²⁺-PKC-stimulated release of free PIP₂ may well regulate the membrane association of other PIP₂-binding proteins, and the findings illustrate the power of single-molecule analysis to elucidate key dynamic and mechanistic features of multiprotein signaling pathways on membrane surfaces.     

Phosphatidylinositol 4,5-Bisphosphate Phospholipase C and Phosphomonoesterase in Dunaliella salina Membranes

In comparison with other cell organelles, the Dunaliella salina plasma membrane was found to be highly enriched in phospholipase C activity toward exogenous [³H]phosphatidylinositol 4,5-bisphosphate (PIP₂). Based on release of [³H]inositol phosphates, the plasma membrane exhibited a PIP₂-phospholipase C activity nearly tenfold higher than the nonplasmalemmal, nonchloroplast `bottom phase' (BP) membrane fraction and 47 times higher than the chloroplast membrane fraction. The majority of phospholipase activity was clearly of a phospholipase C nature since over 80% of [³H]inositol phosphates released were recovered as [³H]inositol trisphosphate (IP₃). These results suggest a plausible mechanism for the rapid breakdown of PIP₂ and phosphatidylinositol 4-phosphate (PIP) following hypoosmotic shock. Quantitative analysis of major [³H]inositol phospholipids during these assays revealed that some of the [³H]-PIP₂ was converted to [³H]phosphatidylinositol 4-monophosphate (PIP) and to [³H]phosphatidyl-inositol (PI) in the BP fraction of membrane remaining after removal of plasmalemma and chloroplasts. This latter fraction is enriched more than fivefold in PIP₂/PIP phosphomonoesterase activity when compared to the plasmalemma or chloroplast membrane fractions. We have also examined some of the in vitro characteristics of the plasma membrane phospholipase C activity and have found it to be calcium sensitive, reaching maximal activity at 10 micromolar free [Ca²⁺]. We also report here that 100 micromolar GTPγS stimulates phosphospholipase C activity over a range of free [Ca²⁺]. Together, these results provide evidence that the plasma membrane PIP₂-phospholipase C of D. salina may be subject to Ca²⁺ and G-protein regulation.     

Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.) : Orientation of Active Site and Activation by Ca and Mg

Polyphosphoinositide-specific phospholipase C activity was present in plasma membranes isolated from different tissues of several higher plants. Phospholipase C activities against added phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP₂) were further characterized in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticum aestivum L. cv Drabant) seedlings. In right-side-out (70-80% apoplastic side out) plasma membrane vesicles, the activities were increased 3 to 5 times upon addition of 0.01 to 0.025% (w/v) sodium deoxycholate, whereas in fractions enriched in inside-out (70-80% cytoplasmic side out) vesicles, the activities were only slightly increased by detergent. Furthermore, the activities of inside-out vesicles in the absence of detergent were very close to those of right-side-out vesicles in the presence of optimal detergent concentration. This verifies the general assumption that polyphosphoinositide phospholipase C activity is located at the cytoplasmic surface of the plasma membrane. PIP and PIP₂ phospholipase C was dependent on Ca²⁺ with maximum activity at 10 to 100 μm free Ca²⁺ and half-maximal activation at 0.1 to 1 μm free Ca²⁺. In the presence of 10 μm Ca²⁺, 1 to 2 mm MgCl₂ or MgSO₄ further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mm Ba²⁺, Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, and Zn²⁺) inhibited the enzyme activity. The stimulatory effect by Mg²⁺ was observed also when 35 mm NaCl was included. Thus, the PIP and PIP₂ phospholipase C exhibited maximum in vitro activity at physiologically relevant ion concentrations. The plant plasma membrane also possessed a phospholipase C activity against phosphatidylinositol that was 40 times lower than that observed with PIP or PIP₂ as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca²⁺, with maximum activity at 1 mm CaCl₂, and could not be further stimulated by Mg²⁺.     

Microwave induced stimulation of32Pi incorporation into phosphoinositides of rat brain synaptosomes

Summary Exposure of synaptosomes to microwave radiation at a power density of 10 mW/sq cm or more produced stimulation of the³²Pi-incorporation into phosphoinositides. The extent of³²Pi incorporation was found to be much more pronounced in phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP₂) as compared to phosphatidylinositol (PI) and phosphatidic acid (PA). Other lipids were also found to incorporate³²Pi but no significant changes in their labeling were seen after exposure to microwave radiation. Inclusion of 10 mM lithium in the medium reduced the basal labeling of PIP₂, PIP and PI and increased PA labeling. Li⁺ also inhibited the microwave stimulated PIP₂, PIP and PI labeling but had no effect on PA labeling. Calcium ionophore, A₂₃₁₈₇, inhibited the basal and microwave stimulated³²Pi labeling of PIP and PIP₂, stimulated basal labeling of PA and PI and had no effect on microwave stimulated PA and PI labeling. Calcium chelator, EGTA, on the other hand, had no effect on basal labeling of PA and PI, stimulated basal PIP and PIP₂ labeling but did not alter microwave stimulated labeling of these lipids. Exposure of synaptosomes to microwave radiation did not alter the chemical concentration of phosphoinositides indicating that the turnover of these lipids was altered. These results suggest that low frequency microwave radiation alter the metabolism of inositol phospholipids by enhancing their turnover and thus may affect the transmembrane signalling in the nerve endings.     

Effects of Ca2+, Mg2+, and depolarizing agents,on the32Pi-labeling and degradation of phosphatidylinositols in rat brain synaptosomes

In isolated synaptosomes from rat brain, 100 M antimycin A and 10 M oxamic acid inhibit the³²Pi-labeling of phosphatidylinositol-4,5-bisphosphate (PIP₂) and phosphatidylinositol-4-phosphate (PIP) by 90% and 95–99% respectively. 10 mM sodium fluoride inhibits the labeling by 50–60% and 10 mM A23187 inhibits the labeling by 63–70%. Phospholipase A₂ inhibits the labeling of PIP₂ and PIP by 93–94% and stimulates their degradation by 84–92%. Depolarization of synaptosomes with 75 mM K⁺ or 100 M veratrine decreases the labeling of PIP₂ and PIP by 66–74%. The decreased labeling results in large part from the Ca²⁺-dependent degradation of³²P-labeled PIP₂ and PIP as shown by pulse-chase experiments in which PIP₂ and PIP were prelabeled with³²Pi. Depolarization of synaptosomes results in the stimulation of⁴⁵Ca²⁺ uptake with the concomitant hydrolysis of PIP and PIP₂. Addition of 1 mM Ca²⁺ accounts for 25% of the enhanced degradation whereas depolarization with 75 mM K⁺ accounts for 75% of the enhanced degradation of PIP₂ and PIP. Depolarization with 100 mM veratrine results in a 223% increase in inositol trisphosphate as evidenced by stimulation of⁴⁵Ca²⁺ uptake. EGTA (10mM) and Mg²⁺ (5–10 mM) inhibit the degradation of PIP and PIP₂ and counteract the action of 1 mM Ca²⁺. Our data demonstrate that⁴⁵Ca²⁺, Mg²⁺, and membrane depolarization play an important role in the turnover of membrane phosphatidylinositols.Abbreviations ATP adenosine triphosphate - Pi inorganic orthophosphate - PIP phosphatidylinositol-4-phosphate - PIP₂ phosphatidylinositol-4,5,-bisphosphate - IP₃ inositol-1,4,5-trisphosphate     

Ca2+ Influx through Store-operated Calcium Channels Replenishes the Functional Phosphatidylinositol 4,5-Bisphosphate Pool Used by Cysteinyl Leukotriene Type I Receptors

Oscillations in cytoplasmic Ca²⁺ concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca²⁺ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), the source of the Ca²⁺-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca²⁺ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li⁺, an inhibitor of inositol monophosphatases that prevents PIP₂ resynthesis. In Li⁺-treated cells, cytoplasmic Ca²⁺ signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca²⁺ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca²⁺ oscillations, and these could not be rescued by inositol or PI4P. In Li⁺-treated cells, recovery of the cytoplasmic Ca²⁺ oscillations in the presence of inositol or PI4P was suppressed when Ca²⁺ influx through store-operated Ca²⁺ channels was inhibited. After rundown of the Ca²⁺ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca²⁺ release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP₂ pools. Our findings show that store-operated Ca²⁺ entry is needed to sustain cytoplasmic Ca²⁺ signaling following leukotriene receptor activation both by refilling the Ca²⁺ stores and by helping to replenish the PIP₂ pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.     

Changes in phosphoinositide metabolism with days in culture affect signal transduction pathways in galdieria sulphuraria

The metabolism of phosphatidylinositol-4,5-bisphosphate (PIP₂) changed during the culture period of the thermoacidophilic red alga Galdieria sulphuraria. Seven days after inoculation, the amount of PIP₂ in the cells was 910 ± 100 pmol g⁻¹ fresh weight; by 12 d, PIP₂ levels increased to 1200 ± 150 pmol g⁻¹ fresh weight. In vitro assays indicated that phosphatidylinositol monophosphate (PIP) kinase specific activity increased from 75 to 230 pmol min⁻¹ mg⁻¹ protein between d 7 and 12. When G. sulphuraria cells were osmostimulated, transient increases of up to 4-fold could be observed in inositol-1,4,5-trisphosphate (IP₃) levels within 90 s, regardless of the age of the cells. In d-12 cells, the increase in IP₃ was preceded by a transient increase of up to 5-fold in specific PIP kinase activity, whereas no such increase was detected after osmostimulation of d-7 cells. The increase in PIP kinase activity before IP₃ signaling in d-12 cells indicates that there is an additional pathway for regulation of phosphoinositide metabolism after stimulation other than an initial activation of phospholipase C. Also, the rapid activation of PIP₂ biosynthesis in cells with already-high PIP₂ levels suggests that the PIP₂ present was not available for signal transduction. By comparing the response of the cells at d 7 and 12, we have identified two potentially distinct pools of PIP₂.     

Sustained phospholipase C stimulation of H9c2 cardiomyoblasts by vasopressin induces an increase in CDP-diacylglycerol synthase 1 (CDS1) through protein kinase C and cFos

Chronic stimulation (24 h) with vasopressin leads to hypertrophy in H9c2 cardiomyoblasts and this is accompanied by continuous activation of phospholipase C. Consequently, vasopressin stimulation leads to a depletion of phosphatidylinositol levels. The substrate for phospholipase C is phosphatidylinositol (4, 5) bisphosphate (PIP₂) and resynthesis of phosphatidylinositol and its subsequent phosphorylation maintains the supply of PIP₂. The resynthesis of PI requires the conversion of phosphatidic acid to CDP-diacylglycerol catalysed by CDP-diacylglycerol synthase (CDS) enzymes. To examine whether the resynthesis of PI is regulated by vasopressin stimulation, we focussed on the CDS enzymes. Three CDS enzymes are present in mammalian cells: CDS1 and CDS2 are integral membrane proteins localised at the endoplasmic reticulum and TAMM41 is a peripheral protein localised in the mitochondria. Vasopressin selectively stimulates an increase CDS1 mRNA that is dependent on protein kinase C, and can be inhibited by the AP-1 inhibitor, T-5224. Vasopressin also stimulates an increase in cFos protein which is inhibited by a protein kinase C inhibitor. We conclude that vasopressin stimulates CDS1 mRNA through phospholipase C, protein kinase C and cFos and provides a potential mechanism for maintenance of phosphatidylinositol levels during long-term phospholipase C signalling.     

Regulation of high molecular weight bovine brain neutral protease by phospholipids in vitro

The activity of the heat stable, glycosylated high molecular weight bovine brain neutral protease (HMW protease) is differentially regulated by phospholipids. While phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) had only marginal stimulatory effect (40–75%) on the activity of HMW protease, lysophoshatidylcholine (lysoPC) and lysophosphatidic acid (lysoPA) activated the enzyme by more than two-fold. Both lysoPC and lysoPA exhibited concentration-dependent saturation kinetics for the activation of HMW protease. Surprisingly, phosphoinositides (phosphatidylinositol, PI; phosphatidylinositol 4-phosphate, PIP; and phosphatidylinositol 4,5-bisphosphate, PIP₂) modulated the activity of protease differently: activation of the enzyme was higher with PIP (90%) as compared to PI (21%), whereas PIP₂ inhibited the enzyme (16%). The inhibition of the protease by PIP₂ was concentration-dependent. During receptor-coupled cell activation, phospholipase A₂ (PLA₂) converts PC and PA to lysoPC and lysoPA, respectively; PI is converted to PIP₂ by successive enzymatic phosphorylation by PI 4-kinase and PIP 5-kinase; and phospholipase C (PLC) degrades PIP₂ to diacylglycerol and inositol 1,4,5-trisphosphate. Therefore, the data suggest that HMW protease may be coupled to cell signal transduction where PLA₂, PI 4-kinase, PIP 5-kinase and PLC are involved.     

The polyphosphoinositides,phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate,are present in nuclei isolated from carrot protoplast

Summary Nuclei were isolated from carrot protoplasts and the distribution of [³H]inositol-labeled phospholipids was analyzed by thinlayer chromatography. Phosphatidylinositol (PI), lysophos-phatidylinositol (LPI), phosphatidylinositol monophosphate (PIP), lysophosphatidylinositol monophosphate (LPIP), and phosphatidylinositol bisphosphate (PIP₂) were 55.7%, 12.3%, 5.0%, 11.5%, and 3.6% of the respective [³H]inositol-labeled lipids recovered from the nuclear fraction. While both the plasma membrane and nuclear fraction contained polyphosphoinositides, the distribution of the phosphoinositides and the amount of inositol-labeled lipid were distinct. For example, the nuclear fraction had a higher percentage of LPI and PIP₂ and less PI and LPIP than the plasma membrane fraction. The amount of [³H]inositol-labeled lipid recovered from the nuclear fraction per mg protein was an order of magnitude lower than that recovered from either the plasma membrane of lower phase fraction isolated by aqueous two-phase partitioning, or from whole cells and protoplasts. In addition, when the ratio of the [³H]inositol-labeled lipid was compared to total [¹⁴C]myristate-labeled lipid recovered there was three to ten fold less [³H] relative to [¹⁴C] in the nuclear fraction.These data indicate that while the polyphosphoinositides are a relatively high percentage of the inositol lipid in the nuclear fraction, the inositol lipid was only a small portion of the total lipid in the nuclei. Despite this low concentration of inositol lipid, when [ ³²P]-ATP was added to the isolated nuclei,³²P-labeled PIP and PIP₂ were synthesized. Thus, the carrot nuclei contained PI and PIP kinase as well as the polyphosphoinositides.Abbreviations PI phosphatidylinositol - LPI lysophosphatidylinositol - PIP phosphatidylinositol monophosphate - LPIP lysophosphatidylinositol monophosphate - PIP₂ phosphatidylinositol bisphosphate - DAG diacylglycerol - IP₃ inositol 1,4,5-trisphosphate     

Phospholipid requirement of Ca2+-stimulated,Mg2+-dependent ATP hydrolysis in rat brain synaptic membranes

The phospholipid requirement for Ca²⁺-stimulated, Mg²⁺-dependent ATP hydrolysis (Ca²⁺/Mg²⁺-ATPase) and Mg²⁺-stimulated ATP hydrolysis (Mg²⁺-ATPase) in rat brain synaptosomal membranes was studied employing partial delipidation of the membranes with phospholipase A₂ (Hog pancreas), phospholipase C (Bacillus cereus) and phospholipase D (cabbage). Treatment with phospholipase A₂ caused an increase in the activities of both Ca²⁺/Mg²⁺-ATPase and Mg²⁺-ATPase whereas with phospholipase C treatment both the enzyme activities were inhibited. Phospholipase D treatment had no effect on Ca²⁺/Mg²⁺-ATPase but Mg²⁺-ATPase activity was inhibited. Inhibition of Mg²⁺-ATPase activity after phospholipase C treatment was relieved with the addition of phosphatidylinositol-4,5-bisphosphate (PIP₂) and to a lesser extent with phosphatidylinositol-4-phosphate (PIP) and phosphatidylcholine (PC). Phosphatidylserine (PS), phosphatidic acid (PA), PIP and PIP₂ brought about the reactivation of Ca²⁺/Mg²⁺-ATPase. Phosphatidylinositol (PI) and PA inhibited Mg²⁺-ATPase activity.K _ms for Ca²⁺ (0.47 M) and Mg²⁺ (60 M) of the enzyme were found to be unaffected after treatment with the phospholipases.     

Prolonged ischemia differently affects phospholipase C acting against phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in brain subsynaptosomal fraction

The effect of 10 min ischemia on the activity of phospholipase C acting against [3H]inositol-phosphatidylinositol (PI) and [3H]inositol-phosphatidylinositol 4,5-bisphosphate (PIP2) in the brain subsynaptosomal fractions was investigated. In the presence of endogenous CaCl2, specific activity of phospholipase C acting on phosphatidylinositol was as follows: synaptic cytosol (SC) greater than synaptic vesicles (SV) greater than synaptic plasma membrane SPM). Brain ischemia activated phospholipase C acting on PI by about 60% and 40% in SV and SPM, respectively. The enzyme of synaptic cytosol was not affected by ischemic insult. Phospholipase C acting against PIP2 in the presence of endogenous calcium expressed the specific activity in the following order: SV greater than SPM greater than SC. After 10 min of brain ischemia, activity of phospholipase C acting on PIP2 was significantly suppressed in all subsynaptosomal fractions by about 50-60%. These results indicate that prolonged ischemia produced activation exclusively of phospholipase C acting against phosphatidylinositol.     

Profiling of phosphoinositide molecular species in human and mouse platelets identifies new species increasing following stimulation

Phosphoinositides are bioactive lipids essential in the regulation of cell signaling as well as cytoskeleton and membrane dynamics. Their metabolism is highly active in blood platelets where they play a critical role during activation, at least through two well identified pathways involving phospholipase C and phosphoinositide 3-kinases (PI3K). Here, using a sensitive high-performance liquid chromatography-mass spectrometry method recently developed, we monitored for the first time the profiling of phosphatidylinositol (PI), PIP, PIP₂ and PIP₃ molecular species (fatty-acyl profiles) in human and mouse platelets during the course of stimulation by thrombin and collagen-related peptide. Furthermore, using class IA PI3K p110α or p110β deficient mouse platelets and a pharmacological inhibitor, we show the crucial role of p110β and the more subtle role of p110α in the production of PIP₃ molecular species following stimulation. This comprehensive platelet phosphoinositides profiling provides important resources for future studies and reveals new information on phosphoinositides biology, similarities and differences in mouse and human platelets and unexpected dramatic increase in low-abundance molecular species of PIP₂ during stimulation, opening new perspectives in phosphoinositide signaling in platelets.     

Phosphatidylinositol 4-Phosphate 5-Kinase α Facilitates Toll-like Receptor 4-mediated Microglial Inflammation through Regulation of the Toll/Interleukin-1 Receptor Domain-containing Adaptor Protein (TIRAP) Location

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP₂), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP₂ is also known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Toll-like receptor (TLR) 4 signaling pathway. Microglia are the primary immune competent cells in brain tissue, and TLR4 is important for microglial activation. However, a functional role for PIP5K and PIP₂ in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5Kα, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5Kα knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1β, and nitric oxide, by lipopolysaccharide. PIP5Kα knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-κB p65 nuclear translocation, and IκB-α degradation. Complementation of the PIP5Kα knockdown cells with wild type but not kinase-dead PIP5Kα effectively restored the LPS-mediated inflammatory response. We found that PIP5Kα and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5Kα rendered TIRAP soluble. Furthermore, in LPS-stimulated control cells, plasma membrane PIP₂ increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP₂. In contrast, PIP5Kα knockdown that reduced PIP₂ levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5Kα promotes TLR4-associated microglial inflammation by mediating PIP₂-dependent recruitment of TIRAP to the plasma membrane.     

Phospholipase C in rabbit thymocytes: subcellular distribution and influences of calcium and GTPγS on the substrate dependence of cytosolic and plasma membrane-associated phospholipase C

The subcellular distribution of phospholipase C (PLC) activity in rabbit thymocytes was examined by measuring the enzyme's activity in different subcellular fractions. PLC activity was determined using exogenously added [³H]PIP₂ as substrate. Approx. 80% of the activity of the cell homogenate was found in the cytosolic fraction. A minor portion of PLC activity was attached to the particulate fraction. This membrane-associated PLC activity was found to be predominantly bound to the plasma membrane. Both PIP₂-cleaving PLCs (the PLC associated with the plasma membrane and the PLC in the cytosol) exhibited maximum activity at pH 5. GTPγS stimulated the cytosolic and the membrane-bound PLC. As revealed by computer analysis of the substrate dependence of both basal and GTPγS-stimulated PLC activity, GTPγS enhanced the V_max of the enzymes. Calcium, at a concentration of 1 mM, decreased PLC activity, as compared to a calcium concentration of 100 nM. The characteristics increase in V_max induced by GTPγS was observed at a concentration of 1 mM calcium and was similar to that at 100 nM. These data suggest that the stimulatory effect of GTPγS is not due to an increased affinity of PLCs to calcium.     

Polyphosphoinositide phospholipase C and evidence for inositol-phosphate-hydrolysing activities in the plasma-membrane fraction from light-grown wheat (Triticum aestivum L.) leaves

The phospholipase C (PLC; EC 3.1.4.3) activity in isolated plasma membranes of light-grown wheat (Triticum aestivum L. cv. Prelude) leaves was investigated. The activity against the polyphosphoinositides was strongly dependent on Ca²⁺ and was affected by the anionic detergent deoxycholate (DOC). In the presence of 20 M Ca²⁺ the PLC activity preferred phosphatidylinositol 4,5-bisphosphate (PIP₂) over phosphatidylinositol 4-monophosphate (PIP) as a substrate. Instead, with 1 mM Ca²⁺ the enzyme clearly favoured PIP. In addition, the PIP₂-PLC activity was increased by Mg²⁺ and in the presence of GTP, guanosine 5-(-thio)-triphosphate as well as ATP, CTP, guanosine 5-diphosphate and guanosine 5-(-thio)-diphosphate. Further analysis showed that a molybdate-sensitive phosphatase activity catalysing the dephosphorylation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) is also associated with the plasma-membrane vesicles. Dephosphorylation of Ins(1,4,5)P₃ was reduced in the presence of GTP or by inclusion of the unspecific phosphatase inhibitor molybdate. The results indicate the presence of a PIP₂-PLC activity and the presence of a molybdate-sensitive phosphatase activity in wheat plasma-membrane vesicles.Abbreviations DOC deoxycholate - IDPase inosine 5-diphosphatase - InsP_s inositol phosphates, the numbering at the end indicates the number of phosphate residues and when their positions on the inositol ring are known they are indicated in parentheses, i.e. - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PIP phosphatidylinositol 4-monophosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PLC phospholipase C This work was financially supported by grant from the Deutsche Forschungsgemeinschaft (DFG). M. C. Arz gratefully acknowledges the support of a Graduiertenstipendium des Landes Nordrhein-Westfalen (Germany). We wish to thank S. Laden and G.E. Grambow for assistance.     

Ca2+ regulation of phosphatidylinositol turnover in the plasma membrane of tobacco suspension culture cells.

The biochemical properties of the enzymes involved in phosphatidylinositol (PI) turnover in higher plants were investigated using the plasma membrane isolated from tobacco suspension culture cells by aqueous two-phase partitioning. Submicromolar concentrations of Ca2+ inhibited PI kinase and phosphatidylinositol 4-phosphate (PIP) kinase and stimulated phospholipase C. Diacylglycerol (DG) kinase was inhibited by Ca2+, but required a higher concentration than the physiological level. From the above results we postulate the following scheme: signal coupled activation of phospholipase C produces IP3 which induces Ca2+ release from the intracellular Ca2+ compartment, the increased cytoplasmic Ca2+ in turn activates phospholipase C and causes a further increase of the cytoplasmic Ca2+ level. This inhibits PI kinase and PIP kinase and brings about a limited supply of PIP2, the substrate of phospholipase C. Consequently, IP3 production decreases and Ca2+ mobilization ceases. Then cytosolic Ca2+ returns to the stationary level by the Ca2+ pump at the plasma membrane and at the endoplasmic reticulum and Ca2+/H+ antiporter at the plasma membrane and at the tonoplast.     

Phosphatidylinositol-4,5-bisphosphate may antecede diacylglycerol as activator of protein kinase C

Phosphatidylserine/calcium-dependent protein kinase C (PKC) from rat brain is activated fifty times more efficiently by phosphatidylinositol-4,5-bisphosphate (PIP2) (Kapp = 0.04 mole% in Triton-lipid micelles) than by diacylglycerol (DG) (Kapp = 2 mole%). Both effector lipids appear to bind to the same site but PIP2 may confer a narrower substrate specificity on the kinase. DG, which together with inositol trisphosphate (IP3) is generated by hydrolysis from PIP2 after cell stimulation, has been considered the natural activator of the kinase but it is likely to be anteceded in this function by PIP2; DG may perhaps retain the function of a back-up activator. The lack of PKC-activation by phosphatidylinositol (PI) or phosphatidylinositol-4-phosphate (PIP) opens the possibility that the Inositide Shuttle, PI reversible PIP reversible PIP2, has a role in controlling the activity of the kinase.