Protein kinase C-stimulated formation of ethanolamine from phosphatidylethanolamine involves a protein phosphorylation mechanism: negative regulation by p21 Ras protein.

Mammalian cells express a phospholipase D (PLD)-like enzyme which forms ethanolamine from phosphatidylethanolamine (PtdEtn) by a protein kinase C-alpha (PKC-alpha)-activated, presently unknown, mechanism. Now we report that addition of a PKC-alpha-enriched purified PKC preparation or recombinant PKC-alpha to a plasma membrane-enriched membrane fraction, isolated from leukemic HL60 cells, greatly ( approximately 6.5-fold stimulation) enhanced PtdEtn hydrolysis if the PKC activator phorbol 12-myristate 13-acetate (PMA) and ATP were both present; this was accompanied by PKC-mediated phosphorylation of several membrane proteins. The combined effects of PKC-alpha, ATP, and PMA on [(14)C]PtdEtn hydrolysis were inhibited by GF 109203X (10 microM), an inhibitor of catalytic activity of PKC. In this membrane fraction, PMA alone also had a smaller ( approximately 3.5-fold) stimulatory effect on PtdEtn hydrolysis which was not affected by adding ATP or GF 109203X to the membranes. These results suggest that PMA can stimulate PtdEtn hydrolysis via a PKC-catalyzed phosphorylation mechanism as well as by a phosphorylation-independent process. Transformation of NIH 3T3 fibroblasts by H-ras reduced the effect of PMA on PtdEtn hydrolysis. Furthermore, in NIH 3T3 fibroblasts, scrape-loaded Y13-259 anti Ras antibody enhanced PMA-stimulated hydrolysis of PtdEtn. These results suggest that activation of the PtdEtn-hydrolyzing PLD enzyme by PKC-alpha is inhibited by p21 Ras.

Generation and remodeling of phospholipid molecular species in rat hepatocytes.

In order to assess the relationship between de novo phospholipid synthesis and remodeling by deacylation-reacylation, we have pulse-labeled glycerolipids by incubating rat hepatocytes in media containing either [U-14C]glycerol or H218O. Further incubation for up to 2 h in the absence of the labeled substrates and analysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) provided information on the remodeling of newly synthesized molecular species by deacylation-reacylation at the sn-1 or sn-2 positions of glycerol. We conclude that de novo synthesis of PC and PE yields primarily four molecular species: 16:0-18:2 (n-6), 16:0-18:1, 16:0-22:6 (n-3), and 18:1-18:2 (n-6). Remodeling occurs in both lipid classes by replacement of the fatty acids at the sn-1 position with stearic acid, 18:0, or at the sn-2 position with arachidonic acid, 20:4 (n-6). A major molecular species of both PC and PE, 18:0-20:4 (n-6), appears to be produced by deacylation-reacylation at both the sn-1 and sn-2 positions and not to be further remodeled. De novo synthesis and remodeling of PC and PE in rat hepatocytes occur at similar rates, involve precursor-product relationships among newly synthesized molecular species, and therefore may be regulated as a single metabolic process.

Preferential inhibition of phorbol ester-induced hydrolysis of phosphatidylethanolamine by N-acetylsphingosine in NIH 3T3 fibroblasts.

It has been reported that in rat fibroblasts cell-permeable ceramide analogs inhibit agonist-induced phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PtdCho). Here we demonstrate that relatively short (30 min) treatments of NIH 3T3 fibroblasts with 15-60 microM concentrations of N-acetylsphingosine result in preferential, although not exclusive, inhibition of phorbol 12-myristate 13-acetate-induced PLD-mediated hydrolysis of phosphatidylethanolamine (PtdEtn). The results suggest that in different cell types the PtdEtn- and PtdCho-hydrolyzing PLD activities are differentially sensitive to the inhibitory effect of ceramide.

Regulation of phospholipase D by sphingosine involves both protein kinase C-dependent and -independent mechanisms in NIH 3T3 fibroblasts.

Previously, the protein kinase C (PKC) inhibitor sphingosine was found to stimulate phospholipase D (PLD)-mediated hydrolysis of both phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) in NIH 3T3 fibroblasts [Kiss & Anderson (1990) J. Biol. Chem. 265, 7345-7350]. Here we examined the possible relationship between the opposite effects of sphingosine on PKC-mediated protein phosphorylation and PLD activation. After treatments for 3-5 min, sphingosine (25 microM) and the PKC activators phorbol 12-myristate 13-acetate (PMA) (100 nM), bryostatin (100 nM) or platelet-derived growth factor (50 ng/ml) synergistically stimulated the hydrolysis of both PtdEtn and PtdCho in NIH 3T3 fibroblasts prelabelled with [14C]ethanolamine or [14C]choline. Inhibition of PMA-induced phospholipid hydrolysis could also be elicited by sphingosine, but this process required prolonged (60 min) treatments of fibroblasts with 40-60 microM-sphingosine. Similarly to sphingosine, the protein phosphatase inhibitor okadaic acid also had either potentiating or inhibitory effects on PMA-stimulated PLD activity, depending on the length of incubation time and the concentration of PMA. Consistent with the presence of an inhibitory component in the overall action of PKC, the PKC inhibitor staurosporine and down-regulation of PKC activity by prolonged (24 h) treatment with PMA similarly enhanced PLD activity. Data suggest that (a) sphingosine may enhance PMA-mediated phospholipid hydrolysis by neutralizing the action of an inhibitory PKC isoform, and that (b) the stimulatory PKC isoform is less sensitive to the inhibitory action of sphingosine.

Effect of parathion and methylparathion on protein content of chicken embryo muscle in vivo.

Chicken eggs were treated with 0.4 per cent solutions of parathion or methylparathion for four or eight days, and the two-dimensional gel electrophoretic protein pattern of cervical muscles of eighteen days old embryos was analyzed. Both compounds significantly decreased the content of alpha-actinin, alpha-tubulin and beta-tubulin after four days treatment, and, in addition, that of three other related proteins (gamma-proteins) after eight days treatment. Under in vitro phosphorylating conditions, both methylparathion and parathion specifically inhibited the phosphorylation of one isoform of beta-tubulin. Data suggest that the muscle-damaging effects of organophosphorous insecticides, such as parathion and its derivates, may be related to the decrease of tissue content of certain cytoskeletal proteins.

Phorbol ester stimulation of sphingomyelin synthesis in human leukemic HL60 cells.

Pulse-chase experiments, performed with 14C-labeled choline, were used to study the possible effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the terminal step of sphingomyelin (CerPCho) synthesis from phosphatidylcholine in intact human promyelocytic leukemic HL60 cells. Addition of TPA for the chase period significantly increased the rate of CerPCho synthesis; maximal stimulation (104%) required only 3 nM TPA. Treatment of cells with TPA for 6 h also increased the mass of CerPCho by 35%. Sphingosine (25 microM) or H7 (100 microM), inhibitors of protein kinase C (PKC) in vitro, inhibited some, but not all effects of TPA on endogenous protein phosphorylation in intact cells, and failed to inhibit TPA-stimulated synthesis of CerPCho. However, bryostatin, mezerein, 1-oleoyl-2-acetylglycerol, and polymyxin B, previously all shown to stimulate PKC in vivo, also stimulated the synthesis of CerPCho. It is suggested that the effect of phorbol ester on CerPCho synthesis is mediated by a subtype of PKC which responds to known activators of enzyme but is not inhibited by H7 or sphingosine.

Temporal changes in intracellular distribution of protein kinase C during differentiation of human leukemia HL60 cells induced by phorbol ester.

Immunocytochemical methods were used to study protein kinase C (PKC) distribution in HL60 cells during the entire course of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation. After an initial translocation of PKC from cytoplasm to plasma membrane, the enzyme was localized close to the nuclear membrane region at day 1 of TPA treatment. PKC was associated with nuclei at day 2 and with nuclei, cytoplasma and plasma membrane at days 3 and 5. Attachment of cells to substratum (day 2) was accompanied by increased phosphorylation of several nuclear proteins. At day 7, the differentiated cells became detached and PKC in these cells was largely cytoplasmic. In view of the crucial role of PKC in cell differentiation, it is expected that changes in its intracellular localization have physiological significance.

Cooperative interactions of protein kinase C and cAMP-dependent protein kinase systems in human promyelocytic leukemia HL60 cells.

Interactions of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) systems were investigated in HL60 cells. It was found that the differentiating effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) were potentiated by dibutyryl cAMP (dbcAMP) or prostaglandin E2 (PGE2). In addition, dbcAMP or PGE2 inhibited TPA-induced binding of PKC to plasma membrane, leading to decreased protein phosphorylation, and promoted subsequent redistribution of enzyme to the nuclear membrane region. The findings are consistent with the hypothesis that PKC and PKA systems regulate cooperatively the phenotypical differentiation of leukemic cells.

Phorbol ester inhibits phosphatidylserine synthesis in human promyelocytic leukaemia HL60 cells. Possible involvement of free radicals and correlation with phosphorylation of nuclear protein 1b.

Treatment of human promyelocytic leukaemia HL60 cells in conditioned medium with 12-O-tetradecanoylphorbol 13-acetate (TPA) for 4 h resulted in 25-30% inhibition of labelling of phosphatidylserine (PS) with [U-14C]serine. PS labelling was 40% lower, and no inhibitory TPA effect was observed when the experiments were performed in fresh medium. Cycloheximide or puromycin also inhibited PS labelling by 38-44%; their inhibitory effects were non-additive with that of TPA and occurred only in conditioned medium. Catalase (CAT) and superoxide dismutase (SOD), both free-radical scavengers, and H7, a protein kinase C inhibitor, reversed to various extents the inhibitory effect of TPA on PS synthesis. On the other hand, chlorobenzoic acid, a free-radical-generating agent, also inhibited PS synthesis by 22% after 4 h treatment when conditioned medium was used. When ethanolamine was added to cells in conditioned medium to quench PS formation through the exchange of free serine with the ethanolamine moiety of phosphatidylethanolamine (PE), PS labelling was decreased by 33% and the inhibitory TPA effect was significantly decreased. On the other hand, ethanolamine had marginal quenching effect on PS labelling when added to cells in fresh medium. TPA increased the phosphorylation of various proteins in the cells, including protein lb (Mr 80,000; pI 5.5) shown to be localized mainly in the nuclear fraction. Chlorobenzoic acid selectively stimulated the phosphorylation of protein lb, whereas CAT and SOD specifically attenuated the TPA-stimulated phosphorylation of this protein. All these agents affected phosphorylation of protein lb only if conditioned medium was used. The findings suggested that net synthesis of PS through the base-exchange mechanism was stimulated in HL60 cells by cell products present in the conditioned medium. TPA inhibited this stimulated PS synthesis by a mechanism which appeared to involve active oxygen species and protein synthesis and might be related to the phosphorylation of protein lb.

Immunocytochemical localization of protein kinase C in resting and activated human neutrophils.

An immunocytochemical method was used to determine possible changes in the subcellular distribution of protein kinase C (PKC) in human neutrophils in response to opsonized latex beads and zymosan. While in resting cells most of the PKC immunoreactivity was localized in the cytoplasm, a redistribution of PKC to the plasma and phagosomal membranes was observed in cells treated with latex beads or zymosan for 5-20 min, suggesting a participation of PKC in endocytosis.

Comparative effects of polymyxin B, phorbol ester and bryostatin on protein phosphorylation, protein kinase C translocation, phospholipid metabolism and differentiation of HL60 cells.

The effects of protein kinase C (PKC) inhibitor polymyxin B (PMB) and PKC activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin on intact HL60 cells were examined. It was found that each of the three agents exhibited similar effects on phosphorylation of certain endogenous proteins, PKC translocation from cytoplasm to plasma membrane and formation of CDP-choline. TPA, however, was the only agent that stimulated phosphatidylcholine formation. Differentiation of HL60 cells was potently induced by TPA; in comparison bryostatin was a relatively weaker inducer and PMB was without effect. The data indicated that the effects of the PKC inhibitor PMB on intact cells could not be predicted by its in vitro activity, and that certain TPA-dependent but PKC-independent reactions might be crucial in HL60 cell differentiation.

Cyclic AMP-like effects of polyamines on phosphatidylcholine synthesis and protein phosphorylation in human promyelocytic leukemia HL60 cells. Comparison with the effects of phorbol ester.

Spermine or putrescine increased cAMP levels through a catalase-sensitive mechanism, resulting in, most notably, a dephosphorylation of protein A (Mr 45,000, pI 5.15) and protein B (Mr 45,000, pI 4.9) and slightly increased phosphatidylcholine (PC) synthesis in HL60 cells. Exogenous dibutyryl cAMP mimicked the polyamine effects. 12-O-Tetradecanoyl phorbol-13-acetate (TPA) also promoted the protein dephosphorylation and PC synthesis, the effects augmented by R59022 and mimicked by exogenous 1-oleoyl-2-acetylglycerol. The effects of spermine (or dibutyryl cAMP) and TPA on PC synthesis were synergistic. It was suggested that cAMP-dependent protein kinase and protein kinase C might mediate, in an independent but inter-related manner, the effects of polyamines and TPA.

Differential effects of various protein kinase C activators on protein phosphorylation in human acute myeloblastic leukemia cell line KG-1 and its phorbol ester-resistant subline KG-1a.

Human myeloid leukemia KG-1 cells are induced to differentiate to macrophage-like cells by tumor-promoting phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Cells from the cloned subline, KG-1a, unlike the parental line, are resistant to the differentiating effect of TPA. In the present studies, we investigated in these cells protein phosphorylation stimulated by various protein kinase C activators, including 1-oleoyl-2-acetylglycerol in the presence of the diacylglycerol kinase inhibitor R59022, TPA, mezerein, and bryostatin. All the agents stimulated, to a greater extent and with a higher potency, phosphorylation of several proteins in KG-1 cells than in KG-1a cells. On the other hand, these agents markedly stimulated phosphorylation of other proteins in KG-1a cells compared to that in KG-1 cells. The findings indicated that the actions of the diacylglycerol, 1-oleoyl-2-acetylglycerol, and the non-metabolizable activators (TPA, mezerein, and bryostatin) were very similar but not fully equivalent; and that KG-1a cells exhibited altered (increased or decreased) phosphorylation patterns, perhaps related to the TPA resistance characteristic of this subline of cells.

Antileukemic agent alkyllysophospholipid regulates phosphorylation of distinct proteins in HL60 and K562 cells and differentiation of HL60 cells promoted by phorbol ester.

The tumor-promoting 12-0-tetradecanoylphorbol-13-acetate (TPA) stimulated phosphorylation of several proteins in block I (including protein Ia) and protein 3 in HL60 cells. The antileukemic agent alkyllysophospholipid (ALP) inhibited the TPA-stimulated phosphorylation of these proteins and the TPA-induced differentiation of the cells. In comparison, TPA only stimulated phosphorylation of protein 3 in K562 cells which, in contrast, were not induced to differentiate by TPA and lacked protein Ia and had a very high basal phosphorylation of protein B. ALP inhibited phosphorylation of protein 3 as well as protein B in K562 cells. The data suggest that the presence of distinct phosphoproteins and regulation of their phosphorylation may be related to the selective susceptibility of the two leukemia cell lines to the maturating effect of TPA and cytotoxicity of ALP.

Biochemical study of muscle samples from chicken embryos affected by Wofatox 50 EC.

On day 12 of incubation 0.4% and 4.0% aqueous emulsions of Wofatox 50 EC (50% methylparathion) were injected into the air space of the chicken egg. The eggs were opened on the day 19 of incubation and samples were obtained from both cervical and femoral muscles. Atrophy was found only in the cervical muscles by light microscopic evaluation. It is known that the inhibition of acetylcholine esterase causes a permanent inflow and accumulation of Ca2+ especially in the cervical muscle due to the increased mass and energy utilization in the last period before hatching. Changes in the activity of creatine kinase were expressed in a decreased creatine and creatine-phosphate content.

Comparative teratological study of insecticide Wofatox 50 EC (50% methyl-parathion) on chicken and pheasant fetuses.

An experimental insecticide Wofatox 50 EC, injected into the embryonated eggs from hens and pheasants at 12th day of incubation, caused a significantly diminished body mass, a high incidence of developmental malformations and embryonic mortalities at higher dose-levels. The lower, in plant protection practice used concentration was no teratogenic or lethal on embryos.

Teratological examination of Wofatox 50 EC (50% methylparathion) on pheasant embryos.

Wofatox 50 EC is a widely used experimental insecticide, which can expose the pheasants (non-target organisms) during the plant protection practice. The test material was employed in 4 different aqueous emulsions, of which the 2 lowest level (0.2 and 0.4%) corresponded to the concentrations used in plant protection. The total volume of injected emulsions was 0.1 ml per egg on the 12th d of incubation. The macroscopic results showed a dose-dependent maldevelopment (generally cervical lordosis and scoliosis, cyllosis and sporadic thoraco-gastroschisis).