Calcium- and calmodulin-regulated breakdown of phospholipid by microsomal membranes from bean cotyledons

Evidence for the involvement of Ca²⁺ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U-¹⁴C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U-¹⁴C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca²⁺, whereas lipolytic acyl hydrolase proved to be insensitive to Ca²⁺. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca²⁺. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC₅₀ values ranging from 10 to 15 micromolar. Thus the Ca²⁺-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca²⁺ on phospholipase D is independent of calmodulin. The role of Ca²⁺ as a second messenger in the initiation of membrane lipid degradation is discussed.     

Autolysis of membrane lipids in potato leaf homogenates: Effects of calmodulin and calmodulin antagonists

Potato leaves contain high levels of lipolytic acyl hydrolase activity which degrades phospholipids and galactolipids during homogenization and organelle isolation. Four calmodulin antagonists (dibucaine, tetracaine, trifluoperazine a and chlorpromazine) were found to inhibit the rate of hydrolysis of endogenous membrane lipids in homogenates of potato leaves. In contrast, the addition of calcium and purified calmodulin stimulated the rate of hydrolysis. These results indicate that a lipolytic acyl hydrolase activity in potato leaves appears to be mediated either directly or indirectly by calcium and calmodulin.     

Catalase activity is modulated by calcium and calmodulin in detached mature leaves of sweet potato

Catalase (CAT) functions as one of the key enzymes in the scavenging of reactive oxygen species and affects the H₂O₂ homeostasis in plants. In sweet potato, a major catalase isoform was detected, and total catalase activity showed the highest level in mature leaves (L3) compared to immature (L1) and completely yellow, senescent leaves (L5). The major catalase isoform as well as total enzymatic activity were strongly suppressed by ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). This inhibition could be specifically and significantly mitigated in mature L3 leaves by exogenous CaCl₂, but not MgCl₂ or CoCl₂. EGTA also inhibited the activity of the catalase isoform in vitro. Furthermore, chlorpromazine (CPZ), a calmodulin (CAM) inhibitor, drastically suppressed the major catalase isoform as well as total enzymatic activity, and this suppression was alleviated by exogenous sweet potato calmodulin (SPCAM) fusion protein in L3 leaves. CPZ also inhibited the activity of the catalase isoform in vitro. Protein blot hybridization showed that both anti-catalase SPCAT1 and anti-calmodulin SPCAM antibodies detect a band at the same position, which corresponds to the activity of the major catalase isoform from unboiled, but not boiled crude protein extract of L3 leaves. An inverse correlation between the major catalase isoform/total enzymatic activity and the H₂O₂ level was also observed. These data suggest that sweet potato CAT activity is modulated by CaCl₂ and SPCAM, and plays an important role in H₂O₂ homeostasis in mature leaves. Association of SPCAM with the major CAT isoform is required and regulates the in-gel CAT activity band.     

Searching for mechanisms of N-methyl-D-aspartate-induced glutathione efflux in organotypic hippocampal cultures

N-Methyl-d-aspartate (NMDA)-receptor stimulation evoked a selective and partly delayed elevated efflux of glutathione, phosphoethanolamine, and taurine from organotypic rat hippocampus slice cultures. The protein kinase inhibitors H9 and staurosporine had no effect on the efflux. The phospholipase A₂ inhibitors quinacrine and 4-bromophenacyl bromide, as well as arachidonic acid, a product of phospholipase A₂ activity, did not affect the stimulated efflux. Polymyxin B, an antimicrobal agent that inhibits protein kinase C, and quinacrine in high concentration (500 µM), blocked efflux completely. The stimulated efflux after but not during NMDA incubation was attenuated by a calmodulin antagonist (W7) and an anion transport inhibitor (DNDS). Omission of calcium increased the spontaneous efflux with no or small additional effects by NMDA. In conclusion, NMDA receptor stimulation cause an increased selective efflux of glutathione, phosphoethanolamine and taurine in organotypic cultures of rat hippocampus. The efflux may partly be regulated by calmodulin and DNDS sensitive channels.     

Regulation of erythrocyte Ca2+ pump activity by protein kinase C

Using either inside-out vesicles (IOV) prepared from human erythrocytes or purified Ca2+-ATPase from the same source, the effects of protein kinase C (Ca2+/phospholipid-dependent enzyme) on Ca2+ transport and Ca2+-ATPase activity were measured. Incubation of IOV with protein kinase C in the presence, but not absence, of either 12-O-tetradecanoylphorbol-13-acetate or diolein led to a Ca2+-dependent stimulation of ATP-dependent calcium uptake. The effect was a 5-7-fold increase of Vmax without a significant change in the apparent Km for Ca2+. By comparison, the effect of calmodulin was a 14-fold stimulation of Vmax and a 4-fold reduction in apparent Km. The effect of protein kinase C and calmodulin on Ca2+ uptake were nearly additive. Stimulation of IOV Ca2+ transport by protein kinase C was entirely reversible by treatment of activated IOV with alkaline phosphatase. Incubation of purified Ca2+-ATPase with protein kinase C in the presence of 12-O-tetradecanoylphorbol-13-acetate or diolein led to a stimulation of Ca2+-dependent ATPase activity. These results indicate that protein kinase C stimulates the activity of the plasma membrane Ca2+ pump by a direct effect on the pump protein.     

Effect of calmodulin antagonists on phospholipase D activity in SH-SY5Y cells

The aim of this study was to investigate the involvement of calmodulin in phospholipase D activation in SH-SY5Y cells. Cells prelabelled with [3H]-palmitic acid were incubated with calmodulin antagonists and/or other compounds. Phosphatidylethanol, a specific marker for phospholipase D activity, and phosphatidic acid were analysed. The calmodulin antagonists, calmidazolium and trifluoperazine, induced an extensive increase in phosphatidylethanol formation, and thus increased basal phospholipase D activity, in a dose- and time-dependent manner. The effect of calmidazolium on carbachol-induced activation of muscarinic receptors was also studied. Calmidazolium did not significantly affect the amount of phosphatidylethanol formed following carbachol addition. However, taking into account the increase in basal activity observed after calmidazolium addition, calmidazolium probably inhibits the muscarinic receptor-induced phospholipase D activation. In addition to phosphatidylethanol, basal phosphatidic acid levels were also increased after calmidazolium and trifluoperazine addition. Incubation with calmidazolium (10 microM) for 10 min induced a two-fold increase in phosphatidic acid. The calmidazolium-induced increase in basal phospholipase D activity was not affected by the protein kinase inhibitors H7 and staurosporine. On the other hand tyrosine kinase inhibitors abolished the calmidazolium-induced activation of phospholipase D. Calmidazolium also induced tyrosine phosphorylation in parallel to the phospholipase D activation. In conclusion, our data indicate that calmodulin antagonists induce phospholipase D activity in SH-SY5Y cells via a tyrosine kinase dependent pathway. This may point to a negative control of phospholipase D by calmodulin although a calmodulin-independent mechanism cannot be excluded. Calmodulin antagonists may be useful tools to further elucidate the mechanisms of phospholipase D regulation.     

Effects of sulfhydryl agents,trifluoperazine, phosphatase inhibitors and tryptic proteolysis on calcineurin isolated from bovine cerebral cortex

Summary Calcineurin was dicovered as an inhibitor of calmodulin stimulated cyclic AMP phosphodiesterase and its ability to act as a calmodulin binding protein largely explains its inhibitory action on calmodulin regulated enzymes. Recent studies establish calcineurin as the enzyme protein phosphatase whose activity is regulated by calmodulin and a variety of divalent metals. In this work, we have investigated the effects of several agents including sulfhydryl agents, trifluoperazine (a calmodulin antagonist), PP_i, NaF and orthovanadate and of tryptic proteolysis on the calcineurin inhibition of cyclic AMP phosphodiesterase (called inhibitory activity) and on protein phosphatase activity. Inhibitors for sulfhydryl groups (pHMB, NEM) inhibited phosphatase activity without any effect on the inhibitory activity. Dithioerythritol completely reversed the inhibition by pHMB. Limited proteolysis of calcineurin caused an activation of basal phosphatase activity with a complete loss of inhibitory activity. Phosphatase activity of the proteolyzed calcineurin was not stimulated by calmodulin. The presence of calmodulin along with calcineurin during tryptic digestion appeared to preserve the stimulation of phosphatase by Ca²⁺-calmodulin. [³H]-Trifluoperazine (TFP) was found to be incorporated irreversibly into calcineurin in the presence of ultraviolet light. This incorporation was evident into the A and B subunits of calcineurin. TFP-caused a decrease in the phosphatase activity and an increase in its inhibitory activity. [³H]-TFP incorporation into the A subunit was drastically decreased in the proteolyzed calcineurin. This was also true when the [³H]-TFP incorporated calcineurin was subjected to tryptic proteolysis. The incorporation into the B unit was essentially unaffected in the trypsinized calcineurin. Phosphatase activity was inhibited by orthovanadate, NaF, PP_i, and EDTA. Inhibitions by these compounds were more pronounced when the phosphatase was determined in the presence of Ca²⁺-cahnodulin than in their absence.     

Calmodulin stimulates polyamine-responsive protein kinase in the absence of Ca2+

A cyclic nucleotide-independent, polyamine-responsive protein kinase from the cytosol of Morris hepatoma 3924A, which phosphorylated heat-stable endogenous substrates and casein in the presence of polyamines (Criss, W.E., Yamamoto, M., Takai, Y., Nishizuka, Y. and Morris, H.P. (1978) Cancer Res. 38, 3540–3545) was observed to be stimulated by an endogenous protein activator. This protein activator was identified to be calmodulin. the polyamine-responsive protein kinase was also stimulated by purified calmodulin, but only in the presence of polyamines such as polylysine. This action of cadmodulin did not require Ca²⁺ for activation of the enzyme; and activation occured in the presence of EGTA. DNA and RNA inhibited the polyamine-responsive protein kinase, either in the presence or absence of Ca²⁺. Purified calmodulin, in the presence of cyclic AMP or cyclic GMP, did not activate the protein kinase. Therefore, polyamines such as polylysine are an absolute requirement for this expression of calmodulin action. The increased enzyme activity by calmodulin was accompanied with an increased V_max and with no changes in the F_m (ATP). High levels of cation, up to 100 mM Mg²⁺, did not effect the action of cadmodulin. These results indicate that tumor cytosolic polyamine-responsive protein kinase is regulated by calmodulin, the latter being increased in the tumor tissue.     

Purification of a phospholipase from Botrytis and its effects on plant tissues

A phospholipase from Botrytis cinerea, grown in pure culture, was purified more than 1000-fold. Whilst it possessed no acyl hydrolase activity toward a variety of p-nitrophenyl fatty acyl derivatives, phosphatidyl choline (lecithin) acted as a substrate; the enzyme being of either type ‘A’ or ‘B’ specificity. When the purified enzyme was applied to washed beetroot discs, betacyanin leakage was induced. Loss of substances which absorb at 260 nm also occurred when washed potato tuber discs were incubated with the enzyme. Incubation with a lysosome-enriched fraction from potato sprout tissues resulted in increased acid phosphatase activity in the incubation supernatant. The phospholipase had no macerating effect on a variety of plant tissues, nor did it cause disruption of isolated protoplasts from these tissues. Studies with mitochondria from mung beans revealed no effects of the enzyme upon the respiratory activity of these organelles. The result suggested that a major site of action of the B.cinerea phospholipase was the lysosomes.     

Identification of pseudo ‘phosphothreonyl-specific’ protein phosphatase T with a fraction of polycation-stimulated protein phosphatase 2A

Protein phosphatase T from rat liver, so termed due to its activity toward [³²P-Thr]casein and its marked preference for the phosphopeptide Arg-Arg-Ala-Thr(P)-Val-Ala over its phosphoseryl derivative (Donella Deana, A., Marchiori, F., Meggio, F. and Pinna, L.A. (1982) J. Biol. Chem. 257, 8565–8568), is shown here to belong to the family of type 2A protein phosphatase according to Cohen's nomenclature (Ingebritsen, T.S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255–261). In particular, protein phosphatase T is endowed with phosphorylase phosphatase activity that is stimulated by protamine, histone H1 and heparin, it is inhibited by spermine, it does not bind to heparin-Sepharose and it readily dephosphorylates the phosphopeptide Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser reproducing the phosphorylation site of the α-subunit of phosphorylase kinase. The M_r of protein phosphatase T determined by gel filtration under non-denaturating conditions is about 150 kDa and its activity ratio toward histone H1 phosphorylated by protein kinase C versus histone H1 phosphorylated by cAMP-dependent protein kinase is unusually high. Some properties of protein phosphatase T, such as its weak binding to DEAE-cellulose and its high stimulation by protamine as compared to a relatively poor stimulation by histone H1, suggest that it may be similar to subtype 2A_o of protein phosphatase 2A.     

Protein phosphorylation in plant mitochondria

Protein kinase activity was detected in osmotically lysed mitochondria isolated from etiolated seedlings of corn, pea, soybean, and wheat, as well as from potato tubers. Ther kinase(s) phosphorylated both endogenous polypeptides and exogenous, nonmitochondrial proteins when supplied with ATP and Mg²⁺. Eight to fifteen endogenous mitochondrial polypeptides were phosphorylated. The major mitochondrial polypeptide labeled in all species migrated during denaturing electrophoresis with an apparent monomeric molecular weight of 47,000. Incorporation of phosphate into endogenous proteins appeared to be biphasic, being most rapid during the first 1 to 2 minutes but slower thereafter. The kinase activity was greatest at neutral and alkaline pH values and utilized ATP with a K_m of approximately 200 micromolar. The kinase was markedly inhibited by CaCl₂ but was essentially unaffected by NaF, calmodulin, oligomycin, or cAMP. These data suggest that plant mitochondrial protein phosphorylation may be similar to protein phosphorylation in animal mitochondria.     

Changes in protein kinase and protein phosphatase properties during the cycle of asparaginase activity in Leptosphaeria michotti

Regulation of the cyclic activity of asparaginase (obtained as a purified protein complex) by a reversible auto-phosphorylation process has been previously reported in the fungus Leptosphaeria michotii (West) Sacc. In the present study, the protein complex was purified in the presence of either a mixture of 3 protein phosphatase inhibitors (fluoride, vanadate and molybdate) or EGTA, during the cycle of asparaginase activity, and the protein kinase and protein phosphatase activities characterized. (I) At the phase of increasing asparaginase activity, a Ca²⁺/calmodulin-dependent kinase activity was identified by (a) its inhibition by calmidazolium, reversed by calmodulin, and its inhibition by EGTA, but not by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole or polylysine (b) an increasing level of calmodulin bound to the complex, as estimated by enzyme-linked immunosorbent assay (ELISA). (2) At the phase of decreasing asparaginase activity, the Ca²⁺-calmodulin-dependent kinase activity disappeared and a little calmodulin remained associated with the complex: phosphorylation of the complex was increased several-fold by 1 nM okadaic acid and 25 nM inhibitor-2, and was not affected by EGTA, indicating a protein phosphatase-2A-like activity. (3) When asparaginase activity was low, a little calmodulin was bound to the complex. The kinase could phosphorylate casein and phosvitin. was inhibited by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole and heparin, stimulated by polylysine and not affected by calmidazolium or EGTA, just as a casein kinase 2. A Ca²⁺-dependent but calmodulin-independent protein phosphatase activity, not affected by okadaic acid and inhibitor-2. was then identified. We postulate the presence in the complex, of (a) only one protein kinase and one protein phosphatase, whose properties could change during the cycle of asparaginase activity: (b) two Ca²⁺/-binding proteins: first calmodulin, which could bind to Ca²⁺ and the casein kinase-2 form to give a Ca²⁺/calmodulin-dependent kinase, which could become Ca²⁺/calmodulin-independent following an auto-phosphorylation process: second a protein homologous to calmodulin, able to bind to the protein phosphatase-2A catalytic subunit to give a protein phosphatase-2B catalytic subunit.     

Abscisic acid and jasmonic acid activate wound-inducible genes in potato through separate, organ-specific signal transduction pathways

Mechanical damage to leaf tissue causes an increase in abscisic acid (ABA) which in turn activates the biosynthesis of jasmonic acid (JA). The resulting higher endogenous JA levels subsequently activate the expression of wound-inducible genes. This study shows that JA induces the expression of different sets of genes in roots and leaves of potato plants. When roots of intact plants were treated with JA, high levels of proteinase inhibitor II (pin2), cathepsin D inhibitor, leucine aminopeptidase and threonine deaminase mRNAs accumulated in the systemic leaves. However, in the treated roots, very low, if any, expression of these genes could be detected. In contrast, a novel, root-specific pin2 homologue accumulated in the JA-treated root tissue which could not be detected in leaves, either systemic or those directly treated with JA. Application of okadaic acid and staurosporine revealed that a protein phosphorylation step is involved in the regulation of this differential response. In leaves, a protein phosphatase is required for the JA-induced expression of pin2 and the other genes analysed. This phosphatase activity is not necessary for the JA-induced expression of a pin2 homologue in roots, suggesting the existence of different transduction pathways for the JA signal in these organs. The requirement of a protein phosphatase activity for JA-mediated gene induction has enabled identification of a JA-independent pathway for ABA induction of pin2 and the other wound-inducible genes. This alternative pathway involves a protein kinase, and appears to be selective for wound-inducible genes. Our data suggest the presence of a complex, organ-specific transduction network for regulating the effects of the plant hormones ABA and JA on gene expression upon wounding.     

Lactoferrin stimulates erythrocyte Na+/K+ -adenosine triphosphatase: effect of some modulators of membrane phosphorylation

We studied the effect of some modulators of signal transduction on the erythrocyte Na+/ K+-ATPase. Go6976 and Go6983 (protein kinase C inhibitors) showed a stimulatory effect and calyculin A (protein phosphatase inhibitor) exerted an inhibitory effect on the Na pump activity. Some of the tested modulators of cell-signaling [protein phosphatase(s), phosphodiesterase, calmodulin and some protein kinases] interfered with the lactoferrin (Lf) stimulatory effect on the sodium pump. Lf itself was able to modulate the effect of some agents upon the pump activity. Moreover, an additive effect of stimulation was found when Lf and some agents were used simultaneously. The summarized results showed that: (i) Lf upregulates the Na+/K+-ATPase in erythrocytes and facilitates the K+ influx into the erythrocytes; (ii) the effect of pump stimulation is mediated by phosphorylation processes. These results suggest a potential opportunity for using Lf alone or together with other agents as a stimulator of the erythrocyte Na+/K+-ATPase.     

Breakdown of Phosphatidylinositol during the Elicitation of Phytoalexin Production in Cultured Carrot Cells

Elicitor-induced production of the phytoalexin, 6-methoxymellein, in cultured carrot cells was appreciably depressed by the calmodulin inhibitors N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and trifluoperazine. An inhibitor of Ca²⁺-phospholipid dependent protein kinase (protein kinase C), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, also inhibited the phytoalexin production in carrot. Both phorbol ester and synthetic diacylglycerol, activators of protein kinase C, showed an ability to induce 6-methoxymellein production even in the absence of elicitor. Phosphatidylinositol-degrading phospholipase activity increased in elicitor-treated carrot cells without a notable lag, and a product of this reaction, inositol trisphosphate, appeared to increase in parallel with the phospholipase activity. These results suggest that breakdown of phosphatidylinositol takes place in the elicitor-treated carrot cells. The messengers liberated from the phospholipid in the plasma membrane may participate in the elicitation process by controlling the activity of protein kinase C-like enzyme(s) and Ca²⁺-mediated processes including calmodulin.     

A calcium-dependent but calmodulin-independent protein kinase from soybean

A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (≈2 micromolar). The protein kinase activity was stimulated 100-fold by ≥10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (≤2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound ⁴⁵Ca²⁺ in the presence of KCl and MgCl₂, which indicates that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity.     

Purification and physiological properties of two lipolytic enzymes of Solanum tuberosum

Two lipolytic enzymes have been separated and partially purified from potato tubers. One enzyme of higher isoelectric value, possessed acyl hydrolase activity toward a number of p-nitrophenyl fatty acyl derivatives, the relative activity depending on the fatty acyl chain length. There was also some activity towards phosphatidyl choline. The other enzyme possessed phospholipase and galactolipase activity, but showed a low acyl hydrolase activity towards p-nitrophenyl fatty acyl derivatives. When applied to plant tissues, the enzyme with the greater acyl hydrolase activity caused rapid ion efflux from discs of potato tuber and beetroot, foflowed by reabsorption of ions by the tissues. The purified phospholipase did not produce this effect but induced acid phosphatase leakage from lysosome-enriched fractions of potato sprout tissue. No maceration of tissue or protoplast disruption was observed when either of the two enzymes were incubated with a variety of plant preparations.     

Dephosphorylation of skeletal muscle phosphorylase,glycogen synthase,and phosphorylase kinase β-subunit by a Mn2+-activated protein phosphatase

An Mn²⁺-activated phosphoprotein phosphatase of M_r = 80,000 from rabbit muscle catalyzes the dephosphorylation of skeletal muscle proteins that are phosphorylated by either phosphorylase kinase or cAMP-dependent protein kinase. Phosphorylase or glycogen synthase labeled by phosphorylase kinase at seryl residues 14 or 7, respectively, are both dephosphorylated by the phosphatase. Phosphorylase a and glycogen synthase compete with one another for the phosphatase. The phosphatase discriminates between different sites labeled by the cAMP-dependent protein kinase: glycogen synthase phosphorylated either to 1.0 or 1.8 mol phosphate/mol, or phosphorylase kinase phosphorylated on its β-subunit serve as substrates for the phosphatase, but the phosphorylase kinase α-subunit, the phosphorylated phosphatase inhibitor 1, or casein do not. Histone fraction IIA, phosphorylated by the catalytic subunit, was a poor substrate even at a concentration of 100 μm. Phosphorylation of the α-subunit of phosphorylase kinase had no influence on the kinetics of dephosphorylation of the β-subunit. Thus, the M_r = 80,000 phosphatase meets the functional definition of a protein phosphatase 1 [Cohen, P. (1978) Curr. Top. Cell. Regul.14, 117–196]. Furthermore, from a comparison of the known phosphorylated sites of these proteins, it appears that the phosphatase discriminates between different sites present in the phosphoproteins tested on the basis of the K_m values for the reactions. It displays a preferential activity toward proteins with a primary structure wherein basic residues are two positions amino-terminal from the phosphoserine, AgrLysX-YSer(P) or LysArgX-YSer(P), rather and one residue away, ArgArgX-Ser(P).     

Dephosphorylation of neuromodulin by calcineurin

Neuromodulin (p57, GAP-43, F1, B-50) is a major neural-specific, calmodulin binding protein found in brain, spinal cord, and retina that is associated with membranes. Phosphorylation of neuromodulin by protein kinase C causes a significant reduction in its affinity for calmodulin (Alexander, K. A., Cimler, B. M., Meirer, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). It has been proposed that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons and that activation of protein kinase C causes the release of free calmodulin at high concentrations near its target proteins. It was the goal of this study to determine whether bovine brain contains a phosphoprotein phosphatase that will utilize phosphoneuromodulin as a substrate. Phosphatase activity for phosphoneuromodulin was partially purified from a bovine brain extract using DEAE-Sephacel and Sephacryl S-200 gel filtration chromatography. The neuromodulin phosphatase activity was resolved into two peaks by Affi-Gel Blue chromatography. One of these phosphatases, which represented approximately 60% of the total neuromodulin phosphatase activity, was tentatively identified as calcineurin by its requirement for Ca2+ and calmodulin (CaM) and inhibition of its activity by chlorpromazine. Therefore, bovine brain calcineurin was purified to homogeneity and examined for its phosphatase activity against bovine phosphoneuromodulin. Calcineurin rapidly dephosphorylated phosphoneuromodulin in the presence of micromolar Ca2+ and 3 microM CaM. The apparent Km and Vmax for the dephosphorylation of neuromodulin, measured in the presence of micromolar Ca2+ and 2 microM CaM, were 2.5 microM and 70 nmol Pi/mg/min, respectively, compared to a Km and Vmax of 4 microM and 55 nmol Pi/mg/min, respectively, for myosin light chain under the same conditions. Dephosphorylation of neuromodulin by calcineurin was stimulated 50-fold by calmodulin in the presence of micromolar free Ca2+. Half-maximal stimulation was observed at a calmodulin concentration of 0.5 microM. We propose that phosphoneuromodulin may be a physiologically important substrate for calcineurin and that calcineurin and protein kinase C may regulate the levels of free calmodulin available in neurons.     

mTOR-dependent suppression of protein phosphatase 2A is critical for phospholipase D survival signals in human breast cancer cells

A critical aspect of tumor progression is the generation of survival signals that overcome default apoptotic programs. Recent studies have revealed that elevated phospholipase D activity generates survival signals in breast and perhaps other human cancers. We report here that the elevated phospholipase D activity in the human breast cancer cell line MDA-MB-231 suppresses the activity of the putative tumor suppressor protein phosphatase 2A in a mammalian target of rapamycin (mTOR)-dependent manner. Increasing the phospholipase D activity in MCF7 cells also suppressed protein phosphatase 2A activity. Elevated phospholipase D activity suppressed association of protein phosphatase 2A with both ribosomal subunit S6-kinase and eukaryotic initiation factor 4E-binding protein 1. Suppression of protein phosphatase 2A by SV40 small t-antigen has been reported to be critical for the transformation of human cells with SV40 early region genes. Consistent with a critical role for protein phosphatase 2A in phospholipase D survival signals, either SV40 small t-antigen or pharmacological suppression of protein phosphatase 2A restored survival signals lost by the suppression of either phospholipase D or mTOR. Blocking phospholipase D signals also led to reduced phosphorylation of the pro-apoptotic protein BAD at the protein phosphatase 2A dephosphorylation site at Ser-112. The ability of phospholipase D to suppress protein phosphatase 2A identifies a critical target of an emerging phospholipase D/mTOR survival pathway in the transformation of human cells.