Synergism of phosphorylation of 46K protein(s) and arachidonate release in the induction of superoxide anion production in guinea pig polymorphonuclear leukocytes

The phosphorylation of 46K protein(s), which was generally observed in parallel with an activation of NADPH oxidase of guinea pig polymorphonuclear leukocytes (PMNL) in our previous studies (N. Okamura et al. (1984) Arch. Biochem. Biophys. 228, 270-277; T. Ohtsuka et al. (1987) J. Biochem. 101, 897-903), was increased by treatment with 1-oleoyl-2-acetylglycerol (OAG), even at low concentrations at which both superoxide anion (O2-) production and arachidonate release were little induced. On the other hand, exposure of PMNL to low concentrations of a calcium ionophore, A23187, stimulated arachidonate release without causing substantial O2- production and increase in phosphorylation of 46K protein(s). Simultaneous addition of the above-mentioned suboptimal concentrations of both OAG and A23187 markedly enhanced O2- production in PMNL. This enhancing effect may be ascribable to the increase in the phosphorylation of 46K protein(s) and arachidonate release, which are induced by the addition of OAG and A23187, respectively. Another arachidonate-releasing agent, N-formyl-methionylleucyl-phenylalanine (FMLP), also stimulated O2- production in accordance with an increase in the arachidonate release and protein phosphorylation. Simultaneous addition of OAG significantly enhanced the FMLP-induced O2- production, presumably by maintaining the 46K protein phosphorylation which would facilitate the effect of arachidonate released by FMLP. Thus, the present results suggest that phosphorylation of 46K protein(s) and arachidonate release synergistically induce O2- production in PMNL, although either of them alone hardly induces the production.     

Stimulation of superoxide anion production in guinea pig polymorphonuclear leukocytes by hypotonic conditions in combination with protein kinase C activators.

Conditions for superoxide anion (O2-) production were examined in guinea pig polymorphonuclear leukocytes (PMNL). When PMNL were suspended in the hypotonic medium, O2- production was significantly enhanced by concurrent treatment with low concentrations of 1-oleoyl-2-acetylglycerol (OAG), a cell-permeable protein kinase C activator. Such hypotonicity or OAG alone had little effect on the production. Other protein kinase C activators also markedly enhanced O2- production in combination with hypotonicity, but not in the isotonic medium. Protein kinase C inhibitors, H-7 and staurosporine, dose-dependently inhibited the production. These observations indicate that protein kinase C participates in such synergistic O2- production with hypotonicity. Phosphorylation of 46-kDa protein(s), which was commonly enhanced in paralleled with an activation of NADPH oxidase in guinea pig PMNL, was increased by treatment with 10 microM OAG, but the phosphorylation was little altered by hypotonic treatment. Intracellular calcium concentration, arachidonate release, and 1,2-diacylglycerol and phosphoinositide concentrations were slightly altered by hypotonic treatment. A change in phosphatidate (PA) production in PMNL was induced by hypotonic treatment either by itself or in combination with OAG treatment. These results suggest that the combination of cell membrane changes by hypotonic treatment accompanied by the increase in PA and 46-kDa protein phosphorylation by protein kinase C provides the conditions required for a marked increase in O2- production. Hypotonicity may be a good tool for studying the mechanism of priming in the activation of NADPH oxidase.     

Significance of phosphorylation/dephosphorylation of 46K protein(s) in regulation of superoxide anion production in intact guinea pig polymorphonuclear leukocytes

Superoxide anion (O2-) production stimulated by concanavalin A (Con A) in guinea pig polymorphonuclear leukocytes (PMNL) was suppressed by addition of methyl-alpha-mannoside, a Con A inhibitor, and resumed upon readdition of Con A. The reversible change in the O2- production was assumed to reflect the change in NADPH oxidase activity measured for the 30,000 X g particulate fraction. The stimulation by Con A of the phosphorylation of 46K protein(s), as observed previously with several membrane-perturbing agents in parallel with an activation of NADPH oxidase in intact guinea pig PMNL (Okamura, N., et al. (1984) Arch. Biochem. Biophys. 228, 270-277), was also suppressed by methyl-alpha-mannoside and resumed upon readdition of Con A. Similar parallelism between the phosphorylation and NADPH oxidase activity was also observed in the case of stimulation by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol 12-myristate 13-acetate (PMA), though both processes were reversible after the stimulation by FMLP but not reversible after that by PMA. Thus, such a parallelism observed in both intact PMNL and 30,000 X g particulate fraction indicates possible involvement of the protein phosphorylation in the regulation of the production of active oxygen metabolites in PMNL.     

Synergism between protein kinase C activator and fatty acids in stimulating superoxide anion production in guinea pig polymorphonuclear leukocytes

Treatment of guinea pig polymorphonuclear leukocytes (PMNL) with various fatty acids elicited superoxide anion (O2-) production and an increase in intracellular Ca2+ [( Ca2+]i). Both responses, however, were seldom observed when PMNL were treated at lower concentrations. But, simultaneous addition of 1-oleoyl-2-acetylglycerol (OAG), a protein kinase C activator, caused an increase in O2- production even at the lower concentrations of fatty acids. In contrast to the synergism in O2- production, [Ca2+]i remained at almost the basal level irrespective of the presence of OAG. Among saturated fatty acids, those with carbon numbers of 14 to 18 were most effective in stimulating O2- production in combination with OAG. Unsaturated fatty acids with a carbon number of 18 were almost equally effective irrespective of the number of double bonds.     

Palmitic acid opens a novel cyclosporin A-insensitive pore in the inner mitochondrial membrane

An assortment of agents can induce mitochondria to undergo a permeability transition, which results in the inner mitochondrial membrane becoming nonselectively permeable to small (<1500 Da) solutes. This mitochondrial permeability transition (MPT) is characterized by a strict dependence on matrix Ca2+ and sensitivity to cyclosporin A (CsA). However, it is becoming increasingly clear that other experimental conditions can elicit increases in mitochondrial permeability that are distinct from this classic MPT. For example, butylated hydroxytoluene (BHT; Sokolove, P. M., and Haley, L. M. (1996) J. Bioenerg. Biomembr. 28, 199-206) and signal peptides (Sokolove, P. M., and Kinnally, K. W. (1996) Arch. Biochem. Biophys. 336, 69-76) promote increases in mitochondrial permeability that are CsA-insensitive. It has been suggested (Gudz, T., Eriksson, O., Kushnareva, Y., Saris, N.-E., and Novgorodov, S. A. (1997) Arch. Biochem. Biophys. 342, 143-156) that BHT might be opening a CsA-insensitive pore by increasing phospholipase A2 activity and thereby producing an accumulation of free fatty acids and lysophospholipids. We have therefore examined the effect of the saturated free fatty acid, palmitic acid (PA), on the permeability of isolated rat liver mitochondria. The following results were obtained: (1) In the absence of additional triggers, PA (20-60 microM) induced concentration-dependent, CsA-insensitive mitochondrial swelling. (2) Swelling required mitochondrial energization. (3) PA-induced swelling was fast and occurred without a lag. (4) Both Ca2+ and Sr2+ supported PA-induced swelling; the site of cation action was the matrix. (5) EGTA and BSA were potent inhibitors of PA-induced swelling. (6) PA opened a pore rather than disrupting mitochondrial membrane structure. (7) The pore opened by PA closed spontaneously. These results suggest that palmitic acid promotes a nonclassic permeability increase that is clearly distinguishable from the occurrence of the MPT.     

A diacylglycerol kinase inhibitor, R 59 022, potentiates superoxide anion production and 46-kDa protein phosphorylation in guinea pig polymorphonuclear leukocytes

A diacylglycerol (DG) kinase inhibitor, R 59 022, potentiated superoxide anion (O2-) production in guinea pig polymorphonuclear leukocytes (PMNL) induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP). R 59 022 also potentiated O2- production induced by 1-oleoyl-2-acetylglycerol, a permeable DG. However, the production induced by phorbol 12-myristate 13-acetate (PMA), a direct activator for protein kinase C, was not potentiated by R 59 022. R 59 022 by itself had no significant effects on unstimulated O2- production. The potentiation of FMLP-induced O2- production by R 59 022 was correlated closely with increased formation of DG and decreased formation of phosphatidic acid, a product of DG kinase. R 59 022 had no effect on the breakdown of phosphoinositides. Phosphorylation of 46-kDa protein(s) by protein kinase C was also examined in relation to O2- production in PMNL. In coincidence with the increase in O2- production, the phosphorylation was potentiated by R 59 022 in the response to FMLP, but not in the response to PMA. In addition, staurosporine, a protein kinase C inhibitor, inhibited increases in both O2- production and phosphorylation of the 46-kDa protein(s) after PMA stimulation. Similar inhibitory effects of staurosporine were also observed upon stimulation with FMLP, irrespective of the presence of R 59 022. These results indicate that retention of DG as a result of the inhibition of further metabolism induces marked stimulation of O2- production via protein kinase C activation in PMNL. These results also provide further evidence for the close relationship between 46-kDa protein phosphorylation by protein kinase C and stimulation of O2- production in PMNL.     

The source of oxygen in the reaction catalysed by collagen lysyl hydroxylase.

The synthetic peptides (Pro-Pro-Gly)5 and (Ile-Lys-Gly)5-Phe were hydroxylated with collagen prolyl hydroxylase and lysyl hydroxylase in an 18O2 atmosphere. The oxygen atoms in the hydroxy groups of hydroxyproline and hydroxylysine were 87% and 6.5% respectively derived from the atmospheric 18O2. The results are consistent with those reported previously for proline hydroxylation in vivo [Fujimoto & Tamiya (1962) Biochem. J. 84, 333-335; Prockop, Kaplan & Udenfriend (1962) Biochem. Biophys. Res. Commun. 9, 192-196; Fujimoto & Tamiya (1963) Biochem. Biophys. Res. Commun. 10, 498-501; Prockop, Kaplan & Udenfriend (1963) Arch. Biochem. Biophys. 101, 499-503] and in vitro [Cardinale, Rhoads & Udenfriend (1971) Biochem. Biophys. Res. Commun. 43, 537-543] and for lysine hydroxylation in vivo [Fujimoto & Tamiya (1963) Biochem. Biophys. Res. Commun. 10, 498-501]. In view of the similarities of these two oxygenase-type hydroxylation reactions the participation of intermediates is proposed, the oxygen atoms of which are exchangeable with those of water. The atmospheric oxygen atoms incorporated into the intermediate must be equilibrated with water oxygen atoms in the slower lysyl hydroxylase reaction.     

Inhibition of neutrophil priming and tyrosyl phosphorylation by cepharanthine, a nonsteroidal antiinflammatory drug.

Receptor-mediated superoxide (O2.-)-generation and tyrosyl phosphorylation of neutrophil proteins, such as 58, 65, 84, 108 and 115 kDa, were enhanced by priming cells with granulocyte colony stimulating factor (G-CSF) [Akimura, K. et al. Arch. Biochem. Biophys. 298: 703-709, 1992]. To elucidate the possible involvement of tyrosyl phosphorylation of neutrophil proteins in the enhancing mechanism of O2.- generation, the effect of cepharanthine, a biscoclaurine alkaloid that inhibits phorbol 12-myristate 13-acetate (PMA)- and receptor-mediated O2.- generation, on the priming of human peripheral neutrophils (HPPMN) was studied. Both enhancement of formyl-methionyl-leucyl- phenylalanine (FMLP)-mediated O2.- generation and tyrosyl phosphorylation of some neutrophil proteins, i.e., 115, 108 and 84 kDa proteins, by HHPMN after treatment with G-CSF were strongly inhibited by cepharanthine in a concentration- and treatment-time-dependent manner. In contrast, inhibition of PMA-mediated O2.- generation by cepharanthine was weak and independent of treatment time. These results suggest that cepharanthine might inhibit the priming step of neutrophil activation concomitantly with its inhibition of the tyrosyl phosphorylation of some neutrophil proteins that might underlie the mechanism for priming of neutrophils with G-CSF.     

Preparation and properties of N6-monobutyryl adenosine 5'-monophosphate. A major hepatic metabolite of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate.

The synthesis and properties of N6-monobutyryl adenosine 5'-monophosphate are described. The properties of synthesized monobutyryl nucleotide have been compared to those of a metabolite recognized in previous studies (Castagna, M. C., Palmer, W.K., and Walsh, D.A. (1977) Arch. Biochem. Biophys. 181, 46-60) as the major radioactive product produced in the liver upon perfusion with N6,O2'-dibutyryl cyclic [3H]adenosine 3':5'-monophosphate. By the criteria of cochromatography on DEAE-cellulose and in three thin layer chromatographic systems and from equivalent rates of alkaline hydrolysis, N6-monobutyryl adenosine 5'-monophosphate has been identified as a major hepatic metabolite of N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate.     

Involvement of protein kinase C in the phosphorylation of 46 kDa proteins which are phosphorylated in parallel with activation of NADPH oxidase in intact guinea-pig polymorphonuclear leukocytes

Two proteins (Mr 46,000, pI 6.4 and 7.0), the phosphorylation of which was increased by any of the membrane-perturbing agents in parallel with activation of NADPH oxidase in intact guinea-pig polymorphonuclear leukocytes in our previous study (Okamura, N., Ohashi, S., Nagahisa, N. and Ishibashi, S. (1984) Arch. Biochem. Biophys. 228, 270-277), were also phosphorylated in a cell-free system prepared from the leukocytes. The in vitro phosphorylation of these two proteins was stimulated by the addition of phosphatidylserine in the presence of higher concentrations of Ca2+ (300-500 microM). The phosphorylation was further increased when protein kinase C partially purified from guinea-pig brain was added to the system. At a low concentration of Ca2+ (about 10 microM), stimulation of the phosphorylation was not attained by phosphatidylserine alone but required the addition of diacylglycerol or phorbol myristate acetate. On the other hand, the increase in the phosphorylation was inhibited by H-7, an inhibitor for protein kinase C. These results indicate that protein kinase C is involved in the phosphorylation of the two proteins, which may be related to the superoxide anion production stimulated by various membrane-perturbing agents.     

Stimulation of protein kinase C and insulin release by 1-oleoyl-2-acetyl-glycerol

The membrane-accessible diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (OAG, 5-500 microM) caused a dose-related activation of protein kinase C in rat islet homogenates. In islet cell membranes exposed to [gamma-32P]ATP, OAG (100 microM) stimulated the net production of labelled phosphatidate and inhibited that of labelled phosphatidylinositol 4-phosphate. In intact islets exposed to 5.6 mM D-glucose, OAG (100 microM) decreased the outflow of 86Rb, increased that of 45Ca and caused a rapid stimulation of insulin release. The secretory response to OAG was dose-related in the 50-500 microM range, being most marked, in relative terms, at a glucose concentration close to the threshold value for stimulation of insulin release by this hexose. It was decreased but not abolished in the absence of CaCl2 and presence of EGTA. At variance with tumor-promoting phorbol esters, OAG failed to potentiate insulin release stimulated by a hypoglycaemic sulphonylurea. Although these findings support the view that activation of protein kinase C by diacylglycerol represents an efficient modality for stimulation of insulin release, they suggest that the effect of OAG upon islet function may not be solely attributable to such an activation.     

Proteolytic degradation of heme-modified hepatic cytochromes P450: A role for phosphorylation, ubiquitination, and the 26S proteasome?

The resident integral hepatic endoplasmic reticulum (ER) proteins, cytochromes P450 (P450s), turn over in vivo with widely varying half-lives. We and others (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992; and Tierney et al., Arch. Biochem. Biophys. 293, 9, 1992) have previously shown that in intact animals, the hepatic P450s of the 3A and 2E1 subfamilies are first ubiquitinated and then proteolyzed after their drug-induced suicide inactivation. Our findings with intact rat hepatocytes and ER preparations containing native P450s and P450s inactivated via heme modification of the protein have revealed that the proteolytic degradation of heme-modified P450s requires a cytosolic ATP-dependent proteolytic system rather than lysosomal or ER proteases (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992). Using purified cumene hydroperoxide-inactivated P450s (rat liver P450s 2B1 or 3A and/or a recombinant human liver P450 3A4) as models, we now document that these heme-modified enzymes are indeed ubiquitinated and then proteolyzed by the 26S proteasome, but not by its 20S proteolytic core. In addition, our studies indicate that the ubiquitination of these heme-modified P450s is preceded by their phosphorylation. It remains to be determined whether, in common with several other cellular proteins, such P450 phosphorylation is indeed required for their degradation. Nevertheless, these findings suggest that the membrane-anchored P450s are to be included in the growing class of ER proteins that undergo ubiquitin-dependent 26S proteasomal degradation.     

Excitation of micelle-solubilized chlorophyll during the peroxidase-catalyzed aerobic oxidation of isonicotinic acid hydrazide

Addition of micelle (hexadecyl-trimethylammoniumbromide)-solubilized chlorophyll alpha to the isoniazid/peroxidase/Mn2+/O2 system promotes light emission, identified as chlorophyll fluorescence. Based on O2 consumption, the quantum yield of chlorophyll excitation to the S1 state exceeds 6 X 10(-6). At least part of the excitation has its origin in the conversion of an intermediate--presumably a diazene--to pyridine-4-carboxaldehyde. On the basis of the present and earlier results [K. Zinner, C. C. C. Vidigal, N. Durán, and G. Cilento (1977) Arch. Biochem. Biophys. 180, 452-458], it is inferred that isoniazid, an important chemotherapeutic and also a carcinogenic agent, can lead to a substantial generation of electronically excited states.     

Rat brain hexokinase: location of the substrate nucleotide binding site in a structural domain at the C-terminus of the enzyme

8-Azido-ATP serves as a substrate for rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1). Irradiation of hexokinase in the presence of this photoactivatable ATP analog results in inactivation of the enzyme. ATP and hexose 6-phosphates (Glc-6-P, 1,5-anhydroglucitol-6-P) previously shown to competitively inhibit nucleotide binding protect the enzyme from photoinactivation; other hexose 6-phosphates do not. Hexoses (Glc, Man) previously shown to enhance nucleotide binding also protect against photoinactivation; other hexoses do not. These effects of hexoses and hexose 6-phosphates can be interpreted in terms of the conformational changes previously shown to result from the binding of these ligands and to influence the characteristics of the nucleotide binding site (M. Baijal and J. E. Wilson (1982) Arch. Biochem. Biophys. 218, 513-524). Limited tryptic cleavage of the enzyme produces three major fragments having molecular weights of about 10K, 40K, and 50K, and thought to represent major structural domains within the enzyme (P. G. Polakis and J. E. Wilson (1984) Arch. Biochem. Biophys. 234, 341-352). Tryptic cleavage of the enzyme, photoinactivated in the presence of 14C-labeled azido-ATP, discloses prominent labeling of the 10K and 40K domains, which are known to originate from the N- and C-terminal regions, respectively. Labeling of the 40K domain is influenced by ligands in a manner that corresponds to the effectiveness of these ligands in protecting against photoinactivation whereas labeling of the 10K domain is not affected by these same ligands. It is concluded that the 40K domain includes the binding site for nucleotide substrates. More refined two-dimensional peptide mapping techniques demonstrate that the predominant site of labeling is a peptide segment, molecular weight approximately 20K, that is located in the central and/or C-terminal region of the 40K domain. Labeling of the 10K domain is attributed to nonspecific interaction of azido-ATP with the hydrophobic sequence shown to be located at the N-terminus of brain hexokinase (P. G. Polakis and J. E. Wilson (1985) Arch. Biochem. Biophys. 236, 328-337).     

Purification and partial characterization of another form of the antiviral protein from the seeds of Phytolacca americana L. (pokeweed).

1. The pokeweed antiviral protein, previously identified in two forms (PAP and PAP II) in the leaves of Phytolacca americana (pokeweed) [Obrig. Irvin & Hardesty (1973) Arch. Biochem. Biophys. 155, 278-289; Irvin, Kelly & Robertus (1980) Arch. Biochem. Biophys. 200, 418-425] is a protein that prevents replication of several viruses and inactivates ribosomes, thus inhibiting protein synthesis. 2. PAP is present in several forms in the seeds of pokeweed. One of them, which we propose to call 'pokeweed antiviral protein from seeds' (PAP-S) was purified in high yield (180 mg per 100 g of seeds) by chromatography on CM-cellulose, has mol.wt. 30 000, and is similar to, but not identical with. PAP and PAP II. 3. PAP-S inhibits protein synthesis in a rabbit reticulocyte lysate with an ID50 (concentration giving 50% inhibition) of 1.1 ng/ml (3.6 x 10(-11) M), but has much less effect on protein synthesis by whole cells, with an ID50 of 1 mg/ml (3.3 x 10(-5) M), and inhibits replication of herpes simplex virus type 1.     

The interaction between Cu(I) superoxide dismutase and hydrogen peroxide

The interaction between superoxide dismutase (SOD) and peroxide, under anaerobic conditions in the presence of an OH radical scavenger, formate, and an indicator, nitro blue tetrazolium, involves five reactions and an equilibrium: (table; see text) Reaction 3 occurs at a rate that is proportional to both peroxide and enzyme with no kinetic evidence for any intermediate peroxide-enzyme complex. Rate studies as a function of pH corroborate previously published work (Fuchs, H. J. R., and Borders, C. L., Jr. (1983) Biochem Biophys. Res. Commun. 116, 1107-1113; Blech, D. M., and Borders, C. L., Jr. (1983) Arch. Biochem. Biophys. 224, 579-586) suggesting that HO2-, and not H2O2, is the active species in this system: k(HO2- + superoxide dismutase-Cu+) = 2.6 x 10(3) M-1 s-1. Evidence is presented which suggests that HO2-, like O2-, reacts at rates that are affected by the electrostatic forces of the enzyme.     

Engineering a disulfide bond in recombinant manganese peroxidase results in increased thermostability

Manganese peroxidase (MnP) produced by Phanerochaete chrysosporium, which catalyzes the oxidation of Mn(2+) to Mn(3+) by hydrogen peroxide, was shown to be susceptible to thermal inactivation due to the loss of calcium [Sutherland, G. R. J.; Aust, S. D. Arch. Biochem. Biophys. 1996, 332, 128-134]. The recombinant enzyme, lacking glycosylation, was found to be more susceptible [Nie, G.; Reading, N. S.; Aust, S. D. Arch. Biochem. Biophys. 1999, 365, 328-334]. On the basis of the properties and structure of peanut peroxidase, we have engineered a disulfide bond near the distal calcium binding site of MnP by means of the double mutation A48C and A63C. The mutant enzyme had activity and spectral properties similar to those of native, glycosylated MnP. The thermostabilities of native, recombinant, and mutant MnP were studied as a function of temperature and pH. MnPA48C/A63C exhibited kinetics of inactivation similar to that of native MnP. The addition of calcium decreased the rate of thermal inactivation of the enzymes, while EGTA increased the rate of inactivation. Thermally treated MnPA48C/A63C mutant was shown to contain one calcium, and it retained a percentage of its original manganese oxidase activity; native and recombinant MnP were inactivated by the removal of calcium from the protein.     

In vivo regulatory phosphorylation site in c(4)-leaf phosphoenolpyruvate carboxylase from maize and sorghum

Reversible seryl-phosphorylation contributes to the light/dark regulation of C₄-leaf phosphoenolpyruvate carboxylase (PEPC) activity in vivo. The specific regulatory residue that, upon in vitro phosphorylation by a maize-leaf protein-serine kinase(s), leads to an increase in catalytic activity and a decrease in malate-sensitivity of the target enzyme has been recently identified as Ser-15 in ³²P-phosphorylated/activated dark-form maize PEPC (J-A Jiao, R Chollet [1990] Arch Biochem Biophys 283: 300-305). In order to ascertain whether this N-terminal seryl residue is, indeed, the in vivo regulatory phosphorylation site, [³²P]phosphopeptides were isolated and purified from in vivo ³²P-labeled maize and sorghum leaf PEPC and subjected to automated Edman degradation analysis. The results show that purified light-form maize PEPC contains 14-fold more ³²P-radioactivity than the corresponding dark-form enzyme on an equal protein basis and, more notably, only a single N-terminal serine residue (Ser-15 in maize PEPC and its structural homolog, Ser-8, in the sorghum enzyme) was found to be ³²P-phosphorylated in the light or dark. These in vivo observations, combined with the results from our previous in vitro phosphorylation studies (J-A Jiao, R Chollet [1989] Arch Biochem Biophys 269: 526-535; [1990] Arch Biochem Biophys 283: 300-305), demonstrate that an N-terminal seryl residue in C₄ PEPC is, indeed, the regulatory site that undergoes light/dark changes in phosphorylation-status and, thus, plays a major, if not cardinal role in the light-induced changes in catalytic and regulatory properties of this cytoplasmic C₄-photosynthesis enzyme in vivo.     

The effects of phorbol ester, diacylglycerol, phospholipase C and Ca2+ ionophore on protein phosphorylation in human and sheep erythrocytes.

Treatment of human or sheep erythrocytes with PMA (phorbol myristate acetate) enhanced [32P]phosphate labelling of membrane polypeptides of approx. 100, 80 and 46 kDa. The 80 kDa and 46 kDa polypeptides coincided with bands 4.1 and 4.9 respectively on Coomassie-Blue-stained gels. Similar but smaller effects were obtained by treating human cells with 1-oleoyl-2-acetyl-rac-glycerol (OAG), exogenous bacterial phospholipase C or ionophore A23187 + Ca2+, each of which treatments would be expected to raise the concentration of membrane diacylglycerol. In contrast, sheep cells, which do not increase their content of diacylglycerol when treated with phospholipase C or A23187 + Ca2+, only showed enhanced phosphorylation with OAG. Neither human nor sheep cells showed any enhanced [32P]phosphate labelling of phosphoproteins when treated with 1-mono-oleoyl-rac-glycerol. It is concluded that diacylglycerol from a variety of sources can activate erythrocyte protein kinase C, but that the most effective diacylglycerol is that derived from endogenous polyphosphoinositides. In contrast with bacterial phospholipase C and A23187, which stimulate synthesis of phosphatidate by increasing the cell-membrane content of diacylglycerol in human erythrocytes, PMA, OAG or 1-mono-oleoyl-rac-glycerol caused no change in phospholipid metabolism.     

The 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae are regulated differentially by phosphorylation via cAMP-dependent protein kinase.

Evidence is presented that demonstrated that the 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae (Morlock, K. R., McLaughlin, J. J., Lin, Y.-P., and Carman, G. M. (1991) J. Biol. Chem. 266, 3586-3593) were regulated differentially by phosphorylation. Purified 45-kDa phosphatidate phosphatase was phosphorylated by cAMP-dependent protein kinase whereas purified 104-kDa phosphatidate phosphatase was not phosphorylated. cAMP-dependent protein kinase catalyzed the phosphorylation of pure 45-kDa phosphatidate phosphatase at a serine residue which resulted in a stimulation (2.4-fold) of phosphatidate phosphatase activity. Alkaline phosphatase catalyzed the dephosphorylation of pure 45-kDa phosphatidate phosphatase which resulted in an inhibition (1.3-fold) of phosphatidate phosphatase activity. Results of studies using mutants (bcy1 and cyr1) defective in cAMP-dependent protein kinase activity corroborated the results of the phosphorylation studies using pure preparations of phosphatidate phosphatase. The 45-kDa phosphatidate phosphatase phosphorylated in vitro and in vivo had phosphopeptides in common. The activation of the GAL10-RAS2val19 allele in mutant cells resulted in an increase in the synthesis of diacylglycerols and triacylglycerols. These results were consistent with the phosphorylation and activation of 45-kDa phosphatidate phosphatase by cAMP-dependent protein kinase in vivo.