Role of phospholipase A2 and prostaglandin E in growth and differentiation of myeloid leukemic cells

Phospholipase A₂ activity and prostaglandin E synthesis have been studied in different clones of myeloid leukemic cells, which differ in their competence to be induced to differentiate by the macrophage and granulocyte differentiation-inducing protein or the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). Clones that could be induced to differentiate by this protein showed a higher basal phospholipase A₂ activity than clones that could not be induced to differentiate by this protein inducer. Cell competence to be induced to differentiate by TPA did not show this correlation, and the clone with the least ability to respond to TPA showed the lowest number of binding sites for [20-³H]phorbol 12,13-dibutyrate. Differentiation induced by the protein was accompanied by a 7–14-fold increase in prostaglandin E synthesis, whereas differentiation induced by TPA did not show this increase. Externally added prostaglandin E₁ did not induce differentiation but inhibited cell proliferation and the degree of inhibition in the different clones was related to the basal phospholipase A₂ activity. The results indicate that increase of prostaglandin E synthesis was not an essential pre-requisite for differentiation, that prostaglandin E seems to be involved in the inhibition of cell proliferation in association with phospholipase A₂, and that the differentiation-inducing protein and TPA can induce differentiation by different pathways. The amount of basal phospholipase A₂ activity was also related to previously found differences in the ability of the clones to develop desensitization to β-adrenergic hormones or prostaglandin E₁.     

Characterization of Campylobacter concisus hemolysins

Campylobacter concisus is an opportunistic pathogen commonly found in the human oral cavity. It has also been isolated from clinical sources including gastroenteritis cases. Both secreted and cell-associated hemolytic activities were detected in C. concisus strains isolated from children with gastroenteritis. The secreted hemolytic activity of C. concisus strains was labile and was detected in variable levels from fresh-culture filtrates only. In addition, another secreted hemolysin/cytotoxin with a molecular weight < 10 kDa was detected in a single C. concisus strain (RCH 12). A C. concisus genomic library, constructed from strain RCH 3 in Escherichia coli XL1-Blue, was screened for hemolytic clones. Subcloning and sequence analysis of selected hemolytic clones identified ORFs for genes that enhance hemolytic activity but do not appear to be related to any known hemolysin genes found in Gram-negative bacteria. In a previous study, a stable cell-associated hemolysin was identified as an outer-membrane phospholipase A (OMPLA) encoded by the pldA gene. In this study, we report cloning of the pldA gene of the clinical strain C. concisus RCH 3 and the complementation of phospholipase A activity in an E. coli pldA mutant.     

Phospholipase D structure and regulation.

The recent identification of cDNA clones for phospholipase D has opened the door to new types of investigations into its structure and regulation. PLD activity has been found to be encoded by at least two different genes that contain catalytic domains that relate their mechanism of action to phosphodiesterases. In vivo roles for PLD suggest that it may be important for multiples steps in regulated secretion and membrane biogenesis.     

Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.

Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma.     

Mammalian phospholipase D structure and regulation.

The recent identification of cDNA clones for phospholipase D1 and 2 has opened the door to new studies on its structure and regulation. PLD activity is encoded by at least two different genes that contain catalytic domains that relate their mechanism of action to phosphodiesterases. In vivo roles for PLD suggest that it may be important for multiple specialized steps in receptor dependent and constitutive processes of secretion, endocytosis, and membrane biogenesis.     

Expression of recombinant human antibody fragments capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom

Phospholipases A(2) are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. This study used a large nonimmune human scFv library named Griffin.1 (MRC, Cambridge, UK) for selection of recombinant antibodies against antigens present in Bothrops jararacussu venom and identification of specific antibodies able to inhibit phospholipase activity. Four clones were identified as capable of inhibiting this activity in vitro. These clones were able to reduce in vivo the myotoxic activity of BthTX-I and BthTX-II PLA(2), but had no effect on the in vitro anticoagulant activity of BthTX-II. This work shows the potential of using recombinant scFv libraries in the search for antibodies that neutralize relevant venom components.     

Mutant of Escherichia coli K-12 Deficient for Detergent-Resistant Phospholipase A

A mutant deficient for detergent-resistant (DR) phospholipase A was isolated from Escherichia coli K-12. Because the enzyme is membrane-bound and the substrate is a lipid, a special procedure was developed for isolating mutants deficient for the enzyme from agar plates. A sodium dodecyl sulfate (SDS)-sensitive mutant was used as a parental strain for the isolation of DR phospholipase A-deficient mutant. Soft agar containing an unsaturated fatty acid auxotroph and SDS was poured over colonies of the parental strain. The cells were easily solubilized with SDS, and phospholipids were efficiently digested by DR phospholipase A from the colonies on an agar plate. Fatty acids released supported the growth of the indicator bacteria. After the cells of the parent were mutagenized with nitrosoguanidine, colonies which could not support the growth of an unsaturated fatty acid auxotroph in the presence of SDS were selected. Four mutants were isolated after in vitro scre[UNK]ning of DR phospholipase A activity of 30 halo-less clones. Since an extract of the parent strain mixed with that of a mutant strain was still active, it was concluded that the inability to hydrolyze phospholipids was not due to the accumulation of inhibitory substance; the activity of DR phospholipase A in the mutant was less than 1% of the parental activity. Physiological studies indicated that DR phospholipase A is not essential for the growth of E. coli.     

Alpha 2-C10 adrenergic receptors expressed in rat 1 fibroblasts can regulate both adenylylcyclase and phospholipase D-mediated hydrolysis of phosphatidylcholine by interacting with pertussis toxin-sensitive guanine nucleotide-binding proteins.

The alpha 2-C10 adrenergic receptor from human platelets was expressed permanently in Rat-1 fibroblasts. A series of clones that varied in expression of the receptor from 0 to 3.5 pmol/mg of membrane protein were isolated. We have demonstrated recently in cells of one of these clones (1C) that the alpha 2-C10 receptor interacts directly with two distinct pertussis toxin-sensitive G-proteins, Gi2 and Gi3 (Milligan, G., Carr, C., Gould, G. W., Mullaney, I., and Lavan, B.E. (1991) J. Biol. Chem. 266, 6447-6455). High affinity GTPase activity in membranes of cells from the various clones was stimulated by the addition of the alpha 2-adrenergic agonist UK14304, defining that the receptor coupled productively to the G-protein signaling system. Maximal stimulation of high affinity GTPase activity correlated with the levels of receptor expressed. Clones expressing the receptor also demonstrated agonist-mediated inhibition of adenylylcyclase. Futhermore, the alpha 2-C10 receptor in one clone (1C), but not other clones, promoted a marked stimulation in the generation of water-soluble products derived from phosphatidylcholine. The concentration of UK14304 required to produce half-maximal regulation of GTPase activity (20-30 nM), of forskolin-amplified adenylylcyclase activity (30-40 nM), and of choline generation (30-40 nM) were similar. Transphosphatidylation experiments with cells of clone 1C indicated that the receptor-mediated hydrolysis of phosphatidylcholine was via the action of a phospholipase D. All of these effects were attenuated by pretreatment of the cells with pertussis toxin. Dose-effect curves of pertussis toxin-treatment demonstrated similar effective concentrations of the toxin in causing endogenous ADP-ribosylation of both Gi2 and Gi3, inhibition of receptor-stimulated GTPase activity, and phospholipase D activity. Receptor activation of phospholipase D activity was not dependent upon prior phospholipase C-dependent activation of protein kinase C, as alpha 2-adrenergic stimulation of inositol phosphate production was negligible and the presence of the selective protein kinase C inhibitor RO-31-8220, at concentrations up to 10 microM, had no effect on UK14304-mediated production of phosphatidylbutanol. These results demonstrate that expression of the alpha 2-C10 receptor in a heterologous system can result in receptor regulation of signaling elements that appear not to be primary targets for the receptor in vivo. Such results are important in respect to recent observations that transfection of a single defined receptor into separate cell lines can lead to the regulation of distinct effector systems (Vallar, L., Muca, C., Magni, M., Albert, P., Bunzow, J., Meldolesi, J. and Civelli, O. (1990) J. Biol. Chem. 265, 10320-10326).(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of recombinant human antibody fragments capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom

Phospholipases A₂ are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. This study used a large nonimmune human scFv library named Griffin.1 (MRC, Cambridge, UK) for selection of recombinant antibodies against antigens present in Bothrops jararacussu venom and identification of specific antibodies able to inhibit phospholipase activity. Four clones were identified as capable of inhibiting this activity in vitro. These clones were able to reduce in vivo the myotoxic activity of BthTX-I and BthTX-II PLA₂, but had no effect on the in vitro anticoagulant activity of BthTX-II. This work shows the potential of using recombinant scFv libraries in the search for antibodies that neutralize relevant venom components.     

Isolation and characterization of rat 3Y1 fibroblast clones overexpressing the src homology region of phospholipase C-gamma 2.

To examine the regulatory function of the src-related SH2 and SH3 (SH2/SH3) region of phospholipase C-gamma 2 (PLC-gamma 2), we expressed this region of rat PLC-gamma 2 cDNA in rat 3Y1 fibroblasts and isolated and characterized a number of clones (approximately 20 clones). An increase of endogenous tyrosine kinase activity was observed in all cell clones that highly expressed a translational product of the SH2/SH3 domain. Moreover, endogenous phosphatidylinositol 4,5-bisphosphate hydrolyzing activity was also enhanced in these clones, and PLC-gamma 1 seemed to be preferentially activated among endogenous PLC isozymes. Genistein, an inhibitor of tyrosine kinase, inhibited this activation of PLC-gamma 1, and tyrosine phosphorylation was observed on PLC-gamma 1 molecules, indicating the involvement of tyrosine kinases in the PLC-gamma 1 activation. These results suggest that the SH2/SH3 region of PLC-gamma would function as a multidirectional regulator which controls at least two major signaling pathways: tyrosine kinase and phosphatidylinositol 4,5-bisphosphate hydrolysis.     

Phosphatidylcholine-specific phospholipase C-mediated induction of phospholipase D activity in Fas-expressing murine cells

We have previously reported that Fas cross-linking resulted in the activation of phosphatidylcholine-specific phospholipase C (PC-PLC) and the subsequent activation of protein kinase C (PKC) and phospholipase D (PLD) in A20 cells. In an attempt to correlate the existence of PC-PLC activity and activation of PLD by Fas activation among various Fas-expressing murine cell lines, we have investigated the effect of anti-Fas monoclonal antibody on PC-PLC and PLD activities in A20, P388D1 and YAC-1 cell lines. Upon treatment of anti-Fas monoclonal antibody to these three cell lines, the activation of PLD was only observed in A20 cells. When the effect of anti-Fas monoclonal antibody on PKC and PC-PLC activities in Fas-expressing clones were investigated, the activation of PKC and PC-PLC was detected only in A20 clones. Results presented here also show that exogenous addition of Bacillus cereus PC-PLC activates PC hydrolysis, PKC and PLD in all three murine cell lines. These findings suggest that the activation of PC-PLC is a necessary requirement for the activation of PLD by Fas cross-linking and cell lines devoid of functional PC-PLC activity could exhibit enhanced PLD activity by exogenous addition of PC-PLC.     

Interaction of p130 with, and consequent inhibition of, the catalytic subunit of protein phosphatase 1alpha

The protein p130 was originally isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-delta1 but which lacks phospholipase C activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode proteins that interact with p130 has now led to the identification of the catalytic subunit of protein phosphatase 1alpha (PP1calpha) as a p130-binding protein. The association between p130 and PP1calpha was also confirmed in vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of p130 with PP1calpha resulted in inhibition of the catalytic activity of the latter in a p130 concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express p130 and of mouse brain extract with antibodies to p130 and to PP1calpha also detected the presence of a complex of p130 and PP1calpha. The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1calpha, was higher in COS-1 cells that stably express p130 than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca(2+) signaling, p130 might also contribute to regulation of protein dephosphorylation through its interaction with PP1calpha.     

Role of phospholipase D in the activation of protein kinase D by lysophosphatidic acid

Protein kinase D was auto-phosphorylated at Ser916 and trans-phosphorylated at Ser744/Ser748 in Rat-2 fibroblasts treated with lysophosphatidic acid. Both phosphorylations were inhibited by 1-butanol, which blocks phosphatidic acid formation by phospholipase D. The phosphorylations were also reduced in Rat-2 clones with decreased phospholipase D activity. Platelet-derived growth factor-induced protein kinase D phosphorylation showed a similar requirement for phospholipase D, but that induced by 4beta-phorbol 12 myristate 13-acetate did not. Propranolol an inhibitor of diacylglycerol formation from phosphatidic acid blocked the phosphorylation of protein kinase D, whereas dioctanoylglycerol induced it. The temporal pattern of auto-phosphorylation of protein kinase D closely resembled that of phospholipase D activation and preceded the trans-phosphorylation by protein kinase C. These results suggest that protein kinase D is activated by lysophosphatidic acid through sequential phosphorylation and that diacylglycerol produced by PLD via phosphatidic acid is required for the autophosphorylation that occurs prior to protein kinase C-mediated phosphorylation.     

Phospholipase activity of bovine milk lipoprotein lipase on phospholipid vesicles: influence of apolipoproteins C-II and C-III.

The effect of human plasma apolipoproteins C-II and C-III on the hydrolytic activity of lipoprotein lipase from bovine milk was determined using dimyristoyl phosphatidylcholine (DMPC) vesicles as substrate. In the absence of apoC-II or C-III, lipoprotein lipase has limited phospholipase activity. When the vesicles were preincubated with apoC-II and then phospholipase activity determined, there was a time dependent release of lysolecithin; activity was dependent upon both apoC-II and lipoprotein lipase concentrations. The addition of apoC-III to DMPC did not stimulate phospholipase activity. We conclude that apoC-II has an activator effect on the phospholipase activity of lipoprotein lipase and that the mechanism is beyond that of simply altering the lateral compressibility of the lipid.     

Human deoxycytidine kinase. Sequence of cDNA clones and analysis of expression in cell lines with and without enzyme activity

Deoxycytidine kinase (dC kinase) is the rate-limiting enzyme in the anabolism of important anticancer and retroviral nucleoside derivatives. Its activity is often decreased in resistance to these drugs. To analyze the structure, function, and control of this clinically important enzyme we isolated 15 cDNA clones for human deoxycytidine kinase from lambda gt11 thymus and Molt 4 libraries. Four clones were sequenced. The largest clone is 2.9 kilobases and codes for a 626-amino acid open reading frame. The DNA and deduced amino acid sequence of the human dC kinase clones are homologous with a previously unidentified murine cDNA clone p3.4J (EMBL:MM34j) reported to be related to granulocyte-macrophage colony-stimulating factor. Deoxycytidine kinase also has cysteine-rich regions that are homologous with thioredoxin, the beta subunit of prolyl 4-hydroxylase, phosphoinositide-specific phospholipase C, thyroid hormone-binding protein, and protein disulfide isomerase. No differences were seen in the amount and size of deoxycytidine kinase protein and mRNA between CCRF/CEM and L1210 leukemic cell lines that express and do not express enzyme activity. Genomic restriction fragments were similar between the active and inactive CCRF/CEM cell lines. These data suggest that the cells deficient in dC kinase activity have a small defect in the structural gene.     

Cloning, expression and biochemical characterization of a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys pallas

A cDNA encoding a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys Pallas with an N-terminus highly homologous to that of BPLA2 and a C-terminus sequence almost the same as that of APLA2 was inserted into a bacterial expression vector and effectively expressed in Escherichia coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing, the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column superose 12. The purified recombinant enzyme with an isoelectric point of pH 6.8 could cross-react with antiserum prepared against acidic phospholipase A2. The enzymatic activity of the expressed basic-acidic hybrid phospholipase A2-II is close to that of denatured-refolded native basic phospholipase A2, and has the same inhibiting effect on platelet aggregation as denatured-refolded acidic phospholipase A2, but lacks the hemolytic activity of denatured-refolded basic phospholipase A2. To study the structural relationships among basic phospholipase A2, acidic phospholipase A2 and basic-acidic hybrid phospholipase A2-II, molecular modeling of basic-acidic hybrid phospholipase A2-II was done. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.     

Beta gamma-subunit activation of G-protein-regulated phospholipase C.

The availability of purified G alpha 11 and the G-protein-regulated phospholipase C from turkey erythrocytes has allowed an examination of the direct effects of G-protein beta gamma-subunit on the components of the inositol lipid signaling system. Reconstitution of purified turkey erythrocyte or bovine brain beta gamma-subunit into phospholipid vesicles containing G alpha 11 inhibited AlF4- induced activation of phospholipase C. However, beta gamma-subunit at higher concentrations increased phospholipase C activity. This stimulatory effect of beta gamma-subunit on phospholipase C did not require the presence of the alpha-subunit. G alpha o had no effect on the catalytic activity of phospholipase C. However, coreconstitution of G alpha o and beta gamma-subunit shifted to the right the concentration-effect curve for beta gamma-subunit-promoted activation of phospholipase C. As was observed with G alpha 11, the increase in activity observed in the presence of beta gamma-subunit occurred as an increase in the maximal activity and with no change in the apparent affinity for Ca2+ for phospholipase C activation. The concentration dependence of G alpha 11 for activation of turkey erythrocyte phospholipase C and bovine brain phospholipase C-beta, as well as the concentration dependence of the two enzymes for activation by G alpha 11, were very similar. In contrast, beta gamma-subunit was a much less effective activator of bovine brain phospholipase C-beta than the turkey erythrocyte enzyme. The observation of direct effects of free beta gamma-subunit on phospholipase C extend the possibilities for receptor-mediated regulation of this signaling pathway.     

G protein regulation of phospholipase C activity in a membrane-solubilized system occurs through a Mg2(+)- and time-dependent mechanism

GTP-binding proteins have been implicated to function as key transducing elements in the mechanism underlying receptor activation of a membrane-associated phospholipase C activity. In the present study, the regulation of phospholipase C activity by GTP-binding proteins has been characterized in a detergent-solubilized system derived from bovine brain membranes. Guanosine-5'-(3-O-thio)triphosphate (GTP-gamma-S) and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) stimulated a dose-dependent increase in phospholipase C activity with half-maximal activation at 0.6 microM and 10 microM, respectively. The maximal degree of stimulation due to Gpp(NH)p or GTP-gamma-S was comparable. 100 microM GTP had only a slight stimulatory effect on phospholipase C activity. Adenine nucleotides, 100 microM adenylyl-imidodiphosphate and ATP, did not stimulate phospholipase C activity, indicating that specific guanine nucleotide-dependent regulation of phospholipase C activity was preserved in the solubilized state. Gpp(NH)p or GTP-gamma-S stimulation of phospholipase C activity was time-dependent and required Mg2+.Mg2+ regulated the time course for activation of phospholipase C by guanine nucleotides and the ability of guanine nucleotides to promote an increase in the Ca2+ sensitivity of phospholipase C. 200 microM GDP-beta-S or 5 mM EDTA rapidly reversed the activation due to GTP-gamma-S or Gpp(NH)p. These findings demonstrate that G protein regulation of phospholipase C activity in a bovine brain membrane- solubilized system occurs through a Mg2+ and time-dependent mechanism. Activation is readily reversible upon addition of excess GDP-beta-S or removal of Mg2+.     

The effect of phospholipases on the active uptake of norepinephrine by synaptosomes isolated from the cerebral cortex of guinea pig

—Phospholipase A (EC 3.1.1.4) and phospholipase C (EC 3.1.4.3) were used for studying the role of phospholipid of synaptosomal membrane on norepinephrine uptake activity. Synaptosomes were isolated from cerebral cortex of guinea pigs and treated with phospholipase A or phospholipase C before the uptake experiments. Treatment of synaptosomes with phospholipase A has resulted in severe inhibition of norepinephrine-uptake. Under similar conditions, the activity of synaptosomal (Na + K)-ATPase (EC 3.6.1.4) was also inhibited by phospholipase A treatment whereas the activity of synaptosomal acetylcholinesterase (EC 3.1.1.8) was not affected. On the other hand, the norepinephrine-uptake was not influenced by phospholipase C treatment. The inhibition of norepinephrine-uptake after phospholipase A treatment may be due to the liberation of lyso-components of phospholipids since a low concentration of lysolecithin as well as other detergents (deoxycholate and sodium dodecyl sulphate) also inhibit the uptake activity. However, electron microscopic examination indicated that the synaptosomal particles still maintain their morphological features after phospholipase A treatment. It is possible that the active uptake of norepinephrine depends upon the fine arrangement of phospholipids present at the active sites of the synaptosomal membrane.