Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts.

The phospholipids in plasma membranes of erythrocytes, as well as platelets, lymphocytes and other cells are asymmetrically distributed, with sphingomyelin and phosphatidylcholine residing predominantly in the outer leaflet of the bilayer, and phosphatidylserine and phosphatidylethanolamine in the inner leaflet. It is known that Ca2+ can disrupt the phospholipid asymmetry by activation of a protein known as phospholipid scramblase, which affects bidirectional phospholipid movement in a largely non-selective manner. As Ca2+ also inhibits aminophospholipid translocase, whose Mg(2+)-ATPase activity is responsible for active translocation of aminophospholipids from the outer to the inner leaflet, it is important to accurately determine the sensitivity of scramblase to intracellular free Ca2+. In the present study we have utilized the favourable Kd of Mag-fura-2 for calcium in the high micromolar range to determine free Ca2+ levels associated with lipid scrambling in resealed human red cell ghosts. The Ca2+ sensitivity was measured in parallel to the translocation of a fluorescent-labelled lipid incorporated into the ghost bilayer. The phospholipid scrambling was found to be half-maximally activated at 63-88 microM free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.     

Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase Cdelta

Phospholipid scramblase induces nonspecific bidirectional movement of phospholipids across the membrane during cell activation and has been proposed to mediate the appearance of phosphatidylserine (PS) in the plasma membrane outer leaflet during apoptosis, a cell surface change that is critical for apoptotic cell removal. We report here that protein kinase C (PKC) delta plays an important role in activated transbilayer movement of phospholipids and surface PS exposure by directly enhancing the activity of phospholipid scramblase. Specific inhibition of PKCdelta by rottlerin prevented both apoptosis- and activation-induced scramblase activity. PKCdelta was either selectively cleaved and activated in a caspase 3-dependent manner (during apoptosis) or translocated to the plasma membrane (in stimulated cells) and could directly phosphorylate scramblase immunoprecipitated from Jurkat cells. Furthermore, reconstitution of PKCdelta and scramblase, but not scramblase or PKCdelta alone in Chinese hamster ovary cells demonstrated enhanced scramblase activity.     

Transbilayer asymmetry of phospholipids in the plasma membrane regulates exocytotic release in mast cells

To investigate the role of the asymmetric distribution of phospholipids of the plasma membrane in exocytosis, we examined the effects of disruption of this asymmetrical distribution of lipids on exocytotic release from mast cells (RBL-2H3). Lipid scramblase, which is activated by divalent cations and catalyzes the transbilayer movement of phospholipids, was overexpressed in mast cells. Exogenous lipid scramblase was expressed in the plasma membrane and the cytoplasm. Activation of scramblase by divalent cations disrupted the asymmetrical distribution of phospholipids in the plasma membrane. Exocytotic release induced by calcium ionophore and phorbol ester was significantly inhibited in the cells transfected with wild-type scramblase. This inhibition was observed with time lag of about 5 min. Furthermore, when the asymmetric distribution of lipids was disrupted before induction of exocytosis, the inhibition of exocytotic release was obvious from the beginning without time lag. These results suggest that the asymmetric distribution of phospholipids in the plasma membrane plays an essential role in fusion between secretory granules and the plasma membrane. This finding also demonstrates that the transbilayer asymmetry of phospholipids regulates exocytosis and gives a new insight into the significance of lipid asymmetry in the plasma membrane.     

Influence of erythrocyte shape on the rate of Ca2+-induced scrambling of phosphatidylserine

Membrane-perturbing agents that cause transformation of biconcave erythrocytes into echinocytes or stomatocytes were used to investigate the influence of erythrocyte shape on the rate of Ca(2+)-induced scrambling of phospholipids. Erythrocytes were treated with a variety of lipid-soluble compounds to induce these shape changes, followed by incubation with calcium and ionomycin to activate lipid scramblase. Prothrombinase activity of the cells was used to monitor the rate of surface exposure of phosphatidylserine, which is taken as a measure of scramblase activity. Echinocytes show an enhanced rate of scrambling, whereas stomatocytes show a reduced rate, relative to normocytes. This phenomenon appears to correlate with enhanced and diminished micro-exovesicle shedding from echinocytes and stomatocytes, respectively. It is concluded that the rate of calcium-induced phosphatidylserine exposure (rate of lipid scrambling) in erythrocytes depends for a considerable part on the cells' ability to form microvesicles.     

Fluoride-dependent calcium-induced platelet procoagulant activity shows that calpain is involved in increased phospholipid transbilayer movement

Treatment of platelets with fluoride (10 mM) was found to result in a transient increase in Ca2+-permeability of the platelet plasma membrane. This phenomenon was used to provide supplementary evidence for the suggestions made earlier (Comfurius et al. (1985) Biochim. Biophys. Acta 815, 143; Verhallen et al. (1987) Biochim. Biophys. Acta 903, 206), that cytoskeletal disrupture by calpain is involved in the process leading to transbilayer movement of phosphatidylserine during expression of platelet procoagulant activity. This was achieved by relating both calpain activity and exposure of phosphatidylserine with platelet procoagulant activity. It was found that only upon addition of extracellular Ca2+ to fluoride-treated platelets, procoagulant activity, expressed as prothrombinase activity, and calpain activity, estimated from protein patterns after gel electrophoresis, were generated. Both Ca2+-inducible prothrombinase activity and calpain activity followed an identical time-course during incubation with fluoride: after a time-lag of about 10 min they sharply increased towards a peak level. Upon further incubation with fluoride, both activities decreased towards a final plateau, still above basal level. The presence of leupeptin during incubation with fluoride was found to inhibit Ca2+-inducible calpain activity and prothrombinase activity in an identical way. Ca2+-inducible exposure of phosphatidylserine, as determined with extracellular phospholipase A2, showed a similar pattern as Ca2+-inducible calpain activity and prothrombinase activity. From the strict parallelism between prothrombinase activity, calpain activity and exposure of phosphatidylserine, it is concluded that calpain plays an important role in the activation-dependent transbilayer movement of phosphatidylserine during expression of platelet procoagulant activity. It is suggested that degradation of the platelet membrane-skeleton by calpain disturbs the structural organization of the lipid bilayer of the platelet plasma membrane leading to enhanced transbilayer movement of phospholipids and appearance of phosphatidylserine at the platelet outer surface.     

Phospholipid scramblase: An update

The best understood consequence of the collapse of lipid asymmetry is exposure of phosphatidylserine (PS) in the external leaflet of the plasma membrane bilayer, where it is known to serve at least two major functions: providing a platform for development of the blood coagulation cascade and presenting the signal that induces phagocytosis of apoptotic cells. Lipid asymmetry is collapsed by activation of phospholipid scramblase(s) that catalyze bidirectional transbilayer movement of the major classes of phospholipid. The protein corresponding to this activity is not yet known. Observations on cells from patients with Scott syndrome, a rare hereditary bleeding disorder resulting from impaired lipid scrambling, have shown that there are multiple activation pathways that converge on scramblase activity.     

Rapid loss and restoration of lipid asymmetry by different pathways in resealed erythrocyte ghosts.

The normal asymmetric distribution of phospholipids across the plasma membrane of erythrocytes can be abolished by lysing and resealing cells in the presence of Ca2+. In the present study, using flow cytometric analysis of the binding of merocyanine 540 to monitor transbilayer phospholipid distribution, Ca(2+)-induced loss of asymmetry is shown to be independent from the aminophospholipid translocase which catalyzes movement of normally internal phospholipids from the outer to the inner leaflet of the membrane. Loss of asymmetry is rapid, temperature-sensitive, and occurs in an uninterrupted, intact bilayer, rather than by diffusion of lipids through the hemolytic pore. Addition of ATP during lysis reverses loss of asymmetry, and this restoration can be blocked by inhibitors of the aminophospholipid translocase. These results suggest that the ATP-dependent translocase is essential for recovery of asymmetry, in turn suggesting that separate mechanisms mediate the loss and the recovery of lipid asymmetry in erythrocytes.     

Mechanisms of suicidal erythrocyte death.

Erythrocyte injury such as osmotic shock, oxidative stress or energy depletion stimulates the formation of prostaglandin E2 through activation of cyclooxygenase which in turn activates a Ca2+ permeable cation channel. Increasing cytosolic Ca2+ concentrations activate Ca2+ sensitive K+ channels leading to hyperpolarization, subsequent loss of KCl and (further) cell shrinkage. Ca2+ further stimulates a scramblase shifting phosphatidylserine from the inner to the outer cell membrane. The scramblase is sensitized for the effects of Ca2+ by ceramide which is formed by a sphingomyelinase following several stressors including osmotic shock. The sphingomyelinase is activated by platelet activating factor PAF which is released by activation of phospholipase A2. Phosphatidylserine at the erythrocyte surface is recognised by macrophages which engulf and degrade the affected cells. Moreover, phosphatidylserine exposing erythrocytes may adhere to the vascular wall and thus interfere with microcirculation. Erythrocyte shrinkage and phosphatidylserine exposure ('eryptosis') mimic features of apoptosis in nucleated cells which however, involves several mechanisms lacking in erythrocytes. In kidney medulla, exposure time is usually too short to induce eryptosis despite high osmolarity. Beyond that high Cl- concentrations inhibit the cation channel and high urea concentrations the sphingomyelinase. Eryptosis is inhibited by erythropoietin which thus extends the life span of circulating erythrocytes. Several conditions trigger premature eryptosis thus favouring the development of anemia. On the other hand, eryptosis may be a mechanism of defective erythrocytes to escape hemolysis. Beyond their significance for erythrocyte survival and death the mechanisms involved in 'eryptosis' may similarly contribute to apoptosis of nucleated cells.     

Abnormal transbilayer mobility of phosphatidylcholine in hereditary pyropoikilocytosis reflects the increased heat sensitivity of the membrane skeleton

We determined whether the membrane defect in hereditary pyropoikilocytosis (HPP) is associated with thermally induced changes in the lipid bilayer, the stability of which was probed by the rate of translocation of phosphatidylcholine (PC) over the two leaflets. [14C]PC was incorporated into the outer leaflet of the lipid bilayer of the intact erythrocytes using a PC-specific phospholipid exchange protein. The transbilayer equilibration of this PC was determined by measuring the time-dependent changes in its accessibility to exogenous phospholipase A2. The rate of transbilayer equilibration of PC was increased in HPP cells at 37 degrees C when compared to normal erythrocytes (rate constants, 0.07 +/- 0.02 and 0.03 +/- 0.01 h-1, respectively). A further dramatic increase in PC transbilayer equilibration was noted in HPP cells incubated at 44 degrees C (rate constant, 0.15 +/- 0.02 h-1). A similar marked acceleration in transbilayer movement of PC was also seen in normal erythrocytes when incubated at 46 degrees C (rate constant, 0.13 +/- 0.03 h-1). Despite the enhanced transbilayer mobility of PC in HPP cells when compared to normal erythrocytes, no major alteration in the asymmetric distribution could be observed when probed with phospholipase A2. Since changes in transbilayer mobility of PC and cell morphology occur in HPP cells at lower temperature than in normal red cells, it may be concluded that the enhanced thermal sensitivity of spectrin is the major factor responsible for these changes. Our results therefore support the view that the structural integrity of the skeletal network is essential for stabilization of the lipid bilayer of the red cell membrane.     

Inhibition and stimulation of phospholipid scrambling activity. Consequences for lipid asymmetry, echinocytosis, and microvesiculation of erythrocytes

An increase of the intracellular Ca(2+) concentration in erythrocytes is known to activate rapid nonspecific bidirectional translocation of membrane-inserted phospholipid probes and to decrease the asymmetric distribution of endogenous membrane phospholipids. These scrambling effects are now shown to be suppressed by pretreatment of cells with the essentially impermeable reagents 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 2,4,6-trinitrobenzenesulfonate. The inhibitory effects are no longer observed during renewed activation of scrambling following a first transient activation by Ca(2+). Assuming the involvement of the human scramblase, this suggests a conformational alteration of this protein during activation by Ca(2+). Marked suppression of scrambling activity is also observed in cells pretreated with the disulfide reducing agent dithioerythritol which can be reverted by the SH oxidizing agent diamide. This indicates the importance of intramolecular and/or intersubunit disulfide bonds for the function of the scramblase. On the other hand, treatment of cells with the SH reagents N-ethylmaleimide and phenylarsine oxide enhances Ca(2+)-activated scrambling and diminution of asymmetry of membrane phospholipids. This suggests an allosteric connection of several protein SH groups to the translocation mechanism. The inhibitors retain their strong suppressive effects. Besides covalent modification, addition of oligomycin highly stimulates and addition of clotrimazole suppresses the Ca(2+)-activated translocation. No evidence for a role of the ATP-binding cassette transporter ABCA1 in the Ca(2+)-activated outward translocation is obtained. Suppression of phospholipid scrambling by dithioerythritol inhibits Ca(2+)-induced spheroechinocytosis and reduces the extent of subsequent microvesiculation. Scrambling of endogenous phospholipids is proposed to induce echinocytosis and to have only a stimulatory effect on microvesiculation.     

Calcium influx factor directly activates store-operated cation channels in vascular smooth muscle cells

Recently, we described a novel 3-pS Ca(2+)-conducting channel that is activated by BAPTA and thapsigargin-induced passive depletion of intracellular Ca(2+) stores and likely to be a native store-operated channel in vascular smooth muscle cells (SMC). Neither Ca(2+) nor inositol 1,4,5-trisphosphate or other second messengers tested activated this channel in membrane patches excised from resting SMC. Here we report that these 3-pS channels are activated in inside-out membrane patches from SMC immediately upon application of Ca(2+) influx factor (CIF) extracted from mutant yeast, which has been previously shown to activate Ca(2+) influx in Xenopus oocytes and Ca(2+) release-activated Ca(2+) current in Jurkat cells. In bioassay experiments depletion of Ca(2+) stores in permeabilized human platelets resulted in the release of endogenous factor, which activated 3-pS channels in isolated inside-out membrane patches excised from SMC and exposed to permeabilized platelets. The same 3-pS channels in excised membrane patches were also activated by acid extracts of CIF derived from human platelets with depleted Ca(2+) stores, which also stimulated Ca(2+) influx upon injection into Xenopus oocytes. Specific high pressure liquid chromatography fractions of platelet extracts were found to have CIF activity when injected into oocytes and activate 3-pS channels in excised membrane patches. These data show for the first time that CIF produced by mammalian cells and yeast with depleted Ca(2+) stores directly activates native 3-pS cation channels, which in intact SMC are activated by Ca(2+) store depletion.     

Regulation of phosphatidylserine exposure in red blood cells

The exposure of phosphatidylserine (PS) on the outer membrane leaflet of red blood cells (RBCs) serves as a signal for eryptosis, a mechanism for the RBC clearance from blood circulation. The process of PS exposure was investigated as function of the intracellular Ca(2+) content and the activation of PKCα in human and sheep RBCs. Cells were treated with lysophosphatidic acid (LPA), 4-bromo-A23187, or phorbol-12 myristate-13 acetate (PMA) and analysed by flow cytometry, single cell fluorescence video imaging, or confocal microscopy. For human RBCs, no clear correlation existed between the number of cells with an elevated Ca(2+) content and PS exposure. Results are explained by three different mechanisms responsible for the PS exposure in human RBCs: (i) Ca(2+)-stimulated scramblase activation (and flippase inhibition) by LPA, 4-bromo-A23187, and PMA; (ii) PKC activation by LPA and PMA; and (iii) enhanced lipid flop caused by LPA. In sheep RBCs, only the latter mechanism occurs suggesting absence of scramblase activity.     

Loss of membrane phospholipid asymmetry during activation of blood platelets and sickled red cells; mechanisms and physiological significance

Membrane phospholipid asymmetry is considered to be a general property of biological membranes. Detailed information is presently available on the non-random orientation of phospholipids in red cell- and platelet membranes. The outer leaflet of the lipid bilayer membrane is rich in choline-phospholipids, whereas amino-phospholipids are abundant in the inner leaflet. Studies with blood platelets have shown that these asymmetries are not maintained when the cells are activated in various ways. Undoing the normal asymmetry of membrane phospholipids in activated blood cells is presumably mediated by increased transbilayer movement of phospholipids. This process, which leads to increased exposure of negatively charged phosphatidylserine at the outer surface, plays an important physiological role in local blood clotting reactions. A similar phenomenon occurs in sickled red cells. Phospholipid vesicles breaking off from reversibly sickled cells contribute similarly to intravascular clotting in the crisis phase of sickle cell disease.The loss of membrane phospholipid asymmetry in activated platelets seems to be strictly correlated with degradation of cytoskeletal proteins by endogenous calpain. It is remarkable that membrane phospholipid asymmetry can be (partly) restored when activated platelets are treated with reducing agents. This leads to disappearance of phosphatidylserine from the outer leaflet where it was previously exposed during cell activation. These observations will be discussed in relation to two mechanisms which have been recognized to play a role in the regulation of membrane phospholipid asymmetry; i.e. the interaction of aminophospholipids to cytoskeletal proteins, and the involvement of a phospholipid-translocase catalyzing outward-inward transbilayer movement of amino-phospholipids.     

Cation channels trigger apoptotic death of erythrocytes

Erythrocytes are devoid of mitochondria and nuclei and were considered unable to undergo apoptosis. As shown recently, however, the Ca(2+)-ionophore ionomycin triggers breakdown of phosphatidylserine asymmetry (leading to annexin binding), membrane blebbing and shrinkage of erythrocytes, features typical for apoptosis in nucleated cells. In the present study, the effects of osmotic shrinkage and oxidative stress, well-known triggers of apoptosis in nucleated cells, were studied. Exposure to 850 mOsm for 24 h, to tert-butyl-hydroperoxide (1 mM) for 15 min, or to glucose-free medium for 48 h, all elicit erythrocyte shrinkage and annexin binding, both sequelae being blunted by removal of extracellular Ca(2+) and mimicked by ionomycin (1 microM). Osmotic shrinkage and oxidative stress activate Ca(2+)-permeable cation channels and increase cytosolic Ca(2+) concentration. The channels are inhibited by amiloride (1 mM), which further blunts annexin binding following osmotic shock, oxidative stress and glucose depletion. In conclusion, osmotic and oxidative stress open Ca(2+)-permeable cation channels in erythrocytes, thus increasing cytosolic Ca(2+) activity and triggering erythrocyte apoptosis.     

p38 MAPK activation and function following osmotic shock of erythrocytes

p38 protein kinase is activated by hyperosmotic shock, participates in the regulation of cell volume sensitive transport and metabolism and is involved in the regulation of various physiological functions including cell proliferation and apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include hyperosmotic shock, which increases cytosolic Ca(2+) activity and ceramide formation. The present study explored whether p38 kinase is expressed in human erythrocytes, is activated by hyperosmotic shock and participates in the regulation of eryptosis. Western blotting was utilized to determine phosphorylation of p38 kinase, forward scatter to estimate cell volume, annexin V binding to depict phosphatidylserine exposure and Fluo3 fluorescence to estimate cytosolic Ca(2+) activity. As a result, erythrocytes express p38 kinase, which is phosphorylated upon osmotic shock (+550 mM sucrose). Osmotic shock decreased forward scatter, increased annexin V binding and increased Fluo3 fluorescence, all effects significantly blunted by the p38 kinase inhibitors SB203580 (2 μM) and p38 Inh III (1 μM). In conclusion, p38 kinase is expressed in erythrocytes and participates in the machinery triggering eryptosis following hyperosmotic shock.     

Regulatory differences between Ca(2+)-ATPase in plasma membranes from chicken (nucleated) and pig (anucleated) erythrocytes

Kinetic and regulatory properties of the plasma membrane Ca(2+)-ATPase activity from chicken (nucleated) erythrocytes were studied and compared to those from pig (anucleated) erythrocytes. In the absence of known activators: (1) Ca(2+) affinity for the Ca(2+)-ATPase activity from nucleated erythrocytes was 12-fold higher than that from pig erythrocytes, and thus the enzyme is sensitive to physiological Ca(2+) concentrations; (2) the enzyme from chicken erythrocytes showed two apparent Km values for ATP, as compared to one apparent Km value displayed by pig erythrocytes; (3) Ca(2+)-ATPase inserted in chicken erythrocyte membranes showed a low sensitivity to activation by phosphatidylinositol-4-phosphate; (4) when p-NPP was used as substrate, the activity of chicken erythrocytes was high, similar to that attained by pig erythrocytes, but barely sensitive to activation by dimethylsulfoxide and calmodulin. ATP hydrolysis was 10-fold lower than that displayed by pig erythrocytes and the maximal velocity was activated three-fold by calmodulin. The enzyme was insensitive to alkaline phosphatase treatment and showed a single phosphorylation band in electrophoresis, ruling out the possibility of previous modulation by endogenous kinases and/or by partial proteolysis. The differences may be attributed to some endogenous modulator, to distinct isoforms, or to a difference in the E(1)/E(2) states of the enzyme.     

Cell volume and the regulation of apoptotic cell death

Apoptosis is a physiological mechanism allowing for the removal of abundant or potentially harmful cells. The hallmarks of apoptosis include degradation of cellular DNA, exposure of phosphatidylserine at the outer leaflet of the cell membrane and cell shrinkage. Phosphatidylserine exposure favours adhesion to macrophages with subsequent phagocytosis of the shrunken apoptotic particles. The interaction of cell volume regulatory mechanisms and apoptosis is illustrated in two different model systems, i.e. (a) lymphocyte apoptosis following stimulation of CD95 receptor and (b) erythrocyte apoptosis upon cell shrinkage. (a) Triggering of CD95 in Jurkat T lymphocytes is paralleled by activation of cell volume regulatory Cl- channels, inhibition of the Na+/H+ exchanger and osmolyte release. The latter coincides with cell shrinkage, DNA fragmentation and phosphatidylserine exposure. CD95 stimulation leads to early inhibition of the voltage gated K+ channel Kv1.3, which may contribute to the inhibition of the Ca2+ release activated Ca2+ channel I(CRAC). (b) Osmotic shock of erythrocytes activates a cell volume regulatory cation conductance allowing the entry not only of Na+ but of Ca2+ as well. Increased cytosolic Ca2+ stimulates a scramblase which disrupts the phosphatidylserine asymmetry of the cell membrane, leading to phosphatidylserine exposure. The cation conductance is further activated by oxidative stress and energy depletion and inhibited by Cl-. Shrinkage of erythrocytes stimulates in addition a sphingomyelinase with subsequent formation of ceramide which potentiates the effect of cytosolic Ca2+ on phosphatidylserine. In conclusion, cell volume-sensitive mechanisms participate in the triggering of apoptosis following receptor stimulation or cell injury.     

Stimulation of phosphatidylserine biosynthesis and facilitation of UV-induced apoptosis in Chinese hamster ovary cells overexpressing phospholipid scramblase 1

Members of the phospholipid scramblase (PLSCR) family play active roles in altering lipid asymmetry at the plasma membrane including phosphatidylserine (PtdSer) exposure on the cell surface. To determine whether PtdSer biosynthesis and externalization are altered by PLSCR activities during apoptosis, Chinese hamster ovary K1 cell lines stably overexpressing PLSCR1 and PLSCR2 were established. PLSCR1 was localized on the plasma membrane, whereas PLSCR2 was predominantly in the nucleus. Cells overexpressing PLSCR1 showed suppressed growth, altered cell morphology, and higher basal levels of cell death. Following UV irradiation, these cells showed earlier and enhanced PtdSer exposure, increased caspase-3 activation, apoptotic nuclear changes, and PARP cleavage indicative of apoptosis. UV irradiation in cells overexpressing PLSCR1 led to a 4-fold stimulation of PtdSer synthesis (accompanied by increased movement of newly made PtdSer into microvesicles) relative to untreated PLSCR1 cells, whereas PtdSer formation in UV-irradiated vector control cells increased only by 2-fold. No differences in these responses were observed between PLSCR2-expressing cells and vector controls. PtdSer synthesis and its transbilayer movement stimulated by PLSCR1 overexpression were blocked by a caspase inhibitor along with progression of apoptosis. Thus, our studies showed that overexpression of PLSCR1 in Chinese hamster ovary K1 cells stimulated caspase-dependent PtdSer externalization and synthesis, implying an up-regulation of PtdSer formation in response to enhanced outward movement of this phospholipid to the cell surface during apoptosis. PLSCR1 also appears to influence progression of UV-induced apoptosis and could be a point of regulation or intervention during programmed cell death.     

Calcium ion-dependent p-nitrophenyl phosphate phosphatase activity and calcium ion-dependent adenosine triphosphatase activity from human erythrocyte membranes

In the presence of ATP and of Mg(2+), human erythrocyte membranes show a phosphatase activity towards p-nitrophenyl phosphate which is activated by low concentrations of Ca(2+). The effect of Ca(2+) is strongly enhanced if either K(+) or Na(+) is also present. Activation of the p-nitrophenyl phosphate phosphatase by Ca(2+) reaches a half-maximum at about 8mum-Ca(2+) and is apparent only when the ion has access to the inner surface of the cell membrane. Ca(2+)-dependent phosphatase activity can only be observed if ATP is at the inner surface of the cell membrane, and the presence of ATP seems to be absolutely necessary, since either its removal or its replacement by other nucleoside triphosphates abolishes the activating effect of Ca(2+). The properties of the (ATP+Ca(2+))-dependent phosphatase are very similar to those of the Ca(2+)-dependent ATPase (adenosine triphosphatase), also present in erythrocyte membranes, which probably is involved in Ca(2+) transport in erythrocytes. The similarities suggest that both activities may be properties of the same molecular system. This view is further supported by the fact that p-nitrophenyl phosphate inhibits to a similar extent Ca(2+)-dependent ATPase activity and ATP-dependent Ca(2+) extrusion from erythrocytes.     

Palmitoylation of phospholipid scramblase 1 controls its distribution between nucleus and plasma membrane

Phospholipid scramblase 1 (PLSCR1) is a Ca(2+)-binding, endofacial plasma membrane protein thought to contribute to the transbilayer movement of phosphatidylserine and other membrane phospholipids that is observed upon influx of calcium into the cytosol. Expression of PLSCR1 is markedly induced by interferon and other cytokines, and PLSCR1-/- bone marrow cells exhibit defective myeloid proliferation and differentiation in response to stimulation by select growth factors, implying that PLSCR1 also functions in cytokine signaling or response pathways. PLSCR1 is multiply palmitoylated and partitions into membrane lipid raft domains. We have now identified the Cys-rich sequence (184)CCCPCC(189) in PLSCR1 as required for palmitoylation of the polypeptide. Mutation of these five cysteines abrogates PLSCR1 trafficking to the plasma membrane and results in virtually all of the expressed protein localizing to the nucleus. Consistent with this observation, cell treatment with the palmitoylation inhibitor, 2-bromo-palmitate, results in a marked redistribution of endogenous PLSCR1 from plasma membrane to nucleus. In a small percentage of untreated cells, predominantly nuclear localization of PLSCR1 is also observed. Furthermore, PLSCR1 is also found in the nucleus following its cytokine-induced expression. These data suggest that under the circumstance of rapid biosynthesis in response to gene induction by cytokines, PLSCR1 traffics into the nucleus, implying a potential nuclear function for this protein.