Magnesium: The missing element in molecular views of cell proliferation control

The quantitative study of regulation of cell growth and proliferation began with the development of the technique for monolayer culture of vertebrate cells in the late 1960s. The basic parameters were defined in the early physiological studies, which continued through the next decade. These included specific and non-specific growth factors and the requirement for continuous exposure to such factors through most of the G1 period for progression to S. In the course of this work, the diversity of biochemical responses and the critical role of increased protein synthesis and accumulation for the onset of DNA synthesis were elucidated. In particular, a central role of free cytosolic Mg2+ in direct regulation of protein synthesis and in ancillary processes as a response to membrane perturbation was established. Eventually, the physiological era was superseded by the molecular era beginning in the 1980s. This work focussed on specific receptors for growth factors that entrained a protein kinase cascade, which terminated in a higher frequency of initiation of protein synthesis. However, the molecular studies virtually ignored the key results of the physiological era. Recent studies of the penultimate molecular steps in the regulatory pathway of protein synthesis, however, have supported a model of growth regulation involving membrane perturbation and MgATP2- concentration, results that integrate the findings of the physiological and molecular eras. The resulting relatively simple "membrane, magnesium mitosis" (MMM) model of proliferation control can explain the seeming paradox of the variety of specific and non-specific growth-enhancing treatments that are mediated by the plasma membrane and which bring about a shared, complex but coordinated growth response that drives cell proliferation.     

Magnesium deprivation reproduces the coordinate effects of serum removal or cortisol addition on transport and metabolism in chick embryo fibroblasts.

A variety of unrelated effectors stimulate or inhibit coordinately the same array of metabolic reactions in chick embryo fibroblasts, including the uptake of 2-deoxy-D-glucose and uridine, and the incorporation of uridine and thymidine into acid insoluble material. The coordinate inhibition of these reactions by omission of serum or addition of cortisol is reproduced quantitatively by lowering the concentration of magnesium (Mg2+) in medium containing 0.2 mM Ca2+. The response times for the utilization of uridine and thymidine following the removal of addition of Mg2+ are similar to those which follow removal or addition of serum. The effect of serum on the incorporation of choline, which is not part of the coordinate response to unrelated effectors, is not reproduced by varying Mg2+ concentrations. The results support the hypothesis that the availability of Mg2+ within the cell plays a central role in the coordinate control of transport, metabolism and growth by external physiological effectors.     

Induction of the nuclear protein ''cyclin'' in quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation with DNA synthesis

The effect of serum and growth factors [platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)] on the synthesis of the nuclear protein cyclin and its correlation with DNA synthesis has been studied in quiescent mouse 3T3 cells by means of quantitative two-dimensional gel electrophoresis. Serum must be present in the medium for at least 8-12 h to induce maximal synthesis of cyclin (6- to 7-fold increase compared with quiescent cells). The stimulation of cyclin synthesis is dose-dependent and correlates directly with DNA synthesis. In addition, partially purified PDGF and FGF also induce cyclin and DNA synthesis in a coordinate way. Both growth factors, like serum, exhibit a similar lag phase to induce maximal cyclin (6- to 7-fold) and DNA synthesis (90% of the cells). Pure PDGF at a concentration as low as 10 ng/ml has the same effect as 10% serum. The coordinate induction of cyclin and DNA synthesis can only be observed with growth factors that induce DNA synthesis. These results strengthen the notion that cyclin is an essential component of the events leading to DNA replication.     

The Escherichia coli cell cycle

This review summarizes present knowledge of the bacterial cell cycle with particular emphasis on Escherichia coli. We discuss data coming from three different types of approaches to the study of cell extension and division: The search for discrete events occurring once per division cycle. It is generally agreed that the initiation and termination of DNA replication and cell septation are discrete events; there is less agreement on the sudden doubling in rate of cell surface extension, murein biosynthesis and the synthesis of membrane proteins and phospholipids. We discuss what is known about the temporal relationship amongst the various cyclic events studied. The search for discrete growth zones in the cell envelope layers. We discuss conflicting reports on the existence of murein growth zones and protein insertion sites in the inner and outer membranes. Elucidation of the mechanism regulating the initiation of DNA replication. The concept of "critical initiation mass" is examined. We review data suggesting that the DNA is attached to the envelope and discuss the role of the latter in the initiation of DNA replication.     

Priming in exocytosis: attaining fusion-competence after vesicle docking

Membrane contact established by tethering or docking mechanisms is not a sufficient condition for membrane fusion. In neural and neuroendocrine cells, only a small fraction of secretory vesicles docked at the plasma membrane are fusion-competent and undergo rapid ATP-independent fusion in response to Ca(2+) elevations. Additional biochemical events termed 'priming' are essential to render vesicles competent for Ca(2+)-triggered fusion. The priming of vesicles is ATP-dependent and a number of ATP-dependent priming reactions have been characterized in permeable neuroendocrine cells. These involve NSF-mediated priming of SNARE protein complexes, the ATP-dependent synthesis of phosphoinositides, and protein kinase-mediated protein phosphorylation. In addition, munc13 is an important protein involved in priming synaptic vesicles. An emphasis in this review is on recent work indicating that priming events identified in the pathways of regulated exocytosis share many features with pre-fusion processes characterized in constitutive fusion pathways.     

The emerging roles of translation factor eIF4E in the nucleus

The emerging field of nuclear eIF research has yielded many surprises and led to the dissolution of some dogmatic/ideological viewpoints of the place of translation in the regulation of gene expression. Eukaryotic initiation factors (eIFs) are classically defined by their cytoplasmic location and ability to regulate the initiation phase of protein synthesis. For instance, in the cytoplasm, the m7G cap-binding protein eIF4E plays a distinct role in cap-dependent translation initiation. Disruption of eIF4E's regulatory function drastically effects cell growth and may lead to oncogenic transformation. A growing number of studies indicate that many eIFs, including a substantial fraction of eIF4E, are found in the nucleus. Indeed, nuclear eIF4E participates in a variety of important RNA-processing events including the nucleocytoplasmic transport of specific, growth regulatory mRNAs. Although unexpected, it is possible that some eIFs regulate protein synthesis within the nucleus. This review will focus on the novel, nuclear functions of eIF4E and how they contribute to eIF4E's growth-activating and oncogenic properties. Both the cytoplasmic and nuclear functions of eIF4E appear to be dependent on its intrinsic ability to bind to the 5' m7G cap of mRNA. For example, Promyelocytic Leukemia Protein (PML) potentially acts as a negative regulator of nuclear eIF4E function by decreasing eIF4E's affinity for the m7G cap. Therefore, eIF4E protein is flexible enough to utilize a common biochemical activity, such as m7G cap binding, to participate in divergent processes in different cellular compartments.     

Initiation of DNA synthesis by human thrombin: relationships between receptor binding, enzymic activity, and stimulation of 86Rb+ influx

Stimulation of amiloride-sensitive sodium (Na+) influx and the subsequent activation of NA+, K+-ATPase by serum or growth factors have been implicated as early events leading to initiation of cell proliferation. We recently demonstrated that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8 to 12 hr after thrombin addition. To further probe the relationship between stimulation of ion influx and initiation of cell proliferation, human alpha-thrombin was converted to gamma-thrombin, nitro-alpha-thrombin, and diisopropylphospho (DIP)-alpha-thrombin. These derivatives retain either the capacity to bind cell surface alpha-thrombin receptors or thrombin esterase activity, but they do not initiate DNA synthesis. At low concentrations of alpha-thrombin or the various thrombin derivatives, only alpha-thrombin stimulates 86Rb+ influx, suggesting a correlation between stimulation of influx and the ability of these derivatives to initiate DNA synthesis. Concentrations of a DIP-alpha-thrombin that saturate the alpha-thrombin receptors (up to 2 micrograms/ml) do not stimulate either the early or late influx of 86Rb+, indicating that DIP-alpha-thrombin binding alone is not sufficient to stimulate ion fluxes. High concentrations of either gamma-thrombin or nitro-alpha-thrombin, however, stimulate both early and late 86RB+ uptake but do not initiate DNA synthesis. These results demonstrate that events leading to both the early and late stimulation of 86Rb+ influx by themselves are not sufficient to initiate cell proliferation. Thus, initiation may require a combination of events that can be independently regulated by different transmembrane signals.     

Inhibition of T-lymphocyte activation by amiloride

The T-lymphocyte activation process involves a series of coordinately coupled biochemical events occurring in response to antigen or mitogen. These events have not been completely characterized. The present studies investigate the mechanism of protein synthesis during the initial phase of T-cell activation. Among the early biochemical changes, induction of protein synthesis was observed as early as 10 minutes after mitogen stimulation of T-lymphocytes. This early protein synthesis was inhibited by cycloheximide but was insensitive to actinomycin-D, indicating the presence of preformed mRNA in resting lymphocytes. Since early protein synthesis parallels the increase in protein kinase C activity in activated T-lymphocytes, these two biochemical events may be related. In the present report, amiloride, an inhibitor of Na+/H+ antiport and protein kinase C, significantly inhibited [3H]leucine and [3H]thymidine incorporation in a dose-dependent manner into phytohemagglutinin (PHA)-stimulated T-lymphocytes. Furthermore, when T-lymphocytes were stimulated by phorbol myristate acetate, a known activator of protein kinase C, a similar inhibition of protein and DNA synthesis by amiloride was observed. The partially purified cytosol fraction isolated from PHA-activated T-lymphocytes showed a 75% decrease in protein kinase C-mediated [32P] incorporation from ATP in the presence of 100 microM amiloride. These results suggest that the T-cell activation process following exposure to mitogens involves early protein synthesis, which may be mediated by protein kinase C.     

Phosphatidic acid modulates DNA synthesis, phospholipase C, and platelet-derived growth factor mRNAs in cultured mesangial cells. Role of protein kinase C

Increases in cell phosphatidic acid content occur in response to a wide variety of agonists, many of which have growth promoting properties. These changes have correlated with calcium flux, enzyme activation, gene induction, or cell proliferation. In the current studies we show that exogenous phosphatidic acid (PA) and phosphatidylserine stimulate phosphoinositide hydrolysis and DNA synthesis in cultured human renal mesangial cells. These phospholipids also induce mRNAs for platelet-derived growth factor (PDGF). The activation of phospholipase C by PA appears to be desensitized via protein kinase C as brief preincubation with phorbol ester abrogates the effect. PA-induced DNA synthesis is only partly mediated via protein kinase C as co-incubation with the inhibitor staurosporine blunts DNA synthesis by only one-third. In contrast, induction of PDGF A-chain mRNA is almost totally inhibited by staurosporine. We propose that changes in endogenous phospholipids such as PA or phosphatidylserine may serve as common signaling pathway for a variety of growth factors. Induction of PDGF proto-oncogenes via protein kinase C may represent one mechanism by which this cell activation occurs.     

A pertussis toxin-sensitive GTP-binding protein plays a role in the G0-G1 transition of rat hepatocytes following establishment in primary culture

Acute spontaneous c-myc gene expression and sustained increase of a GTP-binding protein(s) (G-protein) which is sensitive to islet-activating protein (IAP), pertussis toxin, occurred early during primary culture of adult rat hepatocytes. Following these earlier events, DNA synthesis was demonstrated in response to EGF and insulin. Addition of IAP immediately after plating of primary cultures inhibited c-myc expression and the hormone-induced DNA synthesis. Addition at 24 h or later following cell inoculation, however, produced only weak effects on DNA synthesis, even though the IAP-sensitive G-proteins were completely inactivated. We conclude that the IAP-sensitive G-protein(s) plays a role in the earlier process(es) of the G0-G1 transition, which is essential for the initiation of growth factor-dependent DNA synthesis.     

Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg(2+) ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.     

Functional characterization of DNase X, a novel endonuclease expressed in muscle cells

The activation of endonucleases resulting in the degradation of genomic DNA is one of the most characteristic changes in apoptosis. Here, we report the characterization of a novel endonuclease, termed DNase X due to its X-chromosomal localization. The active nuclease is a 35 kDa protein with 39% identity to DNase I. When incubated with isolated nuclei, recombinant DNase X was capable of triggering DNA degradation at internucleosomal sites. Similarly to DNase I, the nuclease activity of DNase X was dependent on Ca(2+) and Mg(2+) and inhibited by Zn(2+) ions or chelators of bivalent cations. Overexpression of DNase X caused internucleosomal DNA degradation and induction of cell death associated with increased caspase activation. Despite the presence of two potential caspase cleavage sites, DNase X was processed neither in vitro nor in vivo by different caspases. Interestingly, after initiation of apoptosis DNase X was translocated from the cytoplasm to the nuclear compartment and aggregated as a detergent-insoluble complex. Abundant expression of DNase X mRNA was detected in heart and skeletal muscle cells, suggesting that DNase X may be involved in apoptotic or other biological events in muscle tissues.     

Induction of mitochondrial alternative oxidase in response to a cell signal pathway down-regulating the cytochrome pathway prevents programmed cell death

Treatment of tobacco (Nicotiana tabacum L. cv Petit Havana SR1) cells with cysteine (Cys) triggers a signal pathway culminating in a large loss of mitochondrial cytochrome (cyt) pathway capacity. This down-regulation of the cyt path likely requires events outside the mitochondrion and is effectively blocked by cantharidin or endothall, indicating that protein dephosphorylation is one critical process involved. Generation of reactive oxygen species, cytosolic protein synthesis, and Ca(2+) flux from organelles also appear to be involved. Accompanying the loss of cyt path is a large induction of alternative oxidase (AOX) protein and capacity. Induction of AOX allows the cells to maintain high rates of respiration, indicating that the lesion triggered by Cys is in the cyt path downstream of ubiquinone. Consistent with this, transgenic (AS8) cells unable to induce AOX (due to the presence of an antisense transgene) lose all respiratory capacity upon Cys treatment. This initiates in AS8 a programmed cell death pathway, as evidenced by the accumulation of oligonucleosomal fragments of DNA as the culture dies. Alternatively, wild-type cells remain viable and eventually recover their cyt path. Induction of AOX in response to a chemical inhibition of the cyt path (by antimycin A) is also dependent upon protein dephosphorylation and the generation of reactive oxygen species. Common events required for both down-regulation of the cyt path and induction of AOX may represent a mechanism to coordinate the biogenesis of these two electron transport paths. Such coordinate regulation may be necessary, not only to satisfy metabolic demands, but also to modulate the initiation of a programmed cell death pathway responsive to mitochondrial respiratory status.     

Signal transduction mechanisms in the regulation of protein synthesis

Control of polypeptide synthesis plays an important role in cell proliferation and translation rates generally reflect the growth state of the cultured eukaryotic cell. Physiological regulation of protein synthesis is almost always exerted at the level of polypeptide chain initiation, with the binding of mRNA to the small ribosomal subunit a rate-limiting step in many cell systems. Studies have indicated key roles in the regulation of protein synthesis for the structural features of mRNA molecules and phosphorylation of initiation factors which catalyse this process. This review focusses on translational regulation at the level of mRNA binding to the ribosome and the role of phosphorylation of initiation factors in mediating both quantitative and qualitative control. The identity of putative kinases which may mediate these processes is addressed and a possible model for the role of a transient activation of initiation factors in cell growth or differentiation is presented.     

Lipid characteristics of Friend erythroleukaemia cells differentiated by dimethyl sulphoxide or hexamethylenebisacetamide, and of non-inducible clones treated with the inducers.

We have compared the effects of pretreatment of Swiss 3T3 cell with pertussis toxin on the stimulation of DNA synthesis and phosphoinositide hydrolysis in response to a wide variety of mitogens. The toxin substantially inhibited the stimulation of DNA synthesis in response to a phorbol ester or various peptide and polypeptide growth factors irrespective of their ability to activate phosphoinositidase C. Production of inositol phosphates in response to platelet-derived growth factor, fibroblast growth factor and prostaglandin F2 alpha were unaffected by the toxin while bombesin- and vasopressin-stimulated formation of inositol phosphates were inhibited by only 27 and 23% respectively. These results argue against a major role for a pertussis toxin-sensitive G protein in coupling any of these mitogen receptors to activation of a phosphoinositidase C. Furthermore, the results suggest that the widespread inhibitory effects of pertussis toxin on mitogen-stimulated DNA synthesis may be unrelated to the toxin's limited actions on phosphoinositide hydrolysis.     

Superoxide radical formation and associated biochemical alterations in the plasma membrane of brain, heart, and liver during the lifetime of the rat.

Plasma membrane samples from rat brain, heart, and liver were examined for biochemical changes with age. A rise in superoxide radical (SOR) levels was followed by increases in thiobarbituric acid reactive substances and decreases in membrane fluidity with age. The earliest rise in SOR formation appeared in the plasma membrane from the brain. With age, protein synthesis also decreased significantly in tissue homogenates from brain and heart but was unchanged in the liver. Exposure of plasma membrane samples to in vitro-elevated SOR levels stimulated formation of lipid peroxides, as indicated by the thiobarbituric acid test, and resulted in a decrease in membrane fluidity in each tissue and in a decline in protein synthesis in brain and heart. Changes in brain lipid peroxidation and in membrane fluidity in brain and heart as a result of SOR supplementation were further enhanced due to age. In addition, the mechanism of SOR formation was examined in plasma membrane samples from the brain. SOR generation was Ca(2+)-sensitive, blocked by superoxide dismutase or vitamin E and inhibited by both indomethacin, a cyclooxygenase inhibitor, and bromophenacyl bromide, a phospholipase A2 inhibitor. These results show significant increases in SOR formation and biochemical alterations in plasma membranes from brain, heart, and liver in aging rats. SOR formation appears to be enzyme-mediated and elevated levels of this oxygen radical could be involved in membrane breakdown in older rats.     

Increased Activity of Eukaryotic Initiation Factor 2B in PC12 Cells in Response to Differentiation by Nerve Growth Factor

Abstract: Translational rates, and activities and levels of initiation factors 2 and 2B were assessed in rat pheochromocytoma cells upon nerve growth factor (NGF) treatment. Two or 5 days of exposure to NGF caused significant quantitative increases in protein synthesis rate that are deemed necessary for neuronal differentiation. Changes in initiation factor 2 activity, as measured by its capacity to form a ternary complex, occur parallel to the observed changes in protein synthesis. Nevertheless, neither the intracellular levels of the initiation factor 2 nor the degree of phosphorylation of its α subunit can justify this increased activity. Interestingly, initiation factor 2B activity increases parallel to the neurite outgrowth, being significantly higher after 5 days of exposure to NGF, and could be responsible for the elevated rate of protein synthesis. No significant changes in the levels of eukaryotic initiation factor 2B, as determined with two different antibodies against the γ and ε subunits of the factor, were observed, implying that the increased activity should be regulated by factors other than its cellular concentration. Our results support the hypothesis that initiation factor 2B may play a role in the biochemical events controlling the differentiative growth factor-induced signaling pathway in these cells.     

Modulation of vascular smooth muscle cell growth by magnesium-role of mitogen-activated protein kinases

We tested the hypothesis that Mg(2+) influences growth of vascular smooth muscle cells (VSMCs) by modulating cell cycle activation through mitogen-activated protein (MAP) kinase-dependent pathways. Rat VSMCs were grown in culture medium containing normal Mg(2+) (1.02 mmol/L, control) and increasing concentrations of Mg(2+) (2-4 mmol/L) for 1-8 days. Effects of varying extracellular Mg(2+) concentration ([Mg(2+)](e)) on intracellular free Mg(2+) concentration ([Mg(2+)](i)) were assessed using mag-fura. Growth actions of Mg(2+) were evaluated by measuring cell cycle activation, DNA synthesis, and protein synthesis. Expression of cell cycle promoters, cyclin D1, cyclin E, Cdk2, and Cdk4 was assessed by immunoblotting. Phosphorylation of cell cycle inhibitors p21(cip1) and p27(kip1) and MAP kinases, ERK1/2, p38MAP kinase, and JNK was evaluated using phospho-specific antibodies. [Mg(2+)](i) increased in a dose-dependent manner in response to increasing [Mg(2+)](e). These effects were evident within 2 days and maximal responses were obtained after 6 days. High [Mg(2+)](e) induced cell cycle activation with a lower proportion of cells in G(1) phase (75 +/- 1.0%) and a higher fraction of cells in S phase (12 +/- 0.7%) versus control (G(1), 88.5 +/- 1.4%; S, 6.8 +/- 1.2%; P < 0.05). This was associated with increased protein content of cyclin D1 and Cdk4 and decreased activation of p21(cip1) and p27(kip1). In cells exposed to 2 mmol/L Mg(2+), DNA and protein synthesis was increased approximately threefold. Phosphorylation of MEK1/2 and ERK1/2 was enhanced two to threefold in cells grown in 2 mmol/L Mg(2+). These effects were rapid, occurring within 2 days. Phosphorylation of MEK3/6, p38 MAP kinase, and JNK was unaltered by increasing [Mg2](e). PD98059 (10(-5) mol/L), specific MEK1/2 inhibitor, but not SB202190 (10(-5) mol/L) (specific p38 MAP kinase inhibitor), attenuated Mg(2+)-induced growth actions. These data demonstrate the novel findings that cell cycle activation and growth regulation by Mg(2+) occurs via ERK1/2-dependent, p38 MAP kinase-independent pathways.     

Synthesis of sterol and phospholipid induced by the interaction of phytohemagglutinin and other mitogens with human lymphocytes and their relation to blastogenesis and DNA synthesis

Human peripheral blood lymphocytes (PBL) responded to phytohemagglutinin (PHA) and a variety of other mitogens by increased synthesis of sterol and phospholipid. This activity was established within 4–7 hr of the addition of mitogen and was dependent upon the binding of the ligand to the cell membrane. Sterol and phospholipid synthesis reached a peak at approximately 24 hr in association with blastogenic expansion of the lymphocyte membrane and initiation of DNA synthesis. Lipid synthesis and blast transformation occurred independently of replication of the genome since inhibition of DNA synthesis did not reduce the degree of blast transformation and lipid synthesis observed. However, inhibition of sterol synthesis using 20α-hydroxycholesterol resulted in decreased blastogenesis and DNA synthesis, demonstrating that early synthesis of lipid is important for these subsequent events. Human thymocytes responded to T-cell mitogens in the conventional manner as regards synthesis of lipid and blast transformation; however, they did not synthesize DNA. Possible reasons for this incomplete response are discussed. Several nonmitogenic agents which agglutinate lymphocytes were also found to initiate early increases in sterol and phospholipid synthesis, and the possible significance of this observation is considered.     

Effect of insulin secretagogues and potential modulators of secretion on a plasma membrane (Ca2+ + Mg2+)-ATPase activity in islets of Langerhans

Studies were undertaken to determine whether factors which affect insulin secretion may exert their effects by altering the activity of an islet-cell plasma membrane Ca2+ extrusion pump. The insulin secretagogue, D-glucose, and a variety of phosphorylated hexoses, glucose 6-P, glucose 1,6-P, fructose 6-P, and fructose 2,6-P, were evaluated for their effect on an islet-cell plasma membrane (Ca2+ + Mg2+)-ATPase and were found to be ineffective in altering enzyme activity. D-Glucose also did not alter the rate of ATP-dependent Ca2+ uptake into plasma membrane vesicles. Similarly, cAMP, the catalytic subunit of cAMP-dependent protein kinase, arachidonic acid, or prostaglandin E2 did not affect either the plasma membrane (Ca2+ + Mg2+)-ATPase or the rate of ATP-dependent Ca2+ uptake into plasma membrane vesicles. Whereas previous studies have suggested that D-glucose and/or cAMP may inhibit ATPase activities in islets, these results indicate that the agents, i.e., D-glucose and cAMP, which stimulate and/or potentiate insulin secretion from the islet cell, do not modify Ca2+ fluxes by directly regulating the islet-cell plasma membrane (Ca2+ + Mg2+)-ATPase. In contrast, the acidic phospholipids, phosphatidic acid and phosphatidylserine, stimulated the enzyme activity in a concentration-dependent manner whereas phosphatidylcholine had only a minimal effect. The diacylglycerol, dilinolein, stimulated the (Ca2+ + Mg2+)-ATPase activity in the presence of phosphatidylserine, but not in the absence of phospholipids. These effects were independent of phospholipid-stimulated protein phosphorylation in the islet-cell plasma membrane under the conditions of the ATPase assay.